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Animal-mediated pollination is an essential ecosystem service for the production of many fruit trees. To reveal the community composition of flower-visiting wild insects which potentially contribute to fruit production and to examine the effects of geographic location, local meteorological conditions and locally introduced domesticated pollinators on them, we investigated the community composition of insects visiting the flowers (hereafter, "visitors") of apple, Japanese pear and Oriental persimmon for 1‒3 years at 20 sites around Japan. While most of the variation (82%) of the community composition was explained by tree species with a slight contribution by geographic distance (2%), maximum temperature and tree species contributed 62% and 41% of the variation in total abundance of the visitors, respectively. Though the dominant families of the visitors varied spatiotemporally, the community composition of the visitors of apple and Japanese pear clearly differed from that of Oriental persimmon. While Andrenidae and Syrphidae together accounted for 46%‒64% of the visitors of apple and Japanese pear, Apidae represented 57% of the visitors of Oriental persimmon. The taxonomic richness, diversity and evenness of the visitors were best predicted by locally introduced domesticated pollinators and local meteorological conditions of wind speed and maximum temperature. Amongst these selected factors, locally introduced domesticated pollinators could have the largest impact. It seemed to be strongly related to the reduction of taxonomic richness, diversity and evenness of the visitors, accounting for 41‒89% of the variation. Results suggested that the community composition and total abundance of potential pollinators were predominantly determined by tree species and temperature, but locally introduced domesticated pollinators could have a determinantal pressure on the taxonomic diversity of the community., Competing Interests: No conflict of interest to declare Disclaimer: This article is (co-)authored by any of the Editors-in-Chief, Managing Editors or their deputies in this journal., (Shoko Nakamura, Hisatomo Taki, Tomonori Arai, Ken Funayama, Shunsuke Furihata, Yuki Furui, Takamasa Ikeda, Hiromitsu Inoue, Kiyohiko Kagawa, Hidenari Kishimoto, Mitsuko Kohyama, Michiyo Komatsu, Akihiro Konuma, Ken Nakada, Suguru Nakamura, Nobuo Sawamura, Shoji Sonoda, Masahiro Sueyoshi, Seishi Toda, Katsuhiko Yaginuma, Shunsuke Yamamoto, Koki Yoshida, Tomoyuki Yokoi, Masatoshi Toyama.)

Fat atrophy and adipose tissue inflammation can cause the pathogenesis of metabolic symptoms in chronic kidney disease (CKD). During CKD, the serum levels of advanced oxidation protein products (AOPPs) are elevated. However, the relationship between fat atrophy/adipose tissue inflammation and AOPPs has remained unknown. The purpose of this study was to investigate the involvement of AOPPs, which are known as uremic toxins, in adipose tissue inflammation and to establish the underlying molecular mechanism. In vitro studies involved co-culturing mouse-derived adipocytes (differentiated 3T3-L1) and macrophages (RAW264.7). In vivo studies were performed using adenine-induced CKD mice and AOPP-overloaded mice. Fat atrophy, macrophage infiltration and increased AOPP activity in adipose tissue were identified in adenine-induced CKD mice. AOPPs induced MCP-1 expression in differentiated 3T3-L1 adipocytes via ROS production. However, AOPP-induced ROS production was suppressed by the presence of NADPH oxidase inhibitors and the scavengers of mitochondria-derived ROS. A co-culturing system showed AOPPs induced macrophage migration to adipocytes. AOPPs also up-regulated TNF-α expression by polarizing macrophages to an M1-type polarity, and then induced macrophage-mediated adipose inflammation. In vitro data was supported by experiments using AOPP-overloaded mice. AOPPs contribute to macrophage-mediated adipose inflammation and constitute a potential new therapeutic target for adipose inflammation associated with CKD.

Equine proliferative enteropathy (EPE) is an equine infectious disease that can lead to severe weight loss and hyperplasia of the intestinal mucosa due to infection with Lawsonia intracellularis. In this study, we investigated the prevalence of EPE in a major Thoroughbred breeding area: Hidaka district, Hokkaido, Japan. Of the 252 symptomatic horses that we tested, 192 EPE cases (76.2%), including 8 fatal cases, were confirmed from April 2015 to March 2020 by etiological and/or serological investigation. Most of the EPE cases were observed in foals (88.5%), with fewer cases in yearlings (7.3%) and adults (4.2%). Asymptomatic infection was observed in 62.9% of the horses kept with affected horses. These results suggest that EPE is an enzootic disease in Hidaka district., (©2022 The Japanese Society of Equine Science.)

