Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli OX18 from a Fatal Hemolytic Uremic Syndrome Case.

We report a fatal case of hemolytic uremic syndrome with urinary tract infection in Japan caused by Shiga toxin-producing Escherichia coli. We genotypically identified the isolate as OX18:H2. Whole-genome sequencing revealed 3 potentially pathogenic lineages (OX18:H2, H19, and H34) that have been continuously isolated in Japan.

Vibrio cholerae Infection in Japan Not Associated with Overseas Travel.

A 74-year-old Japanese man who was taking antacids presented with profuse diarrhea. Stool culture revealed Vibrio cholerae O1 strain, serogroup Ogawa, biotype El tor. He recalled he had consumed some sashimi but denied any history of travelling abroad, and another cholera case with almost the same strain was reported at the same time in a remote prefecture in the Kanto area. This is a rare case of travel-unrelated cholera in Japan, and it illustrates the importance of suspecting cholera in all patients presenting with large volumes of watery diarrhea in Japan, especially in those who are taking antacids, regardless of their international travel history.

Assessment of the local clonal spread of Streptococcus pneumoniae serotype 12F caused invasive pneumococcal diseases among children and adults.

BACKGROUND: We conducted active surveillance to elucidate the distribution of Streptococcus pneumoniae serotypes causing invasive pneumococcal disease (IPD) and clarified the genetic relatedness among the isolates in Kobe City, Japan. METHODS: Forty-five IPD-causing S. pneumoniae strains were analyzed from March 2016 to May 2018 through active surveillance in Kobe City, Hyogo, Japan. Serotypes were determined by multiplex serotyping PCR and the Quellung reaction with pneumococcal antisera. Fourteen Sp12F strains were subjected to whole-genome sequencing (WGS). RESULTS: Among 45 isolates, the most frequent serotypes were 12F (n=14, 31%), 24F (n=5, 11%), and 10A (n=4, 9%). Multilocus sequence typing (MLST) analysis of 14 isolates of Sp12F divided them into ST4846 (n=4) and ST6495 (n=10). WGS showed clonality of the 10 isolates of ST6495, with only 13 single nucleotide polymorphisms in the genomes. Meanwhile, ST4846 strains in Kobe differed from only the outbreak strains of Sp12F ST4846 in Tsuruoka, Japan, reported on 2018. CONCLUSIONS: Serotype monitoring showed Sp12F to be the predominant serotype in Kobe, and WGS revealed the clonal spread of Sp12F ST6495 in this city. Thus, the spread of Sp12F could become a serious public health problem in Japan, warranting thorough monitoring in future.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.

Long-term monitoring of fecal steroid hormones in female snow leopards (Panthera uncia) during pregnancy or pseudopregnancy.

Knowledge of the basic reproductive physiology of snow leopards is required urgently in order to develop a suitable management conditions under captivity. In this study, the long-term monitoring of concentrations of three steroid hormones in fecal matter of three female snow leopards was performed using enzyme immunoassays: (1) estradiol-17ß, (2) progesterone and (3) cortisol metabolite. Two of the female animals were housed with a male during the winter breeding season, and copulated around the day the estradiol-17ß metabolite peaked subsequently becoming pregnant. The other female was treated in two different ways: (1) first housed with a male in all year round and then (2) in the winter season only. She did not mate with him on the first occasion, but did so latter around when estradiol-17ß metabolite peaked, and became pseudopregnant. During pregnancy, progesterone metabolite concentrations increased for 92 or 94 days, with this period being approximately twice as long as in the pseudopregnant case (31, 42, 49 and 53 days). The levels of cortisol metabolite in the pseudopregnant female (1.35 µg/g) were significantly higher than in the pregnant females (0.33 and 0.24 µg/g) (P<0.05). Similarly, during the breeding season, the levels of estradiol-17ß metabolite in the pseudopregnant female (2.18 µg/g) were significantly higher than those in the pregnant females (0.81 and 0.85 µg/g) (P<0.05). Unlike cortisol the average levels of estradiol-17ß during the breeding season were independent of reproductive success.The hormone levels may also be related to housing conditions and the resulting reproductive success in female leopards. The female housed with a male during the non-breeding season had high levels of cortisol metabolites and low levels of estradiol-17ß in the breeding season, and failed to become pregnant. This indicates that housing conditions in snow leopards may be an important factor for normal endocrine secretion and resulting breeding success.

