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Relationships play a very important role in studies on quantitative genetics. In traditional breeding, pedigree records are used to establish relationships between animals; while this kind of relationship actually represents one kind of relatedness, it cannot distinguish individual specificity, capture the variation between individuals or determine the actual genetic superiority of an animal. However, with the popularization of high-throughput genotypes, assessments of relationships among animals based on genomic information could be a better option. In this study, we compared the relationships between animals based on pedigree and genomic information from two pig breeding herds with different genetic backgrounds and a simulated dataset. Two different methods were implemented to calculate genomic relationship coefficients and genomic kinship coefficients, respectively. Our results show that, for the same kind of relative, the average genomic relationship coefficients (G matrix) were very close to the pedigree relationship coefficients (A matrix), and on average, the corresponding values were halved in genomic kinship coefficients (K matrix). However, the genomic relationship yielded a larger variation than the pedigree relationship, and the latter was similar to that expected for one relative with no or little variation. Two genomic relationship coefficients were highly correlated, for farm1, farm2 and simulated data, and the correlations for the parent-offspring, full-sib and half-sib were 0.95, 0.90 and 0.85; 0.93, 0.96 and 0.89; and 0.52, 0.85 and 0.77, respectively. When the inbreeding coefficient was measured, the genomic information also yielded a higher inbreeding coefficient and a larger variation than that yielded by the pedigree information. For the two genetically divergent Large White populations, the pedigree relationship coefficients between the individuals were 0, and 62 310 and 175 271 animal pairs in the G matrix and K matrix were greater than 0. Our results demonstrated that genomic information outperformed the pedigree information; it can more accurately reflect the relationships and capture the variation that is not detected by pedigree. This information is very helpful in the estimation of genomic breeding values or gene mapping. In addition, genomic information is useful for pedigree correction. Further, our findings also indicate that genomic information can establish the genetic connection between different groups with different genetic background. In addition, it can be used to provide a more accurate measurement of the inbreeding of an animal, which is very important for the assessment of a population structure and breeding plan. However, the approaches for measuring genomic relationships need further investigation.
The co-occurrence of hepatic cystic echinococcosis (CE) and alveolar echinococcosis (AE) is extremely rare. Here, we present the clinical manifestations and treatment outcomes of three cases with co-occurring CE and AE in the liver. Computed tomography (CT), magnetic resonance imaging and 18FFluorodeoxyglucose Positron Emission Tomography-CT were used for preoperative diagnosis. Specimens were taken intraoperatively and sent for pathological studies to confirm the coexistence of CE and AE by laminated membrane, daughter cysts or germinal layer and infiltration structure. Albendazole was prescribed after operation for 12 months. All patients were completely recovered and showed no recurrence at last follow-up. Therefore, surgical intervention and postoperative application of albendazole are recommended for patients with concurrence of hepatic AE and CE.
The COllaborative project of Development of Anthropometrical measures in Twins (CODATwins) project is a large international collaborative effort to analyze individual-level phenotype data from twins in multiple cohorts from different environments. The main objective is to study factors that modify genetic and environmental variation of height, body mass index (BMI, kg/m2) and size at birth, and additionally to address other research questions such as long-term consequences of birth size. The project started in 2013 and is open to all twin projects in the world having height and weight measures on twins with information on zygosity. Thus far, 54 twin projects from 24 countries have provided individual-level data. The CODATwins database includes 489,981 twin individuals (228,635 complete twin pairs). Since many twin cohorts have collected longitudinal data, there is a total of 1,049,785 height and weight observations. For many cohorts, we also have information on birth weight and length, own smoking behavior and own or parental education. We found that the heritability estimates of height and BMI systematically changed from infancy to old age. Remarkably, only minor differences in the heritability estimates were found across cultural–geographic regions, measurement time and birth cohort for height and BMI. In addition to genetic epidemiological studies, we looked at associations of height and BMI with education, birth weight and smoking status. Within-family analyses examined differences within same-sex and opposite-sex dizygotic twins in birth size and later development. The CODATwins project demonstrates the feasibility and value of international collaboration to address gene-by-exposure interactions that require large sample sizes and address the effects of different exposures across time, geographical regions and socioeconomic status.