Porcine circovirus 2 (PCV2) causes porcine circovirus-associated disease, and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) severely affects the pig breeding industry. Both viruses target the macrophages in lymphoid tissues. Various porcine pathogens enter via the nasal cavity, and the nasopharynx-associated lymphoid tissue (NALT) acts as the mucosal immune system. However, the pathological analysis has not progressed. This study aimed to histologically examine the NALT of pigs with suspected PCV2 and PRRSV infections. Six pigs were subjected to necropsy, and their NALT, tonsils, and mesenteric lymph nodes were collected. Macrophages, lymphocytic depletion, multinucleated giant cells, intracytoplasmic inclusion bodies, and neutrophil infiltration increased in the NALT. In situ hybridization revealed positive signals for PCV2 in the NALT of all pigs and PRRSV in the NALT of three pigs. PCV2-positive macrophages were mainly identified in the follicles, whereas PRRSV-positive tissues were found primarily around the crypt and directly below the epithelium. Quantitative PCR revealed 10 8 -10 10 copies of PCV2 DNA/µL and 10 2 -10 4 copies of PRRSV DNA/µL in the NALT. Therefore, both PCV2 and PRRSV were detected in the NALT of pigs. In conclusion, the infection and replication of both viruses in the NALT and tonsils may suppress host immunity and promote co-infection with other pathogens.

Background: Mycoplasma bovis causes various diseases such as bronchopneumonia, otitis media, arthritis, and mastitis in cattle. Mycoplasma bovis is often isolated from the deep pharynges of healthy cattle and is generally considered not to cause clinical symptoms while in the upper respiratory tract. In mycoplasma infections, adhesion to the host cells is a crucial step. In recent years, five new adhesins, NOX, α-enolase, TrmFO, P27, and VpmaX, have been reported in M. bovis strains from pneumonia cases. However, the presence of these adhesins in wild isolates has not been established., Aim: This study aimed to investigate the presence of these adhesin genes in wild isolates isolated from cattle nasal cavities and lesion sites (pneumonia, otitis media, arthritis, and mastitis) in various regions in Japan and clarify the relationship between adhesion and the symptoms caused by M. bovis infection., Methods: A total of 141 M. bovis wild isolates isolated from nasal cavities (healthy or sick cattle), lungs with pneumonia, ears with otitis media, joint fluids of arthritic animals, and milk of mastitic animals. Mycoplasma bovis type strain PG45 was also used. Specific polymerase chain reaction reactions were performed to detect nox , α-enolase , trmFO , P27 , and vpmaX , which are adhesins of M. bovis ., Results: This study reports 139 M. bovis wild isolates were positive for nox , α-enolase , trmFO , P27 , and vpmaX , while two isolates each lacked α-enolase or P27 genes. Mycoplasma bovis PG45 also had all five adherens genes., Conclusion: Almost all M. bovis wild isolates possessed all nox , α-enolase , trmFO , P27 , and vpmaX genes regardless of the lesion site or region of origin. This means no relationship was found between the presence of the five adhesins and lesion sites in M. bovis and M. bovis isolated from the nasal cavities of asymptomatic cattle have the same numbers and types of adhesins as isolates from symptomatic lesion sites (pneumonia, otitis media, arthritis, and mastitis). This suggests that not only M. bovis isolates from pulmonary lesions, but also M. bovis existing in the nasal cavity has the potential to causes symptoms in the host., Competing Interests: The authors declare that there is no conflict of interest.