Development of an enzyme immunoassay for urinary pregnanediol-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

In order to enable monitoring of the reproductive status of the female giant panda after observation of estrus behavior, we developed an enzyme immunoassay (EIA) system for urinary pregnanediol-3-glucuronide (PdG), a progesterone metabolite, using commercial reagents and examined the changes in the urinary concentration of PdG in a female giant panda that showed pseudopregnancy and suspicious pseudopregnancy in 6 consecutive years. The developed EIA system had good reproducibility (intra- and interassay CVs 6.1% and 16.3%, respectively), good parallelism between the standard curve and the dose response curve of serial diluted samples and positive correlation (r=0.836) with the data for PdG in the same samples measured by gas chromatography. Urinary PdG in the female panda showed two phases of increase. The first elevation was observed immediately after estrus with the levels of PdG below 100 ng/Crmg, while the second phase was characterized by a drastic elevation above 100 ng/Crmg until the level began to decrease at the end of pseudopregnancy or suspicious pseudopregnancy. The length of the second phase had wider range than that of the first phase. In the present study, a new EIA assay system for urinary PdG in the female giant panda was developed, and we found that the length of the second phase is unstable in the pseudopregnant and suspicious pseudopregnant giant panda, in contrast with the unstable length of the first phase caused by delayed implantation in the pregnant giant panda.

Light and scanning electron microscopic study on the tongue and lingual papillae of the common hippopotamus, Hippopotamus amphibius amphibius.

We observed the three-dimensional structures of the external surface and connective tissue cores CTCs, after exfoliation of the epithelium of the lingual papillae (filiform, fungiform, and foliate papillae) of the common hippopotamus (Hippopotamus amphibius amphibius) using scanning electron microscopy and conventional light microscopy. Following unique features were found; typical vallate papillae with a circumferential furrow were not observable. Instead, numerous large fungiform papillae were rather densely distributed on the posterior of the lingual prominence. Taste buds were observable only on the dorsal epithelium. Serous lingual gland was not seen in the lamina propria; however, mucous-rich mixed lingual glands were found and in a few of orifices were seen on the large fungiform tops. Lingual prominence was diminished their width. Rather long and slender conical papillae were distributed on the lingual prominence and were similar to nonruminant herbivore, that is donkey. Beside this narrow lingual prominence, lateral slopes were situated with numerous short spine-like protrusions. After removal of the epithelium, CTCs of lateral slopes exhibited attenuated flower bud structures. Large-conical papillae were situated on the root of the tongue. These large conical papillae were not seen among ruminants and seen on the lingual root of omnivores and carnivores. It implies that lingual structure of common hippopotamus possessed mixed characteristics between Perissodactyls, Ruminantia, and nonherbivores such as Suiformes because of their unique evolutionally taxonomic position. Moreover, adaptation for soft grass diet and associating easier mastication may be also affecting these mixed morphological features of the tongue.

Light and scanning electron microscopic study on the lingual papillae and their connective tissue cores of the Cape hyrax Procavia capensis.