Laser interaction with an ultra-thin pre-structured target is investigated with the help of both two-dimensional and three-dimensional particle-in-cell simulations. With the existence of a periodic structure on the target surface, the laser seems to penetrate through the target at its fundamental frequency even if the plasma density of the target is much higher than the laser’s relativistically critical density. The particle-in-cell simulations show that the transmitted laser energy behind the pre-structured target is increased by about two orders of magnitude compared to that behind the flat target. Theoretical analyses show that the transmitted energy behind the pre-structured target is actually re-emitted by electron ‘islands’ formed by the surface plasma waves on the target surfaces. In other words, the radiation with the fundamental frequency is actually ‘surface emission’ on the target rear surface. Besides the intensity of the component with the fundamental frequency, the intensity of the high-order harmonics behind the pre-structured target is also much enhanced compared to that behind the flat target. The enhancement of the high-order harmonics is also related to the surface plasma waves generated on the target surfaces.
Estimating the feed intake of grazing herbivores is critical for determining their nutrition, overall productivity and utilization of grassland resources. A 17-day indoor feeding experiment was conducted to evaluate the potential use of Medicago sativa as a natural supplement for estimating the total feed intake of sheep. A total of 16 sheep were randomly assigned to four diets (four sheep per diet) containing a known amount of M. sativa together with up to seven forages common to typical steppes. The diets were: diet 1, M. sativa + Leymus chinensis + Puccinellia distans; diet 2, species in diet 1 + Phragmites australis; diet 3, species in diet 2 + Chenopodium album + Elymus sibiricus; and diet 4, species in diet 3 + Artemisia scoparia + Artemisia tanacetifolia. After faecal marker concentrations were corrected by individual sheep recovery, treatment mean recovery or overall recovery, the proportions of M. sativa and other dietary forages were estimated from a combination of alkanes and long-chain alcohols using a least-square procedure. Total intake was the ratio of the known intake of M. sativa to its estimated dietary proportion. Each dietary component intake was obtained using total intake and the corresponding dietary proportions. The estimated values were compared with actual values to assess the estimation accuracy. The results showed that M. sativa exhibited a distinguishable marker pattern in comparison to the other dietary forage species. The accuracy of the dietary composition estimates was significantly (P < 0.001) affected by both diet diversity and the faecal recovery method. The proportion of M. sativa and total intake across all diets could be accurately estimated using the individual sheep or the treatment mean recovery methods. The largest differences between the estimated and observed total intake were 2.6 g and 19.2 g, respectively, representing only 0.4% and 2.6% of the total intake. However, they were significantly (P < 0.05) biased for most diets when using the overall recovery method. Due to the difficulty in obtaining individual sheep recovery under field conditions, treatment mean recovery is recommended. This study suggests that M. sativa, a natural roughage instead of a labelled concentrate, can be utilized as a dietary supplement to accurately estimate the total feed intake of sheep indoors and further indicates that it has potential to be used in steppe grassland of northern China, where the marker patterns of M. sativa differ markedly from commonly occurring plant species.
A multichannel calorimeter system is designed and constructed which is capable of delivering single-shot and broad-band spectral measurement of terahertz (THz) radiation generated in intense laser–plasma interactions. The generation mechanism of backward THz radiation (BTR) is studied by using the multichannel calorimeter system in an intense picosecond laser–solid interaction experiment. The dependence of the BTR energy and spectrum on laser energy, target thickness and pre-plasma scale length is obtained. These results indicate that coherent transition radiation is responsible for the low-frequency component (
