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Summary: Avian coccidiosis is a widespread intestinal disease found in poultry that causes substantial economic losses. To extensively investigate the molecular mechanism of drug resistance in Eimeria tenella, we analyzed the sporozoites and second-generation merozoites of drug-sensitive (DS), diclazuril-resistant (DZR) strain, and salinomycin-resistant (SMR) strains of E. tenella through transcriptome sequencing. Whole genome sequencing analyses were performed on resistant strains at different concentrations—11 sensitive strains, 16 field diclazuril-resistant strains, and 15 field salinomycin-resistant strains of E. tenella. Co-analysis indicated that the ABC transporter protein showed differential expression and base mutations in the two resistant strains compared with the DS strain. KEGG pathway analysis demonstrated that the expression of pABAS and HPPK-DHPS, which are associated with the folate biosynthetic pathway, showed downregulation only in the DZR strain with respect to the DS strain. Several key enzymes in the glycolytic pathway were differentially expressed between DS and SMR strains.

Abstract Solanum pimpinellifolium, the closest wild relative of the domesticated tomato, has high potential for use in breeding programs aimed at developing multi-pathogen resistance and quality improvement. We generated a chromosome-level genome assembly of S. pimpinellifolium LA1589, with a size of 833 Mb and a contig N50 of 31 Mb. We anchored 98.80% of the contigs into 12 pseudo-chromosomes, and identified 74.47% of the sequences as repetitive sequences. The genome evaluation revealed BUSCO and LAI score of 98.3% and 14.49, respectively, indicating high quality of this assembly. A total of 41,449 protein-coding genes were predicted in the genome, of which 89.17% were functionally annotated. This high-quality genome assembly serves as a valuable resource for accelerating the biological discovery and molecular breeding of this important horticultural crop.

Swine coccidiosis is a widespread disease caused by species of the apicomplexan parasites Eimeria and Cystoisospora. Coccidiosis is a common cause of diarrhea in suckling piglets worldwide that directly reduces piglets’ immunity and increases the risk of infection with other enteropathogens, leading to increased clinical infection and mortality and consequent economic losses in the global pig industry. We searched the available literature to date, including English and Chinese articles, using six electronic bibliographic databases, including PubMed, ScienceDirect, Web of Science, CNKI, VIP Chinese Journal Database, and Wanfang Data. A standard approach for conducting scoping reviews was used to identify studies on the species and distribution of genus Sus coccidia worldwide. A quality assessment was done for each study reviewed and relevant information reported in the identified studies was collated, categorized, and summarized. A total of 149 publications and references were eligible for the final review. The distribution of 18 species of genus Sus coccidia recorded in 63 countries was collated. These included 15 Eimeria and 3 Cystoisospora species. C. suis was found in 48 countries, E. debliecki in 45 countries, E. scabra in 33 countries, E. polita in 31 countries, E. suis in 28 countries, E. perminuta in 26 countries, E. porci in 24 countries, E. neodebliecki and E.spinosa in 21 countries each, E.guevarai in 5 countries, C. almataensis in 4 countries, E. betica in 2 countries, and E. almataensis, E. ibrahimovae, E. residualis, E. szechuanensis, E. yanglingensis, and C. sundarbanensis were each found in only 1 country. Each species was listed according to its scientific name, host name, finding location, and geographical distribution. This review reflects the distribution and infection of genus Sus coccidia worldwide and provides more complete basic information to aid our understanding of the species and geographical distribution of coccidia in the genus Sus.

Coccidiosis, caused by different species of Eimeria parasites, is an economically important disease in poultry and livestock worldwide. This study aimed to investigate the changes in the ileal microbiota and fecal metabolites in chickens after repeated infections with low-dose E. mitis. The chickens developed solid immunity against a high dose of E. mitis infection after repeated infections with low-dose E. mitis. The composition of the ileal microbiota and the metabonomics of the Eimeria-immunized group and the control group were detected using 16S rRNA sequencing and liquid chromatography–mass spectrometry (LC-MS). The relative abundance of Neisseria, Erysipelotrichaceae, Incertae sedis, Coprobacter, Capnocytophaga, Bifidobacterium, and the Ruminococcus torques group declined in the Eimeria-immunized chickens, whereas Alloprevotella, Staphylococcus, Haemophilus, and Streptococcus increased. Furthermore, 286 differential metabolites (including N-undecylbenzenesulfonic acid, 1,25-dihydroxyvitamin D3, gluconic acid, isoleucylproline, proline, and 1-kestose) and 19 significantly altered metabolic pathways (including galactose metabolism, ABC transporters, starch and sucrose metabolism, the ErbB signaling pathway, and the MAPK signaling pathway) were identified between the Eimeria-immunized group and the control group. These discoveries will help us learn more about the composition and dynamics of the gut microbiota as well as the metabolic changes in chickens infected with Eimeria spp.