Adequate management and conservation of instream thermal habitats requires an understanding of the control that different landscape features exert on water temperatures. Previous studies have extensively explored the influence of spatial scale on these relationships. However, the effect of temporal scale remains poorly understood. Here, we use paired air-water mean daily and monthly summer temperatures collected over four years from 130 monitoring stations in Japanese mid- to low-order streams to investigate whether perceived effects of different environmental controls on water temperature are dependent on the timescale of the temperature data, and whether those dependencies are related to the spatial scale at which these controls operate. We found a clear pattern for the significant cooling effect, high relative importance and strong dominance exerted by the riparian forest cover on daily temperatures at the reach scale becoming dampened by concomitant increases associated to the proportion of volcanic geology on monthly temperatures at the catchment scale. These results highlight the importance of contextualizing the effects of environmental controls on water temperatures to the timescale of the analysis. Such dependencies are particularly important for the management and conservation of instream thermal habitats in a rapidly warming world., (© 2022. The Author(s).)

Growth promoting antibiotics are used in modern animal husbandry to promote growth and avoid infections. Negative effects of these antibiotics on human health are a big concern and they need to be replaced. Probiotics are expected to be a good replacement for growth promoting antibiotics. In this study, we evaluated effects of probiotics on growth and immune status of liver and secondary lymphoid organs of piglets. Body weights of probiotic-fed piglets were heavier than those of control piglets (p < 0.05) at days 30 and 45 of the experiment. Relative populations of CD4+ and IgM+ cells isolated from the liver were significantly increased (p < 0.05 and p < 0.01, respectively) in probiotic-fed piglets compared to control piglets. CD4+CD8+ T cells were significantly decreased (p < 0.05) in jejunal Peyer’s patches of treated piglets. Phagocytosis of MHC class II+ cells isolated from the liver of probiotic-fed piglets was significantly higher (p < 0.05) than that of control piglets. Phagocytosis of granulocytes isolated from the liver and peripheral blood of probiotic-fed piglets were also higher than those of control piglets. These results indicate excellent effects on growth and immune status of piglets. In conclusion, probiotics have beneficial effects on the growth and health of piglets and could be good replacement for growth promoting antibiotics.

In Japan, many pig producers buy antimicrobials using veterinarians' prescriptions. Even if veterinarians prescribe antimicrobials for treatments and metaphylaxis, producers may be using them for prophylaxis on farms with a high incidence of chronic diseases. To reduce the frequency of antimicrobial use, we applied the concept of medication adherence and launched an intervention in eight farrow-to-finish farms. For the intervention farms, we attempted to implement measures for preventing the repeated onset of chronic infections that interfered with adherence, confirmed the need to continue medications, and discontinued medications if possible. For each farm, these interventions began at different time points after 2017 and continued until 2020. We evaluated the effectiveness of this intervention based on the amount of active ingredients/population correction unit and the total number of defined daily doses for the weight group/slaughter pig. The ratio of antimicrobial use for both indicators in the year 2020 compared with the start of the intervention decreased on all farms. Among the eight non-intervention farms, six showed an increase in both indicators. These results suggest that interventions for chronic diseases administered by producers and veterinarians lead to high adherence to prescriptions and responsible and prudent use of antimicrobials on pig farms., (© 2022 Japanese Society of Animal Science.)

When a large-scale epidemic of porcine epidemic diarrhea (PED) occurred in 2013 in Japan, feedback feeding (feeding feces and gut tissues of infected piglets) was attempted to impart immunity to sows and immunize nursing piglets via breastfeeding. This study evaluated the effect of feedback feeding on PED control at 172 farms in Kagoshima and Miyazaki Prefectures. Univariable and multivariable generalized linear models were used to analyze the associations between conduct of feedback feeding and damage from the outbreak (outbreak period and the number of piglet deaths) at the farm level. The within-farm outbreak period shortened over time after the regional outbreak began on Kyushu Island (P=0.009) and was longer on large-scale farms (mean 66.0 days, P=0.003) than small-scale farms (29.4 days) and on farms that used feedback feeding (145.2 days, P=0.059) than those that did not (66.0 days). The number of dead piglets decreased over time since the first regional case (P<0.001) and was higher at farrow-to-finish farms (3.8 piglets/sow, P<0.001) than reproduction farms (0.7 piglets/sow). The effect of feedback feeding on the number of dead piglets was not significant, but its interaction term with farm style had a significant effect (5.0 more piglet deaths at reproduction farms than fallow-to-finish farms, P=0.001). These results suggest that feedback feeding made the damage from PED worse, though it was well established at a later stage of the regional PED epidemic.