We examined the epithelial surface and connective tissue cores (CTCs) of each lingual papilla on the Paenungulata, Cape hyrax (Procavia capensis), by scanning electron microscopy and light microscopy. The tongue consisted of a lingual apex, lingual body and lingual root. Filiform, fungiform and foliate papillae were observed on the dorsal surface of the tongue; however, fungiform papillae were quite diminished on the lingual prominence. Moreover, no clearly distinguishable vallate papillae were found on the tongue. Instead of vallate papillae, numerous dome-like large fungiform papillae were arranged in a row just in front of the rather large foliate papillae. Foliate papillae were situated in the one-third postero-lateral margin of the lingual body. The epithelium of filiform papillae was covered by a keratinized layer with kerato-hyaline granules, whereas weak keratinization was observed on the interpapillary epithelium. The external surface of the filiform papillae was conical in shape. CTCs of the filiform papillae were seen as a hood-like core with a semicircular concavity in the anterior portion of each core. Large filiform papillae were distributed on the lingual prominence. The CTCs of large filiform papillae after exfoliation of their epithelium consisted of a concave primary core and were associated with several small protrusions. The surface of fungiform papillae was smooth and dome-like. After removal of the epithelium, CTCs appeared as a flower bud-like primary core and were associated with several protrusions that were arranged on the rim of the primary core. Several taste buds were found on the top of the dorsal part of the epithelium of both fungiform and large fungiform papillae. Well-developed foliate papillae were seen and numerous taste buds could be observed in the lateral wall of the epithelium in a slit-like groove. The morphological characteristics of the tongue of the Cape hyrax had similarities with other Paenungulata such as Sirenia. However, three-dimensional characteristics, especially CTCs of lingual papillae, exhibited multiple similarities with rodents, insectivores and artiodactyls.

Development and evaluation of a rapid enzyme-immunoassay system for measurement of the urinary concentration of estrone-3-glucuronide in a female giant panda (Ailuropoda melanoleuca).

To detect estrus for reproductive management, and to determine the relationship between urinary estrogen and estrous behavior, in a female giant panda, we developed and evaluated a rapid enzyme immunoassay (EIA) system for urinary Estrone-3-glucuronide (E1G) using commercial reagents. The developed EIA system took only around 3 hours, including all procedures to obtain a result. It indicated good reproducibility (intra-assay CV of 5.16%, interassay CV of 15.4%) and sensitivity (lowest standard concentration was 0.0156 ng/ml) for measurement of the urinary concentrations of E1G in the giant panda. There was a positive correlation (r=0.934) with the data for estrone (E1) in the same samples, as measured by radioimmunoassay (RIA) performed in a commercial laboratory. The changes in the E1G concentrations were almost synchronous with the changes in E1 assayed by RIA in urine collected during 4 consecutive estrous seasons. The dynamics of urinary E1G measured by this system highly correlated with the occurrence of the presenting estrous behavior in the giant panda. The above results indicate that this assay system may be normally, rapidly and practically used for measurement of the urinary concentration of E1G in the giant panda.

Comparative morphological studies on the stereo structure of the lingual papillae of selected primates using scanning electron microscopy.

A scanning electron microscope was used to observe the lingual papillae and their connective tissue cores (CTCs) in five primates (tupai, tamarin, crab-eating monkey, mandrill, and human). There were some slender protrusions rising from the top of the filiform papilla in all five types of primate. After removing the epithelium the filiform CTC from the tupai, tamarin and crab-eating monkey displayed a U-shaped arrangement of rod-shaped protrusions. The filiform CTC from the crab-eating monkey also had a columnar base. The human filiform CTC consisted of a primary columnar base, numerous short rod-shaped secondary protrusions from its upper periphery, and a few central protrusions. The filiform CTC from the Mandrill was fundamentally similar to that of the human, however, its base was shorter. The fungiform CTC from the tupai was column shaped, with several depressions for taste buds on the top. There were three vallate papillae in the tupai, tamarin, and mandrill, approximately four in the monkey, and between five and twelve in the human. Moderately developed foliate papillae were found in the tamarin, monkey, mandrill and human. The tupai, however, possessed a finger-like lateral organ instead. The lingual root area of the tupai, tamarin, crab-eating monkey and mandrill was relatively small with a smooth surface. Only the human had a tonsil-structure, which was located on the surface of its larger lingual root.