This paper introduces a reduced Gaussian kernel filtered-x least mean square (RGKxLMS) algorithm for a nonlinear active noise control (NANC) system. This algorithm addresses the computational and storage challenges posed by the traditional kernel (i.e., KFxLMS) algorithm. Then, we analyze the mean weight behavior and computational complexity of the RGKxLMS, demonstrating its reduced complexity compared to existing kernel filtering methods and its mean stable performance. To further enhance noise reduction, we also develop the historical error correction RGKxLMS (HECRGKxLMS) algorithm, incorporating historical error information. Finally, the effectiveness of the proposed algorithms is validated, using Lorenz chaotic noise, non-stationary noise environments, and factory noise.

Necrotic enteritis (NE), caused by Clostridium perfringens, represents a major economic challenge to poultry production. This study evaluated the effects of four plant extracts—Astragalus, pomegranate peel, Sophora flavescens, and Artemisia annua—in combination with B. subtilis, as potential antibiotic alternatives for controlling NE. Using an experimentally induced NE model produced by coinfection with Eimeria maxima and C. perfringens, 288 chickens were divided into 12 groups to assess growth performance, intestinal lesions, and inflammatory markers. Compared with the enramycin group, the pomegranate peel extract group had a 9.9% increase in body weight gain (BWG), significant enhancement in claudin-2 expression, and reduced production of inflammatory factors. Compared with the infected control group, the combination of A. annua extract and B. subtilis resulted in the lowest intestinal lesion scores. The claudin-2, occludin, and zonula occludens-1 expression levels in the group treated with S. flavescens extract combined with B. subtilis increased to the highest levels compared to those in the healthy control group. These findings emphasize the potential of plant extracts, particularly pomegranate peel and A. annua with B. subtilis, as natural alternatives for NE management in poultry, providing a promising approach to reducing reliance on antibiotics.

BackgroundThis study aimed to investigate the knowledge, attitude, and practice (KAP) of human papillomavirus (HPV) and self-sampling among adult women.MethodsThe cross-sectional, questionnaire-based study included adult women at Shanghai Pudong Hospital from October 14, 2022, to March 31, 2023. The questionnaire contained demographic information, knowledge, attitude and practice dimensions. Factors associated with KAP and self-sampling were identified by multivariate logistic regression.ResultsA total of 1843 valid questionnaires were collected. The average knowledge, attitude, and practice score was 10.09 ± 5.60, 26.76 ± 3.80, and 6.24 ± 2.20, respectively. Urban residents (estimate = 0.705, p

With the advancements in deep learning and synthetic aperture radar (SAR) technology, an increasing number of individuals are utilizing deep-learning techniques to detect ships in SAR images. However, the efficiency of SAR ship detection is affected by complex background interference and various ship sizes. Addressing these challenges, this article proposes a balanced feature enhanced attention model. First, we introduce a novel attention feature fusion network (WEF-Net) tailored for SAR multiscale ship detection. WEF-Net effectively balances the information across different backbone layers and harmonizes semantic information from various levels of the feature pyramid through aggregation and averaging. Next, we embed the receiving field extension module in WEF-Net to learn the context information and generate the global characteristics of the receiving field balance. In addition, it can extract features from multiple scales to enhance the detection capability of the model for ships of different scales. At the same time, acknowledging the impact of surrounding complex background interference on the detector, we redesigned the ELAN module by combining convolution and attention. This enhancement enables the model to better attend to target position information during feature fusion, suppress the surrounding complex background interference, and highlight the ship's feature information. Finally, owing to the prevalence of small targets in SAR images, we employ an optimized loss function to bolster the model's performance in detecting small targets. This approach accelerates training convergence, reduces instances of missed detection on small targets, and enhances overall detection performance across multiple scales. Experimental results demonstrate that our model achieves detection accuracies of 98%, 93.1%, and 76.9% on the SAR ship detection dataset, high-resolution SAR image dataset, and large-scale SAR ship detection dataset, respectively, effectively discerning ship targets amid complex backgrounds in SAR images.