A key piece of information for ecosystem management is the relationship between the environment and population genetic structure. However, it is difficult to clearly quantify the effects of environmental factors on genetic differentiation because of spatial autocorrelation and analytical problems. In this study, we focused on stream ecosystems and the environmental heterogeneity caused by groundwater and constructed a sampling design in which geographic distance and environmental differences are not correlated. Using multiplexed ISSR genotyping by sequencing (MIG-seq) method, a fine-scale population genetics study was conducted in fluvial sculpin Cottus nozawae, for which summer water temperature is the determinant factor in distribution and survival. There was a clear genetic structure in the watershed. Although a significant isolation-by-distance pattern was detected in the watershed, there was no association between genetic differentiation and water temperature. Instead, asymmetric gene flow from relatively low-temperature streams to high-temperature streams was detected, indicating the importance of low-temperature streams and continuous habitats. The groundwater-focused sampling strategy yielded insightful results for conservation., (© 2021. The Author(s), under exclusive licence to The Genetics Society.)

Forest conversion is one of the greatest global threats to biodiversity, and land-use change and subsequent biodiversity declines sometimes occur over a variety of underlying geologies. However, how forest conversion and underlying geology interact to alter biodiversity is underappreciated, although spatial variability in geology is considered an integral part of sustaining ecosystems. We aimed to examine the effects of forest conversion to farmland, the underlying geology, and their interaction on the stream fishes' diversity, evenness, and abundance in northeastern Japan. We disentangled complex pathways between abiotic and biotic factors with structural equation modeling. Species diversity of stream fishes was indirectly shaped by the interaction of land use and underlying geology. Diversity declined due to nutrient enrichment associated with farmlands, which was mainly the result of changes in evenness rather than by changes in species richness. This impact was strongest in streams with volcanic geology with coarse substrates probably because of the differential responses of abundant stream fishes to nutrient enrichment (i.e., dominance) and the high dependency of these fishes on large streambed materials during their life cycles. Our findings suggest that remediation of deforested or degraded forest landscapes would be more efficient if the interaction between land use and underlying geology was considered. For example, the negative impacts of farmland on evenness were larger in streams with volcanic geology than in other stream types, suggesting that riparian forest restoration along such streams would efficiently provide restoration benefits to stream fishes. Our results also suggest that land clearing around such streams should be avoided to conserve species evenness of stream fishes., (© 2020 Society for Conservation Biology.)

Background and Aims: Interspecific difference in pollinators (pollinator isolation) is important for reproductive isolation in flowering plants. Species-specific pollination by fungus gnats has been discovered in several plant taxa, suggesting that they can contribute to reproductive isolation. Nevertheless, their contribution has not been studied in detail, partly because they are too small for field observations during flower visitation. To quantify their flower visitation, we used the genus Arisaema (Araceae) because the pitcher-like spathe of Arisaema can trap all floral visitors., Methods: We evaluated floral visitor assemblage in an altitudinal gradient including five Arisaema species. We also examined interspecific differences in altitudinal distribution (geographic isolation) and flowering phenology (phenological isolation). To exclude the effect of interspecific differences in altitudinal distribution on floral visitor assemblage, we established ten experimental plots including the five Arisaema species in high- and low-altitude areas and collected floral visitors. We also collected floral visitors in three additional sites. Finally, we estimated the strength and contribution of these three reproductive barriers using a unified formula for reproductive isolation., Key Results: Each Arisaema species selectively attracted different fungus gnats in the altitudinal gradient, experimental plots and additional sites. Altitudinal distribution and flowering phenology differed among the five Arisaema species, whereas the strength of geographic and phenological isolations were distinctly weaker than those in pollinator isolation. Nevertheless, the absolute contribution of pollinator isolation to total reproductive isolation was weaker than geographic and phenological isolations, because pollinator isolation functions after the two early-acting barriers in plant life history., Conclusions: Our results suggest that selective pollination by fungus gnats potentially contributes to reproductive isolation. Since geographic and phenological isolations can be disrupted by habitat disturbance and interannual climate change, the strong and stable pollinator isolation might compensate for the weakened early-acting barriers as an alternative reproductive isolation among the five Arisaema species., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In Japan, reducing the use of antimicrobials in pig production is a significant issue. However, there are no published reports concerning porcine disease treatment, as related to the age of the pigs and the indications (e.g., organ system) in Japan. In this study, we analyzed the prescription records of 17 farrow-to-finish farms from 2014 to 2018 in southern Kyushu, Japan. The farms' antimicrobial usage was calculated as the active ingredient per population correction unit (PCU) or the number of treated pigs per PCU using the defined daily dose. All data were analyzed according to the indications and production stages (sows, suckling pigs, post-weaning pigs, and fattening pigs). In terms of active ingredients/PCU, tetracyclines were the most commonly used (43.2-59.3%), and the largest amounts of antimicrobials administered through feeds were for treating the respiratory organs of fattening pigs. In terms of the number of treated pigs/PCU, tetracyclines were most frequently used (16.3-31.1%), and a high frequency of antimicrobials administered through feeds was used for the treatment of respiratory organs in post-weaning pigs. In this study, it was confirmed that tetracyclines were used frequently as a herd treatment for respiratory diseases in post-weaning and fattening pigs in southern Kyushu, Japan. The findings suggest that it is necessary to improve the treatment and prevention of respiratory diseases in post-weaning and fattening pigs in order to reduce the frequencies of antimicrobial treatments.

Extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing Klebsiella spp. have become a major health problem, leading to treatment failure in humans and animals. This study aimed to evaluate the presence of ESBL/AmpC-producing Klebsiella spp. isolated from racehorses in Japan. Feces samples from 212 healthy Thoroughbred racehorses were collected from the Japan Racing Association Training Centers between March 2017 and August 2018. ESBL/AmpC-producing Klebsiella spp. were isolated using selective medium containing 1 µg/mL cefotaxime. All isolates were subjected to bacterial species identification (MALDI-TOF MS), antimicrobial susceptibility test (disk diffusion test), characterization of resistance genes (PCR), conjugation assay, and genetic relatedness (multilocus sequence typing/MLST). Twelve ESBL/AmpC-producing Klebsiella pneumoniae (ESBL/AmpC-KP) were isolated from 3.3% of horse samples. Antimicrobial resistance profiling for 17 antimicrobials showed all ESBL/AmpC-KP were multidrug-resistant (MDR). Only 1 isolate was confirmed as an ESBL producer ( bla CTX-M-2- positive), whereas the other 11 isolates were plasmid-mediated AmpC (pAmpC) producers ( bla CMY positive). On the basis of MLST analysis, the ESBL-KP isolate was identified as sequence type (ST)-133 and four different STs among AmpC-KP isolates, ST-145, ST-4830, ST-4831, and ST-4832, were found to share six of the seven loci constituting a single-locus variant. This is the first study to show K. pneumoniae carrying MDR pAmpC isolated from a racehorse.

In our previous study, extended spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) were isolated from healthy Thoroughbred racehorse feces samples in Japan. Some ESBL genes were predicted to be located on the conjugative plasmid. PCR-based replicon typing (PBRT) is a useful method to monitor and detect the association of replicons with specific plasmid-borne resistant genes. This study aimed to evaluate the plasmid replicon associated with ESBLEC isolated from healthy Thoroughbred racehorses at Japan Racing Association Training Centers in Japan. A total of 24 ESBLECs isolated from 23 (10.8%) individual Thoroughbred racehorse feces samples were used in this study. ESBL gene transfer was performed using a conjugation assay. Then, replicon types of ESBLEC isolates and their transconjugants were determined using PBRT. Pulsed-field gel electrophoresis (PFGE) was performed to look at the clonality of the ESBLECs isolates. ESBLECs were detected from 10.8% of healthy Thoroughbred racehorses. The bla CTX-M-2 was identified as the dominant type of ESBL gene, followed by bla CTX-M-1 and bla TEM-116 . In this study, only the bla CTX-M-2 and the IncI1 plasmid were transferred to transconjugants. The PFGE results showed that ESBL genes were distributed in diversity of ESBLECs. This finding suggested that the IncI1 plasmid was associated with the dissemination of bla CTX-M-2 in Thoroughbred racehorses in Japan.

