Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats.

To investigate the pathogenicities of P. pneumotropica (Pp) and V-factor dependent Pasteurellaceae (VFDP) in immunodeficient rats, experimental infections of F344-rnu rats were performed using 3 strains (ATCC 35149, CNP 160 and RPZ) of Pp and 4 strains (V6, V7, V8 and V9) of VFDP. Four animals per experimental group were inoculated twice on day 0 and post-inoculation day (PID) 14 with bacterial suspension intranasally. Two animals from each group were sacrificed on PID 60 and 120, and examined. In the animals inoculated with strains of Pp, sneezing was observed in some animals inoculated with strains ATCC 35149 and CNP 160 until PID 31. No clinical signs were observed in other animals. The strains were mainly isolated from the nasal cavity and trachea on PID 60, and the nasal cavity, trachea and lung on PID 120. Inflammation and necrosis of nasal cavity mucosa were observed in all animals inoculated with strains ATCC 35149 and CNP 160 in a histopathologic examination. No histopathological changes were observed in any other animal. In the animals inoculated with strains of VFDP, neither clinical disorder nor histopathological change was observed. The strains were mainly isolated from the trachea on PID 60, and from the trachea and lungs on PID 120. From these results, the pathogenicity of Pp in immunodeficient rats appears to differ by strain, and VFDP appears to be non-pathogenic in immunodeficient rats.     

Identification of an Astrovirus Commonly Infecting Laboratory Mice in the US and Japan

Mice (Mus musculus) are the most commonly used laboratory animals. Viral metagenomics on tissues of immunodeficient mice revealed sequences of a novel mammalian astrovirus. Using PCR, we screened mice from 4 breeders, 4 pharmaceutical companies, 14 research institutes and 30 universities in the US and Japan. Mice from one US breeder tested positive while none from Japanese breeders were positive for MuAstV. Mice in over half of the universities (19/30), institutes (7/14) and pharmaceutical animal facilities (2/4) investigated revealed the presence of MuAstV. Nine mice strains tested positive including both immunodeficient strains (NSG, NOD-SCID, NSG-3GS, C57BL6-Timp-3 ^−/−, and uPA-NOG) and immunocompetent strains (B6J, ICR, Bash2, BALB/c). Our data indicates that MuAstV has a wide geographical, institutional and host strain distribution. Comparison of the MuAstV RdRp sequences showed numerous mutations indicating ongoing viral divergence in different facilities. This study demonstrates the need for metagenomic screening of laboratory animals to identify adventitious infections that may affect experimental outcomes.     

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.     

Prevalence of Helicobacter in Laboratory Micein Thailand

Prevalence of Helicobacter is mostly unknown in laboratory animals in Thailand. The 221 mice feces/cecum from 8 universities, 2 pharmaceutical companies and 3 research institutions in Thailand were surveyed for the prevalence and distribution of Helicobacter species by using the Electrochemical DNA chip. Helicobacter were detected 23/46 samples in Specific Pathogen Free (SPF) and 168/175 in conventional condition. Prevalence of Helicobacter were 98%, 96%, 92% and 78% in South (n=40), Northeast (n=40), North (n=25) and Central area (n=116), respectively. Only Central area holds SPF facility resulting in Helicobacter prevalence that seems to be lower than other areas. Three species of Helicobacter were detected in feces/cecum samples by sequence analysis: H. rodentium (67.0%, 148 samples), Helicobacter sp. MIT 01-6451 (15.4%, 34 samples), and unidentified Helicobacter species (14.1%, 9 samples). The results suggested that H. rodentium is the most common species of Helicobacter in laboratory mice in Thailand.     

Analysis of Late-Onset Ovarian Insufficiency after Ovarian Surgery: Retrospective Study with 75 Patients of Post-Surgical Ovarian Insufficiency