Avian coccidiosis, caused by several species of Eimeria, is a widespread and economically important poultry disease that inflicts severe losses in the poultry industry. Understanding the interplay between Eimeria and gut microbiota is critical for controlling coccidiosis and developing innovative treatments to ensure good poultry health. In the present study, chickens were immunized six times with a low dose of Eimeria tenella, resulting in complete immunity against Eimeria infection. The results of fecal microbiota transplantation showed that the gut microbiota of immunized chickens induced a certain degree of resistance to coccidial infection. To investigate the types of intestinal microbiota involved in the development of resistance to Eimeria, the intestinal contents and fecal samples from both immunized and unimmunized groups were collected for 16S rRNA gene sequencing. The results showed that, at the genus level, the abundance of the Eubacterium coprostanoligenes group, Erysipelatoclostridium, Shuttleworthia, and Colidextribacter was significantly increased in the intestinal content of immunized chickens, whereas the abundance of Eisenbergiella was significantly decreased. In fecal samples, the abundance of Clostridiaceae and Muribaculaceae significantly increased, whereas that of Bacillales significantly decreased. These findings will help to elucidate the interactions between E. tenella and the gut microbiota of chickens, providing a basis for isolating E. tenella-resistant strains from the gut microbiome and developing new vaccines against coccidiosis.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.

With the exponential growth of network resources, recommendation systems have become successful at combating information overload. In intelligent recommendation systems, the prediction of click-through rates (CTR) plays a crucial role. Most CTR models employ a parallel network architecture to successfully capture explicit and implicit feature interactions. However, the existing models ignore two aspects. One limitation observed in most models is that they focus only on the interaction of paired term features, with no emphasis on modeling unary terms. The second issue is that most models input characteristics indiscriminately into parallel networks, resulting in network input oversharing. We propose a disentangled self-attention neural network based on information sharing (DSAN) for CTR prediction to simulate complex feature interactions. Firstly, an embedding layer transforms high-dimensional sparse features into low-dimensional dense matrices. Then, the disentangled multi-head self-attention learns the relationship between different features and is fed into a parallel network architecture. Finally, we set up a shared interaction layer to solve the problem of insufficient information sharing in parallel networks. Results from experiments conducted on two real-world datasets demonstrate that our proposed method surpasses existing methods in predictive accuracy.

Eimeria tenella, an intestinal parasite, has brought huge economic losses to the poultry industry. The prevalence and severity of the development of drug resistance has increased the challenge of coccidiosis control. We previously identified the enolase 2 of E. tenella (EtENO2) was differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In this study, the expression of EtENO2 in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains was analyzed by quantitative real-time PCR (qRT-PCR) and western blots. EtENO2 was highly expressed in several drug-resistant strains compared with the DS strain. The qRT-PCR showed that the transcription level of EtENO2 in the field-isolated resistant strains was upregulated compared with the DS strain. The enzyme activity results indicated that the catalytic activity of EtENO2 in the drug-resistant strains was higher than in the DS strain. In addition, qRT-PCR and western blots showed that the expression level of EtENO2 was higher in second generation merozoites (SM) and unsporulated oocysts (UO) than that in sporozoites (SZ) and sporulated oocysts (SO). Immunofluorescence localization revealed that EtENO2 was distributed throughout SZ and SM and on the surface of the parasites. After the SZ invasion DF-1 cells, it was also observed on the parasitophorous vacuole membrane. Our secretion experiments found that EtENO2 could be secreted outside the SZ. This study indicated that EtENO2 might be related to the interaction between E. tenella and host cells and be involved in the development of E. tenella resistance to some anticoccidial drugs.