The objectives of this study were to assess abortion risk (AR) and the number of piglets that died during suckling periods per litter (DP) in farms infected with porcine epidemic diarrhea (PED) in relation to herd immunization procedures. Data were obtained from 91 farms in Japan that had PED infection during 2013 to 2014. The 91 PED-positive farms were asked the number of abortions that occurred and DP for 3 months (1 month before PED outbreak (previous month), 1 month after PED outbreak (the month of PED), and from 1 month after PED outbreak to 2 months after PED outbreak (following month)). AR in each month was calculated as the number of abortions divided by sow inventory. Both AR and DP in the month of PED were higher than those in the previous and following months (p < .05). Farms that performed a herd immunization procedure had higher AR and DP in the month of PED than those that did not perform the procedure (p < .05). In summary, PED occurrence increased AR and DP., (© 2020 Japanese Society of Animal Science.)

The objective of the present study was to assess the productivity of pigs to investigate the time-dependent change in productivity and compare productivity according to breeding company group and porcine reproductive and respiratory syndrome (PRRS) status on commercial swine farms in southern Kyushu, Japan. Data used in the present study were 245 annual productivity records from 2014 to 2018 obtained from 72 farms. Average sow inventory was 481.2 sows. Mean numbers of pigs weaned per sow per year and market pigs sold per sow per year were 23.7 and 21.6, respectively. Pigs born alive per litter increased from 11.1 to 11.9 pigs from 2014 to 2018 (p < .05). Farms using domestic breeding companies had similar numbers in 2016 to those using international breeding companies, but fewer pigs in 2014, 2015, 2017, and 2018 (p < .05). Farms with an unknown or unstable PRRS status had fewer pigs born alive per litter and pigs weaned per sow per year than those with stable or negative PRRS statuses (11.2 ± 0.06 vs. 11.8 ± 0.08 pigs and 22.6 ± 0.38 vs. 25.0 ± 0.22 pigs, respectively; p < .05). These results can be used to establish feasible targets and standards of performance to identify problem areas and improve production., (© 2020 Japanese Society of Animal Science.)

In this study, we investigated the occurrence of antimicrobial resistance in commensal Escherichia coli isolated from healthy Thoroughbred (TB) racehorses in Japan. A total of 212 fecal samples were individually collected from TB racehorses from March 2017 to August 2018 at Japan Racing Association training centers. E. coli was isolated by using selective agar media, deoxycholate-hydrogen sulfide-lactose (DHL) and eosin methylene blue (EMB). A total of 417 E. coli isolates were examined against 10 antimicrobial agents by using the broth microdilution method. The 417 E. coli isolates were phylogenetically grouped using a multiplex polymerase chain reaction. The highest proportion of resistance was observed for streptomycin (30.9%, 129/417) followed by ampicillin (19.4%, 81/417), trimethoprim (15.8%, 66/417), tetracycline (8.4%, 35/417), chloramphenicol (2.6%, 11/417), kanamycin (1.2%, 5/417), nalidixic acid (0.5%, 2/417), cefazolin (0.2%, 1/417), colistin (0.2%, 1/417), and gentamycin (0%). Multidrug-resistant (MDR) E. coli was detected in 7.9% (33/417) of isolates. The proportions of resistance against ampicillin, streptomycin, kanamycin, and chloramphenicol and of multidrug-resistant phenotypes in E. coli belonging to phylogenetic group B2 were significantly higher than those of other groups. This study clarified the distribution of antimicrobial-resistant (AMR) E. coli in Japanese racehorses. A continuous monitoring program for antimicrobial resistance is required to control the spread of AMR bacteria in racehorses., (©2020 The Japanese Society of Equine Science.)