1 THz) of BTR. It is also observed that a large-scale pre-plasma primarily enhances the high-frequency component (
3 THz) of BTR.
Rabies is one of the major public health problems in China, and the mortality rate of rabies remains the highest among all notifiable infectious diseases. A meta-analysis was conducted to investigate the post-exposure prophylaxis (PEP) vaccination rate and risk factors for human rabies in mainland China. The PubMed, Web of Science, Chinese National Knowledge Infrastructure, Chinese Science and Technology Periodical and Wanfang databases were searched for articles on rabies vaccination status (published between 2007 and 2017). In total, 10 174 human rabies cases from 136 studies were included in this meta-analysis. Approximately 97.2% (95% confidence interval (CI) 95.1–98.7%) of rabies cases occurred in rural areas and 72.6% (95% CI 70.0–75.1%) occurred in farmers. Overall, the vaccination rate in the reported human rabies cases was 15.4% (95% CI 13.7–17.4%). However, among vaccinated individuals, 85.5% (95% CI 79.8%–83.4%) did not complete the vaccination regimen. In a subgroup analysis, the PEP vaccination rate in the eastern region (18.8%, 95% CI 15.9–22.1%) was higher than that in the western region (13.3%, 95% CI 11.1–15.8%) and this rate decreased after 2007. Approximately 68.9% (95% CI 63.6–73.8%) of rabies cases experienced category-III exposures, but their PEP vaccination rate was 27.0% (95% CI 14.4–44.9%) and only 6.1% (95% CI 4.4–8.4%) received rabies immunoglobulin. Together, these results suggested that the PEP vaccination rate among human rabies cases was low in mainland China. Therefore, standardised treatment and vaccination programs of dog bites need to be further strengthened, particularly in rural areas.
The mechanical properties of thin-walled plate with close-packed film cooling holes are studied based on the equivalent solid material concept. The equivalent principals of the method of equivalent strain energy, homogenization theory and uniform static deformation are considered. A simplification method of square penetration pattern for pitch and diagonal direction loading is presented. The goodness of fit is calculated to determine the optimal method. The tensile deformation, bending deflection, rotation displacement and maximum Mises equivalent stress of simplification plate models are in good agreement with plate models with close-packed film cooling holes. For square penetration pattern for pitch direction loading, the equivalent errors of Mises equivalent stress are all less than 10% when the ligament efficiency is more than 0.6.
Taihu pig breeds are the most prolific breeds of swine in the world, and they also have superior economic traits, including high resistance to disease, superior meat quality, high resistance to crude feed and a docile temperament. The formation of these phenotypic characteristics is largely a result of long-term artificial or natural selection. Therefore, exploring selection signatures in the genomes of the Taihu pigs will help us to identify porcine genes related to productivity traits, disease and behaviour. In this study, we used both intra-population (Relative Extend Haplotype Homozygosity Test (REHH)) and inter-population (the Cross-Population Extend Haplotype Homozygosity Test (XPEHH); F-STATISTICS, FST) methods to detect genomic regions that might be under selection process in Taihu pig breeds. As a result, we found 282 (REHH) and 112 (XPEHH) selection signature candidate regions corresponding to 159.78 Mb (6.15%) and 62.29 Mb (2.40%) genomic regions, respectively. Further investigations of the selection candidate regions revealed that many genes under these genomic regions were related to reproductive traits (such as the TLR9 gene), coat colour (such as the KIT gene) and fat metabolism (such as the CPT1A and MAML3 genes). Furthermore, gene enrichment analyses showed that genes under the selection candidate regions were significantly over-represented in pathways related to diseases, such as autoimmune thyroid and asthma diseases. In conclusion, several candidate genes potentially under positive selection were involved in characteristics of Taihu pig. These results will further allow us to better understand the mechanisms of selection in pig breeding.
Phenoloxidase (PO) plays a key role in melanin biosynthesis during insect development. Here, we isolated the 2310-bp full-length cDNA of PPO1 from Zeugodacus tau, a destructive horticultural pest. qRT-polymerase chain reaction showed that the ZtPPO1 transcripts were highly expressed during larval–prepupal transition and in the haemolymph. When the larvae were fed a 1.66% kojic acid (KA)-containing diet, the levels of the ZtPPO1 transcripts significantly increased by 2.79- and 3.39-fold in the whole larvae and cuticles, respectively, while the corresponding PO activity was significantly reduced; in addition, the larval and pupal durations were significantly prolonged; pupal weights were lowered; and abnormal phenotypes were observed. An in vitro inhibition experiment indicated that KA was an effective competitive inhibitor of PO in Z. tau. Additionally, the functional analysis showed that 20E could significantly up-regulate the expression of ZtPPO1, induce lower pupal weight, and advance pupation. Knockdown of the ZtPPO1 gene by RNAi significantly decreased mRNA levels after 24 h and led to low pupation rates and incomplete pupae with abnormal phenotypes during the larval-pupal interim period. These results proved that PO is important for the normal growth of Z. tau and that KA can disrupt the development of this pest insect.