Background Oxidative stress refers to the imbalance between oxidants and antioxidants in organisms and often induces hepatic inflammation. Supplementing exogenous superoxide dismutase is an effective way to alleviate oxidative stress; however, the effects and mechanisms by which superoxide dismutase alleviates hepatic inflammation remain unclear. Methods This study established a Kunming mouse model to verify and investigate the oxidative stress and hepatic inflammation-alleviating effects of the superoxide dismutase oral supplement that was prepared by our research group in a previous study. Results The superoxide dismutase product significantly restored the body weight and liver alanine transaminase, aspartate aminotransferase, superoxide dismutase, catalase, glutathione, and glutathione peroxidase levels of oxidative stress induced mice. Moreover, exogenous superoxide dismutase significantly inhibited interleukin 1β and interleukin 6 mRNA expression in the livers of mice with hepatic inflammation. Transcriptomic analysis indicated that superoxide dismutase had a significant inhibitory effect on Endog expression, alleviating oxidative stress damage, and mediating liver cell apoptosis by regulating the expression of Rab5if, Hnrnpab, and Ifit1. Conclusion Our research verified the oxidative stress remediation effects of superoxide dismutase and its therapeutic role against hepatic inflammation. This study can lay a foundation for investigating the mechanism by which superoxide dismutase alleviates hepatic disease.

A key goal in current research on industrial curdlan production is the expansion of carbon sources for fermentation. In this study, a recombinant bacterial strain, sp-AmyAXCC, capable of fermenting and synthesizing curdlan using dextrin as a carbon source, was produced via heterologous expression of IPTG-inducible α-amylase from Xanthomonas campestris NRRL B-1459 in Agrobacterium tumefaciens ATCC31749. External expression of the enzyme was confirmed by western blotting, and the expression levels of exogenous proteins during the fermentation process were monitored. Additionally, the properties of the curdlan product were characterized using attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction. The recombinant strain produced curdlan at a titer of 30.40 ± 0.14 g/L, gel strength of 703.5 ± 34.2 g/cm2, and a molecular weight of 3.58 × 106 Da, which is 33% greater than the molecular weight of native curdlan (2.69 × 106 Da). In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L with a yield of 0.56 g/g, and a productivity rate of 0.62 g/L/h at 108 h. The results of this study expand the substrate spectrum for Agrobacterium fermentation in curdlan production and provides guidance for further industrialization of curdlan production.

Background: Ovarian cancer is a highly lethal gynecologic malignancy. ARHGAP10, a member of Rho GTPase-activating proteins, is a potential tumor suppressor in ovarian cancer. However, its role and the involved mechanism need further examination. Here, we investigated whether ARHGAP10 is also associated with ferroptosis. Methods: Lentivirus infection was used for gene overexpression or silencing. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to assess mRNA and protein levels, respectively. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. Lipid reactive oxygen species level was measured by flow cytometry. A tumorigenicity assay was performed to evaluate tumor growth in vivo, and sections of mouse tumor tissues were examined by immunofluorescence microscopy. Chromatin Immunoprecipitation (ChIP) assay was used to assess the binding of H3K9ac to the promoter region of ARHGAP10. Results: ARHGAP10 overexpression promoted ferroptosis in ovarian cancer cells, resulting in decreased cell viability, and increased lipid reactive oxygen species (ROS) level. Further, it decreased and increased GPX4 and PTGS2 expression, respectively, and also induced suppression of tumor growth in mice. Fer-1, a potent inhibitor of ferroptosis, suppressed the above effects of ARHGAP10. Contrarily, ARHGAP10 silencing alleviated ferroptosis in ovarian cancer cells, which was reversed by RSL3, a ferroptosis-inducing agent. Lastly, sodium butyrate (SB) was found to transcriptionally regulate ARHGAP10, thereby also contributing to the ferroptosis of ovarian cancer cells. Conclusions: Our results suggest that SB/ARHGAP10/GPX4 is a new signaling axis involved in inducing ferroptosis in ovarian cancer cells and suppressing tumor growth, which has potential clinical significance.

In order to realize the generating machining of worm gears， the worm gear hob is used on the NC transformed ordinary milling machine. The proportional relationship between the rotation of one turn of the worm gear hob and the rotation of one tooth of the worm gear workpiece is realized by extending the function of thread instruction and taper thread instruction. By distributing the allowable cumulative error of worm gear pitch evenly on several arcs of worm gear pitch circle， the accuracy of worm gear pitch and transmission is greatly improved， and thus the transmission stability is improved. The practice shows that this method not only achieves high machining accuracy of worm gears， but also expands the application range of NC transformation.

Abstract With the rapid development of radar, the phenomenon of Doppler frequency shift is not rare. Frequency shift needs to be considered in order to accurately calculate the mutual interference between signals. Meanwhile, to reduce interference to the received signal, each user's frequency hopping sequence shall have wide frequency hopping gap. The authors introduce the time‐frequency two‐dimensional Hamming correlation function and extend the bounds for wide‐gap frequency‐hopping sequences. The Li–Fan–Yang–Wang bounds are the special cases of our bounds for frequency shift is zero.