Premise: The genus Arisaema (Araceae) has rapidly diversified in Japan, and multiple species often coexist in the field. Although Japanese Arisaema species hybridize from artificial crossing, hybrid individuals are rare in mixed populations; suggesting the presence of effective pre-pollination barriers. We examined the following reproductive barriers between A. sikokianum and A. tosaense: habitat, phenology, and pollinator isolations., Methods: Habitat isolation was examined by interspecific comparisons of microhabitat conditions at a mixed site and of altitude at the sampling site of herbarium specimens. Phenological isolation was evaluated by comparing seasonal transition in apparent spathe condition and frequency of insect visitation. Pollinator isolation was examined by comparing floral visitor assemblages between the two Arisaema species. To avoid overestimation of pollinator isolation due to seasonal changes in insect assemblages, we also compared visitor assemblages between natural and late-flowering A. sikokianum, where the latter was experimentally introduced and blooming with a natural A. tosaense population., Results: Microhabitat conditions and sampling elevations of herbarium specimens overlapped between the two Arisaema species. At the population level, A. sikokianum and A. tosaense flowered for 39 and 52 days, respectively, with 13 days overlap. Insect visitation in A. sikokianum decreased before the seasonal overlap. Floral visitor assemblages differed between the two Arisaema species, while the difference between natural and late-flowering A. sikokianum was less distinct., Conclusions: Phenological and pollinator isolation contribute to reproductive isolation between the two Arisaema species and should enable the two species to coexist in this area., (© 2019 Botanical Society of America.)

Background/aim: Osteosarcoma is the most malignant type of bone tumor. Patients with osteosarcoma metastases have a poorer prognosis than those without metastases. Thus, the prognosis of osteosarcoma patients with metastases must be improved., Materials and Methods: The present study investigated the inhibitory effects of 6-hydroxythiobinupharidine isolated from Nuphar pumilum on migration of LM8 murine osteosarcoma cells by a migration assay and also examined the expression of proteins related to actin dynamics by western blot. The present study also developed an automatic cell counting system using machine learning to count migrated cells by Fiji and Trainable Weka Segmentation., Results: 6-Hydroxythiobinupharidine inhibited migration of LM8 osteosarcoma cells in a dose-dependent manner, and decreased protein expression of Lin11, Isl-1, and Mec-3 domain kinase 1 (LIMK1) and the levels of phosphorylated Cofilin., Conclusion: 6-Hydroxythiobinupharidine suppressed migration of LM8 osteosarcoma cells by decreasing expression of LIMK1. 6-Hydroxythiobinupharidine could be potentially used as an anti-metastatic compound., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC) have become a major health concern in both human and veterinary medicine. These bacteria could become a critical problem in equine medicine due to the limited number of antimicrobial drugs available. However, there are no previous reports of ESBLEC isolated from horses in Japan. The objectives of this study were to investigate the occurrence of ESBLEC isolated from feces in healthy Thoroughbred racehorses in Japan. Feces samples were collected from 147 healthy Thoroughbred racehorses by equine veterinarians at the Japan Racing Association (103 from Miho Training Center and 44 from Ritto Training Center) between March 2017 and April 2018. Samples were screened for ESBLECs using MacConkey agar supplemented with 1 µg/ml cefotaxime. Detection of ESBL genes was performed by PCR and confirmed by DNA sequencing. Horizontal transmission was demonstrated by conjugation assay. In this study, 24 ESBLECs were isolated from twelve horse feces samples (8.2%). All ESBLECs harbored bla CTX-M-2 , and both bla TEM-1 and bla CTX-M-2 were detected in nine isolates (37.5%). ESBLECs showed resistance to all β-lactam antibiotics (100%) tested, followed by trimethoprim (66.7%), streptomycin (62.5%), tetracycline (25.0%), and oxytetracycline (25.0%). Horizontal transmission was successfully demonstrated by conjugation assay in eight of 13 isolates, and bla CTX-M-2 was detected by PCR in all transconjugants. This study showed that racehorses in Japan are potential reservoirs of ESBLECs., (©2019 The Japanese Society of Equine Science.)

Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis is characterized by hypoproteinemia. There are currently no reliable reports that provide a reference value for the total serum protein (TP) concentration to clinically diagnose EPE. The objective of this study was to statistically determine the reference value. Feces and sera of 99 foals with EPE-like clinical signs and of 35 healthy foals were obtained. The samples were used for specific-gene detection of L. intracellularis, TP measurement, and specific-antibody detection against L. intracellularis. Based on these results, the optimal reference value for the TP concentration as a clinical diagnostic index of EPE was found to be ≤ 4.8 g/dl. This clinical diagnostic index will provide an effective approach for diagnosing EPE., (©2019 The Japanese Society of Equine Science.)