Cucumber powdery mildew is a destructive foliar disease caused by Podosphaera xanthii (formerly known as Sphaerotheca fuliginea) that substantially damages the yield and quality of crops. The control of this disease primarily involves the use of chemical pesticides that cause serious environmental problems. Currently, numerous studies have indicated that some plant extracts or products potentially have the ability to act as natural pesticides to control plant diseases. It has been reported that turmeric (Curcuma longa L.) and its extract can be used in agriculture due to their insecticidal and fungicidal properties. However, the most effective fungicidal component of this plant is still unknown. In the current study, the crude extract of C. longa L. was found to have a fungicidal effect against P. xanthii. Afterwards, eight fractions (Fr.1–Fr.8) were gradually separated from the crude extract by column chromatography. Fraction 1 had the highest fungicidal effect against this pathogen among the eight fractions. The active compound, (+)-(S)-ar-turmerone, was separated from Fr 1 by semi-preparative high-performance liquid chromatography and identified based on its 1H nuclear magnetic resonance (NMR) and 13C NMR spectrum data. The EC50 value of (+)-(S)-ar-turmerone was found to be 28.7 µg/ml. The compound also proved to have a curative effect. This is the first study to report that the compound (+)-(S)-ar-turmerone has an effect on controlling this disease. These results provide a basis for developing a new phytochemical fungicide from C. longa L. extract.
In vivo and in vitro trials were conducted to assess the effects of tributyrin (TB) supplementation on short-chain fatty acid (SFCA) concentrations, fibrolytic enzyme activity, nutrient digestibility and methanogenesis in adult sheep. Nine 12-month-old ruminally cannulated Small Tail ewes (initial body weight 55 ± 5.0 kg) without pregnancy were used for the in vitro trial. In vitro substrate made to offer TB at 0, 2, 4, 6 and 8 g/kg on a dry matter (DM) basis was incubated by ruminal microbes for 72 h at 39°C. Forty-five adult Small Tail ewes used for the in vivo trial were randomly assigned to five treatments with nine animals each for an 18-d period according to body weight (55 ± 5.0 kg). Total mixed ration fed to ewes was also used to offer TB at 0, 2, 4, 6 and 8 g/kg on a DM basis. The in vitro trial showed that TB supplementation linearly increased apparent digestibility of DM, crude protein, neutral detergent fibre and acid detergent fibre, and enhanced gas production and methane emissions. The in vivo trial showed that TB supplementation decreased DM intake, but enhanced ruminal fermentation efficiency. Both in vitro and in vivo trials showed that TB supplementation enhanced total SFCA concentrations and carboxymethyl cellulase activity. The results indicate that TB supplementation might exert advantage effects on rumen microbial metabolism, despite having an enhancing effect on methanogenesis.
Superior and inferior rice grains have different weights and are located on the upper primary branch and lower secondary branches of the panicle, respectively. To study differences in germination vigour of these two types of grain, a number of factors were investigated from 0 to 48 h of germination. The present study demonstrated that in inferior grains the starch granule structure was looser at 0 h, with full water absorption at 48 h, while in superior grains the structure was tight and dense. Relative water content increased, and dry matter decreased, more rapidly in inferior grains than in superior ones. Abscisic acid and gibberellin levels, as well as α-amylase activity, also changed more rapidly in inferior grains, while soluble sugar content and amylase coding gene expression increased more rapidly in inferior than superior grains during early germination. The expression of OsGAMYB was higher in inferior grains at 24 h but higher in superior grains at 48 h. The phenotypic index of seedlings was higher in seedlings from superior grains at the two-leaf stage. However, the thousand-grain weight and yield per plant in superior and inferior plants showed no significant difference at harvest. The present study indicates that inferior grains germinate faster than superior ones in the early germination stage. Although inferior grains produced weaker seedlings, it is worthwhile using them in rice production due to their comparative yield potential over that of superior grains.
The triplite LiFeSO4F displays both the highest potential ever reported for an Fe-based compound, as well as a comparable specific energy with that of popular LiFePO4. The synthesis is still a challenge because the present approaches are connected with long time, special equipments or organic reagents, etc. In this work, the triplite LiFeSO4F powder was synthesized through an ambient two-step solid-state route. The reaction process and phase purity were analyzed, coupled with structure refinement and electrochemical test.