The primary objectives of the present study are to determine the period of onset of ovarian insufficiency after surgery and to confirm potential risk factors for ovarian insufficiency after surgery for the removal of benign ovarian cysts. Data were obtained from 75 patients who underwent surgery for benign ovarian cysts prior to the onset of ovarian insufficiency. Our analysis included 835 ovarian insufficiency patients who were referred to our institution from July 2003 to July 2013. Several epidemiological parameters of ovarian insufficiency after surgery (age at operation, period of onset of ovarian insufficiency, operation procedure, and pathological diagnosis) were investigated. Of the 835 patients who had ovarian insufficiency, 75 patients (9.0%) underwent ovarian surgery before the onset of ovarian insufficiency. Of those 75 patients, 66 patients (88.0%) underwent cystectomy. For the majority of the 75 patients the surgical indication was the presence of endometriotic cysts (57 patients; 76.0%). Twelve patients (16.0%) underwent multiple surgeries (all bilateral cystectomies). The mean age of the patients at the time of surgery was 27.8±5.5 years-old, and the mean period of onset of ovarian insufficiency was 5.8±3.8 years. In patients with cystectomy, the patient''s age at the time of surgery and period of onset of ovarian insufficiency was well-correlated (coefficient of correlation; hemilateral endometriotic cystectomy: −0.64, bilateral endometriotic cystectomy: −0.61, and multiple endimetriotic cystectomy: −0.40). We found that cystectomy of endometriotic cysts is the potential risk factor for ovarian insufficiency after surgery, at times, the onset of ovarian insufficiency long after cystectomy. Therefore, it is important to monitor ovarian reserve for an extended period of time after ovarian surgery. It is particularly important to monitor ovarian reserve long-term for patients who wish to conceive in the future and to suggest a variety of infertility treatments appropriate for their ovarian reserve.     

Life history of Juniperus sabina L. adapted to the sand shifting environment in the Mu Us Sandy Land,China: A review

We have reviewed publications on the physiological and ecological features of the growth and regeneration processes of Juniperus sabina L. which grows in semiarid sandy land in the Ordos plateau in northern China where desertification has progressed over time. J. sabina is a key native plant species used for ecological restoration in this region. The life history of J. sabina in this sandy land that has been revealed through this review includes several unique features: (1) both vegetative and seed propagations are observed, but seed propagation is not successful in the location where the mature J. sabina stands. Instead, seed propagation can occur at a different place with different landscapes from the mature stands. (2) Nurse plants play an important role in providing the microclimatic environment necessary for the growth of J. sabina seedlings and young plants. (3) During the horizontal and vertical growth processes of the J. sabina patch, the root system was affected by burial in shifting sand and consequently acquired greater access to the water supply in deeper soil horizons, which could support larger growth. These characteristics suggested that the regeneration by seed propagation and growth strategy of J. sabina in this region was strongly affected by sand movement and the landscape that is generated by sand movement.

     

Comparison of oxygen consumption in drosophilid flies from different climates

Oxygen consumption at rest was studied in drosophilid species from cool‐temperate, warm‐temperate and subtropical regions to assess whether adaptations to different climates are associated with changes in metabolic rates. In experiments at 23°C using 8‐day‐old males of 28 species, body mass was revealed to be a significant predictor of oxygen consumption. No significant relation was detected between mass‐adjusted oxygen consumption and latitudinal distribution or thermal tolerance by either conventional regression analysis or a phylogenetically based method. The effect of temperature on oxygen consumption was studied with experiments at 15, 18, 23 and 28°C using 8‐ and 24‐day‐old males of four species of each of the montium species subgroup and the subgenus Drosophila. In these experiments, it was confirmed that temperature was a significant predictor of mass‐adjusted oxygen consumption. In both lineages, mass‐adjusted oxygen consumption was not higher in cool‐temperate species than in subtropical species. Thus, adaptations to colder climates are not associated with elevation of metabolic rates in these drosophilid species. The results of the present study also indicate that oxygen consumption is not related to the capacity to walk quickly.     

Higher diversity and abundance of denitrifying microorganisms in environments than considered previously

Denitrification is an important process in the global nitrogen cycle. The genes encoding NirK and NirS (nirK and nirS), which catalyze the reduction of nitrite to nitric oxide, have been used as marker genes to study the ecological behavior of denitrifiers in environments. However, conventional polymerase chain reaction (PCR) primers can only detect a limited range of the phylogenetically diverse nirK and nirS. Thus, we developed new PCR primers covering the diverse nirK and nirS. Clone library and qPCR analysis using the primers showed that nirK and nirS in terrestrial environments are more phylogenetically diverse and 2–6 times more abundant than those revealed with the conventional primers. RNA- and culture-based analyses using a cropland soil also suggested that microorganisms with previously unconsidered nirK or nirS are responsible for denitrification in the soil. PCR techniques still have a greater capacity for the deep analysis of target genes than PCR-independent methods including metagenome analysis, although efforts are needed to minimize the PCR biases. The methodology and the insights obtained here should allow us to achieve a more precise understanding of the ecological behavior of denitrifiers and facilitate more precise estimate of denitrification in environments.