Large dams provide vital protection and services to humans. However, an increasing number of large dams worldwide are old and not operating properly. The removal of large dams has excellent potential to restore habitat connectivity and flow regimes; therefore, projecting the related ecological consequences is an emerging need for water resource and ecosystem management. However, no modeling methods are currently available for such projections at the basin scale. We devised a scheme that integrates changes in flow regimes and habitat network structure into a basin-scale impact assessment of removal of large dams and applied it to the Nagara-Ibi Basin, Japan. We used a graph-theoretical approach and a hydrological model, to quantify changes in habitat availability for 11 freshwater fishes at the basin scale under multiple removal scenarios. We compared these results with the change predicted using a conventional scheme that considered only changes to the habitat network due to dam removal. Our proposed scheme revealed that an increase in flow variability associated with dam removal projected both positive and negative effects on basin-scale habitat availability, depending on the focal species, endangered species had a negative response to dam removal. In contrast, the conventional approach projected only positive effects for all species. This difference in the outcomes indicates that large-dam removal can have negative and positive effects on watershed restoration due to changes in flow regimes. Our results also suggest the effect of removal of large dams may depend on the dams and their locations. Our study is the first step in projecting ecological trade-offs associated with the removal of large dams on riverscapes at the basin scale and provides a foundation for future process-based watershed restoration., (© 2018 Society for Conservation Biology.)

Background: Since late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013-2016, were sequenced and analyzed., Results: Phylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics., Conclusions: These results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease.

Swine edema disease is caused by Shiga toxin (Stx) 2e-producing Escherichia coli (STEC). Addition of highly concentrated zinc formulations to feed has been used to treat and prevent the disease, but the mechanism of the beneficial effect is unknown. The purpose of the present study was to investigate the effects of highly concentrated zinc formulations on bacterial growth, hemolysin production, and an Stx2e release by STEC in vitro. STEC strain MVH269 isolated from a piglet with edema disease was cultured with zinc oxide (ZnO) or with zinc carbonate (ZnCO 3 ), each at up to 3,000 ppm. There was no effect of zinc addition on bacterial growth. Nonetheless, the cytotoxic activity of Stx2e released into the supernatant was significantly attenuated in the zinc-supplemented media compared to that in the control, with the 50% cytotoxic dose values of 163.2 ± 12.7, 211.6 ± 33.1 and 659.9 ± 84.2 after 24 hr of growth in the presence of ZnO, ZnCO 3 , or no supplemental zinc, respectively. The hemolytic zones around colonies grown on sheep blood agar supplemented with zinc were significantly smaller than those of colonies grown on control agar. Similarly, hemoglobin absorbance after exposure to the supernatants of STEC cultures incubated in sheep blood broth supplemented with zinc was significantly lower than that resulting from exposure to the control supernatant. These in vitro findings indicated that zinc formulations directly impair the factors associated with the virulence of STEC, suggesting a mechanism by which zinc supplementation prevents swine edema disease.

Since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (PEDV) infection have reemerged and swept rapidly across Japan, resulting in significant economic losses. In this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field PEDV variants with large genomic deletions. The sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28-714 (10-238) on the S gene (protein). Among 17 PEDV samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact S gene. These variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent PEDV infection in the farms. Full-length S and ORF3 genes of eight variants derived from 2 samples were characterized. This is the first report of mixed infections caused by various genotypes of PEDV and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease., Competing Interests: The authors have declared that no competing interests exist.

We improved a loop-mediated isothermal amplification (LAMP) assay permitting sensitive and rapid Mycoplasma bovis detection. A total of 55 bacterial strains were examined in this study, including 33 M. bovis strains, 14 non-M. bovis mycoplasmas and eight non-mycoplasma bacterial strains. M. bovis was successfully detected by the LAMP assay within 60 min without cross-reaction to any other bacteria. Furthermore, a total of 135 nasal swab samples were tested directly using our LAMP assays, the previously reported LAMP assay, conventional PCR assay without pre-culture and comparing standard culture methods. The improved LAMP assay showed sensitivity and specificity of 97.2% and 90.9%, respectively (with a kappa coefficient of 0.8231), and the sensitivity of our revised LAMP assay was increased compared to existing methods.