Branched-chain volatile fatty acids (BCVFA) supplements could promote lactation performance and milk quality by improving ruminal fermentation and milk fatty acid synthesis. This study was conducted to evaluate the effects of BCVFA supplementation on milk performance, ruminal fermentation, nutrient digestibility and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows. A total of 36 multiparous Chinese Holstein cows averaging 606±4.7 kg of BW, 65±5.2 day in milk (DIM) with daily milk production of 30.6±0.72 kg were assigned to one of four groups blocked by lactation number, milk yield and DIM. The treatments were control, low-BCVFA (LBCVFA), medium-BCVFA (MBCVFA) and high-BCVFA (HBCVFA) with 0, 30, 60 and 90 g BCVFA per cow per day, respectively. Experimental periods were 105 days with 15 days of adaptation and 90 days of data collection. Dry matter (DM) intake tended to increase, but BW changes were similar among treatments. Yields of actual milk, 4% fat corrected milk, milk fat and true protein linearly increased, but feed conversion ratio (FCR) linearly decreased with increasing BCVFA supplementation. Milk fat content linearly increased, but true protein content tended to increase. Contents of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 and C15:0 fatty acids in milk fat linearly increased, whereas other fatty acids were not affected with increasing BCVFA supplementation. Ruminal pH, ammonia N concentration and propionate molar proportion linearly decreased, but total VFA production and molar proportions of acetate and butyrate linearly increased with increasing BCVFA supplementation. Consequently, acetate to propionate ratios linearly increased. Digestibilities of DM, organic matter, CP, NDF and ADF also linearly increased. In addition, mRNA expressions of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding factor 1 and fatty acid-binding protein 3 linearly increased, mRNA expressions of acetyl-coenzyme A carboxylase-α, fatty acid synthase and stearoyl-CoA desaturase quadratically increased. However, lipoprotein lipase mRNA expression was not affected by treatments. The results indicated that lactation performance and milk fat synthesis increased with BCVFA supplementation by improving ruminal fermentation, nutrient digestibility and mRNA expressions of genes related to milk fat synthesis.
Genomic selection has become increasingly important in the breeding of animals and plants. The response variable is an important factor, influencing the accuracy of genomic selection. The de-regressed proof (DRP) based on traditional estimated breeding value (EBV) is commonly used as response variable. In the current study, simulated data from 16th QTL-MAS Workshop and real data from Chinese Holstein cattle were used to compare accuracy and bias of genomic prediction with two methods of calculating DRP. Our results with simulated data showed that the correlation between genomic EBV and true breeding value achieved using the Jairath method (DRP_J) was superior to that achieved using the Garrick method (DRP_G) for simulated trait 1 but the reverse was true for simulated trait 3, and these two methods performed comparably for simulated trait 2. For all three simulated traits, DRP_J yielded larger bias of genomic prediction. However, DRP_J outperformed DRP_G in both accuracy and unbiasedness for four milk production traits in Chinese Holstein. In the estimation of genomic breeding value using genomic BLUP model, two methods for weighting diagonal elements of incidence matrix associated with residual error were also compared. With increasing the proportion of genetic variance unexplained by markers, the accuracy of genomic prediction was decreased and the bias was increased. Weighting by the reliability of DRP produced accuracy comparable to the evaluation where the proportion of genetic variance unexplained by markers was considered, but with smaller bias in general.
The origins and phylogeny of different sheep breeds has been widely studied using polymorphisms within the mitochondrial hypervariable region. However, little is known about the mitochondrial DNA (mtDNA) content and phylogeny based on mtDNA protein-coding genes. In this study, we assessed the phylogeny and copy number of the mtDNA in eight indigenous (population size, n=184) and three introduced (n=66) sheep breeds in China based on five mitochondrial coding genes (COX1, COX2, ATP8, ATP6 and COX3). The mean haplotype and nucleotide diversities were 0.944 and 0.00322, respectively. We identified a correlation between the lineages distribution and the genetic distance, whereby Valley-type Tibetan sheep had a closer genetic relationship with introduced breeds (Dorper, Poll Dorset and Suffolk) than with other indigenous breeds. Similarly, the Median-joining profile of haplotypes revealed the distribution of clusters according to genetic differences. Moreover, copy number analysis based on the five mitochondrial coding genes was affected by the genetic distance combining with genetic phylogeny; we also identified obvious non-synonymous mutations in ATP6 between the different levels of copy number expressions. These results imply that differences in mitogenomic compositions resulting from geographical separation lead to differences in mitochondrial function.