Abstract Background Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa, which has caused great economic loss to the poultry breeding industry. Host vimentin is a key protein in the process of infection of many pathogens. In an earlier phosphorylation proteomics study, we found that the phosphorylation level of host vimentin was significantly regulated after Eimeria tenella sporozoite infection. Therefore, we explored the role of host vimentin in the invasion of host cells by sporozoites. Methods Chicken vimentin protein was cloned and expressed. We used qPCR, western blotting, and indirect immunofluorescence to detect levels of mRNA transcription, translation, and phosphorylation, and changes in the distribution of vimentin after E. tenella sporozoite infection. The sporozoite invasion rate in DF-1 cells treated with vimentin polyclonal antibody or with small interfering RNA (siRNA), which downregulated vimentin expression, was assessed by an in vitro invasion test. Results The results showed that vimentin transcription and translation levels increased continually at 6–72 h after E. tenella sporozoite infection, and the total phosphorylation levels of vimentin also changed. About 24 h after sporozoite infection, vimentin accumulated around sporozoites in DF-1 cells. Treating DF-1 cells with vimentin polyclonal antibody or downregulating vimentin expression by siRNA significantly improved the invasion efficiency of sporozoites. Conclusion In this study, we showed that vimentin played an inhibitory role during the invasion of sporozoites. These data provided a foundation for clarifying the relationship between Eimeria and the host. Graphical Abstract

ABSTRACT: Mesocarp color is an important agronomic trait of Cucurbita maxima and is determined mainly by the contents and compositions of the carotenoids. The two inbred lines with significant differences in fruit flesh color were used in the study, the orange ‘312-1’ and white ‘98-2’. Changes in seven carotenoid contents and compositions in the flesh of fruit produced by inbred lines ‘312-1’ and ‘98-2’ were analyzed during fruit development. The expression of eight key carotenoid biosynthesis genes in the fruit flesh were investigated during five fruit development stage in two inbred lines. As the flesh color intensified, the orange flesh color of ‘312-1’ was determined mainly by the increased contents of lutein, β-carotene and zeaxanthin and the lack of carotenoid accumulation led to the formation of white flesh in ‘98-2’ fruit. The expression of the LCY-e and CHYb genes was significantly stronger in ‘312-1’ than in ‘98-2’, and their expression was strongly correlated with lutein and β-carotene contents during fruit development. This study provides a deep understanding of the molecular mechanism of carotenoid biosynthesis in fruit flesh and provides a basis for additional studies on the highly refined improvement of squash quality.

This paper discusses the modeling and solving of orbital pursuit–evasion games (OPEGs) under J2 perturbation. The optimal long-range maneuver method under J2 perturbation is designed, and it is proved that the effect of eccentricity can be ignored when transfer times and the ΔV budgets are fixed. It is discovered that when the inclination between the initial and target orbit is equal and is between 10° and 25°, the whole maneuver process can be simplified to a fixed-inclination transfer. Subsequently, a long-term OPEG model is provided under the assumption of fixed inclination, zero eccentricity, and impulsive thrust. Winning conditions of OPEGs under J2 perturbation are then carefully derived, with long-time OPEG (J2 dominated) and short-time OPEG (traditional) separated, and typical tactics of both sides formulated and verified. These studies are further extended to the “Arrival time matching game” for maintaining/avoiding resonant arrival time under J2 perturbation, and the advantages and disadvantages of both sides are analyzed. The models and strategies obtained in this paper can be potentially used in practical applications of OPEGs, especially for evaders that have low thrust–weight ratios and are weak in traditional short-term OPEGs.

This paper explores the blocking capabilities of a spacecraft deployed near Earth–Moon L1 libration point against another spacecraft attempting lunar gravity assist, based on the ΔV required for interception. The study demonstrates that a pursuer at L1 libration point can effectively block low-energy evaders with minimal ΔV expenditure, creating a blockade against their use of gravity assists. However, blocking against high-energy evaders is relatively weaker. Pursuers on Lyapunov orbits can execute blockades that L1 pursuers cannot, albeit with lower mission-capable rates. The paper discusses mission-capable rates for different Lyapunov orbits and evader energies, revealing that each Lyapunov orbit has its unique optimal blocking energy, decreasing as the Lyapunov orbit size expands. In addition, the paper proposes a strategy for evaders to bypass blockades by sacrificing a portion of their ΔV and verifies it numerically. The analysis covers the cost and benefits of the L1 libration point-related blockade, the importance of the mission-capable rate, and the possibility of lunar-orbit blocking. These findings provide insights for future research on orbital games in the Earth–Moon system and orbital blockade.

In quasi-synchronous frequency-hopping multiple access (QS-FHMA) systems, low-hit-zone (LHZ) frequency-hopping sequence (FHS) sets have been well-applied to reduce mutual interference (MI). In this paper, we propose three constructions of LHZ FHS sets with new parameters via interleaving techniques. The obtained sequences can be verified that they are optimal with respect to the Peng–Fan–Lee bound.

Ammonia, one of the most polluted gases in poultry houses, has always been an urgent problem to solve. Exposure to ammonia can threaten the respiratory tract, induce inflammation, and decrease growth performance. To date, there are few studies investigating the effects of ammonia on skeletal muscle growth. In this experiment, a total of 144 broilers were randomly divided into two groups, and 0 ppm and 35 ppm atmospheric ammonia were administered in the chambers. The trial lasted for 21 days. The breast muscle, thigh muscle, dressed weight, and serum biochemical indexes were measured. The skeletal muscle fibre morphology was observed using light microscopy, and the expressions of genes associated with skeletal muscle development and myosin heavy chain genes were assessed. After 7 days of ammonia exposure, the broilers’ weight in the ammonia group decreased. On the 21st day of the experiment, in the ammonia group, the breast muscle weight, thigh muscle weight, and dressed weight decreased, the blood urea nitrogen content increased, skeletal muscle fibre diameter shortened, the expression of myostatin increased, and the expression of myosin heavy chain-FWM and myosin heavy chain-FRM decreased significantly. This article suggests that 35 ppm atmospheric ammonia seriously affects the skeletal muscle gain rate of broilers, and the myostatin pathway could be a potential regulation of the growth rate of muscle fibre under ammonia exposure.

The widespread use of drugs has exacerbated the resistance of Eimeria tenalla to anti-coccidial drugs. Using RNA-seq, we previously found the ATPase ASNA1 homolog of E. tenella (EtASNA1) was differentially expressed in resistant strains and drug sensitive (DS) strain. In our study, we used western blotting and quantitative real-time PCR (qRT-PCR) to analyze the translational and transcriptional levels of EtASNA1 in a diclazuril-resistant (DZR) strain, maduramicin-resistant (MRR) strain, salinomycin-resistant (SMR) strain, and DS strain and found EtASNA1 was highly expressed in three drug-resistant strains. The qRT-PCR and western blotting results also showed that the expression levels of EtASNA1 increased with increasing drug concentration, and the transcription levels of the DZR strains isolated from the field were higher than those of the DS strain. In addition, we used in vivo and in vitro tests to analyze the changes of EtASNA1 expression after DZR, MRR, and DS strain infections in chickens, and in vitro inoculation of DF-1 cells in the presence of drugs. The addition of drugs caused expression to be upregulated. The results of qRT-PCR and western blotting also showed that the expression levels of EtASNA1 in second-generation merozoites (SM) and unsporulated oocysts (UO) were significantly higher than those in the other two developmental stages. The immunofluorescence localization of EtASNA1 indicated that the protein was distributed throughout the sporozoites (SZ) and SM, except for the refractile bodies of SZ. In vitro inhibition experiments showed that anti-EtASNA1 antibody incubation significantly inhibited SZ invasion of DF-1 cells. The above results showed that EtASNA1 may be related to host cell invasion of E. tenella and may be involved in the development of E. tenella resistance to some drugs.