The effects of obesity on reproduction have been widely reported in humans and mice. The present study was designed to compare the reproductive performance of lean and fat chicken lines, divergently selected for abdominal fat content. The following parameters were determined and analyzed in the two lines: (1) reproductive traits, including age at first egg and total egg numbers from generations 14 to 18, absolute and relative testicular weights at 7, 14, 25, 30, 45 and 56 weeks of age, semen quality at 30, 45 and 56 weeks of age in generation 18, and fertility and hatchability from generations 14 to 18; (2) reproductive hormones at 7, 14, 25, 30, 45 and 56 weeks of age in generation 18; (3) and the relative mRNA abundance of genes involved in reproduction at 7, 14, 25, 30, 45 and 56 weeks of age in generation 18. In females, birds in the lean line laid more eggs from the first egg to 40 weeks of age than the birds in the fat line. In male broilers, the birds in the lean line had higher absolute and relative testicular weights at 7, 14 and 25 weeks of age, but lower absolute and relative testicular weights at 56 weeks of age than the birds in the fat line. Male birds in the lean line had greater sperm concentrations and larger numbers of motile and morphologically normal sperms at 30, 45 and 56 weeks of age than the birds in the fat line. Fertility and hatchability were also higher in the lean line than in the fat line. Significant differences in the plasma levels of reproductive hormones and the expression of reproduction-associated genes were also found at different ages in the lean and fat birds, in both males and females. These results suggest that reproductive performance is better in lean birds than in fat birds. In view of the unique divergent lines used in this study, these results imply that selecting for abdominal fat deposition negatively affects the reproductive performance of birds.
Human cystic echinococcosis is a widespread, chronic, endemic, helminthic zoonosis caused by larval tapeworms of the species Echinococcus granulosus. At present, there is no rational and effective therapy for patients with echinococcosis. The present study evaluated whether the combination of alkaloids from Sophora moorcroftiana seeds (SMSa2) and Bacillus Calmette–Guérin (BCG) was effective in the treatment of experimental echinococcosis. After 20 weeks of secondary infection with protoscoleces, mice were randomly allocated to five groups and treated for 6 weeks by daily intragastric administration of albendazole (ABZ, 100 mg/kg), SMSa2 (100 mg/kg), BCG (abdominal subcutaneous injection at 5 × 106 CFU), SMSa2 + BCG (100 mg/kg SMSa2 and 5 × 106 CFU BCG) or normal saline (untreated group), respectively. The results indicated a significant reduction in the weight of hydatid cysts in the SMSa2 + BCG group compared with the untreated, SMSa2 and BCG groups. The rate of inhibition of hydatid cyst growth in the SMSa2 + BCG group (76.1%) was obviously increased compared with that in the SMSa2 (25.7%) and BCG (26.6%) groups, respectively. Compared with the untreated control, the SMSa2 + BCG group showed a non-significant increase in serum interleukin-4 (IL-4). Furthermore, the serum levels of interferon-γ (IFN-γ) between the untreated and SMSa2 + BCG groups were not statistically different. Therefore, the combination of alkaloids from S. moorcroftiana seeds and BCG can reduce cyst burden and is an effective therapeutic regimen against echinococcosis.
Diarrhea is a common cause of morbidity and mortality and the incidence of diarrhea in the world has changed little over the past four decades. To assess the prevalence of and healthcare practices for diarrhea, a cross-sectional study was conducted in Pudong, Shanghai, China. In October 2014, a total of 5324 community residents were interviewed. Respondents were asked if they had experienced diarrhea (defined as ⩾3 passages of watery, loose, bloody, or mucoid stools within a 24-h period) in the previous month prior to the interview. The monthly prevalence of diarrhea was 4·1% (95% CI: 3·3–4·8), corresponding to an incidence rate of 0·54 episodes per person-year. The proportion of individuals with diarrhea who sought healthcare was 21·2% (95% CI: 13·4–29·0). Diarrhea continues to impose a considerable burden on the community and healthcare system in Pudong. Young age and travel were identified as predictors of increased diarrhea occurrence.