Abstract Background Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species. These parasites have complex and diverse life-cycles that require invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp). Methods Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C. The localization of EtEsp was analyzed with an immunofluorescence assay (IFA). An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites. Results The interaction between EtAMA1 and EtEsp was confirmed with BiFC and by GST pull-down. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life-cycle. IFA showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells. Conclusion In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in sporozoite invasion of host cells and is maybe involved in the growth of the parasite. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

The presence of gastric cancer stem cells (GCSCs) marks the onset of gastric carcinoma. The sonic hedgehog (SHH) pathway plays a vital role in the maintenance of GCSC characteristics. Apatinib has been approved in China for advanced gastric cancer (GC) treatment. However, whether apatinib can target GCSCs and affect the SHH pathway remains unclear. The present study aimed to investigate the underlying mechanism of apatinib’s antitumor effects on GC. The expression levels of GCSC markers and number of CD133+ cells were significantly elevated in the sphere-forming cells. Apatinib effectively suppressed GCSC traits by inhibiting tumorsphere formation and cell proliferation, suppressing GCSC markers expression and CD133+ cell number, and inducing apoptosis. Apatinib downregulated the activation of the SHH pathway; while upregulation of the SHH pathway attenuated the inhibitory effects of apatinib on GCSCs. Moreover, apatinib treatment significantly delayed tumor growth and inhibited GCSC characteristics in the xenograft model. Our data suggested that apatinib exhibited inhibitory effects on GCSCs by suppressing SHH pathway both in vitro and in vivo, thus providing new insights into the therapeutic application of apatinib in GCSC suppression and advanced gastric cancer treatment.

Thermoacidophilic Alicyclobacillus strains attract great interests as the resource of thermostable or acidic enzymes. In this study, a putative gene encoding superoxide dismutase (AaSOD) was identified in a thermoacidophilic Alicyclobacillus strain. With a 16-fold activity observed, the AaSOD activity expressing in the medium of manganese enrichment was much higher than that in the iron medium. In addition, the purified AaSOD can be reconstituted exclusively with either Fe2+ or Mn2+, with its Mn-bound protein showing 25-fold activity than that of Fe-bound form. The optimal temperature for AaSOD reaction was 35°C, and was highly stable at any certain temperature up to 80°C. Of particular interest, the enzyme is found to be very stable across a wide pH range spanning from 2.0 to 10.0, which confers its robust stability in the acidic stomach environment and implies striking potentials as food additive and for medical use.

The fungus Stemphylium lycopersici (S. lycopersici) is an economically important plant pathogen that causes grey leaf spot disease in tomato. However, functional genomic studies in S. lycopersici are lacking, and the factors influencing its pathogenicity remain largely unknown. Here, we present the first example of genetic transformation and targeted gene replacement in S. lycopersici. We functionally analyzed the NLP gene, which encodes a necrosis- and ethylene-inducing peptide 1 (Nep1)-like protein (NLP). We found that targeted disruption of the NLP gene in S. lycopersici significantly compromised its virulence on tomato. Moreover, our data suggest that NLP affects S. lycopersici conidiospore production and weakly affects its adaptation to osmotic and oxidative stress. Interestingly, we found that NLP suppressed the production of reactive oxygen species (ROS) in tomato leaves during S. lycopersici infection. Further, expressing the fungal NLP in tomato resulted in constitutive transcription of immune-responsive genes and inhibited plant growth. Through gene manipulation, we demonstrated the function of NLP in S. lycopersici virulence and development. Our work provides a paradigm for functional genomics studies in a non-model fungal pathogen system.

Oxidative stress, which could be evoked by numerous inducements including mycotoxins like deoxynivalenol (DON), cause severe damages to organisms. Antioxidants are promising protectants against oxidative stress that could be applied in pharmaceutical, cosmetic, and food and feed industries. In this study, a thermostable and acidophilic superoxide dismutase (AaSOD) was used to develop an antioxidant product that can potentially protect organisms from oxidative stress related damages. The enzyme was successfully expressed as an extracelluar protein in Bacillus subtilis with a high yield. To obtain a feasible protocol for industrial production of AaSOD, the fermentation mediums that are commonly used for culturing B. subtilis were screened, the feasibility of expressing AaSOD without antibiotic as selection pressure was confirmed, and the effect of using lactose as an inducer instead of isopropyl-β-d-thiogalactoside (IPTG) was investigated. Batch fermentation was conducted to validate the optimized conditions for AaSOD production, and 6530 U mL-1 of SOD activity was obtained in the fermentation broth. The dry powder product of AaSOD with an activity of 22202 U g-1 was prepared by spray-drying and was administrated on zebrafish to test its function as a protectant against DON, and thus gained a significant redress of the reactive oxygen species (ROS) accumulation induced by DON. Taken together, this study provides a feasible protocol to prepare the AaSOD-based antioxidant product that is potentially applied in livestock industry.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

The analogues of biphenol A (BPA), including bisphenol S (BPS) and bisphenol B (BPB), are commonly used to replace the application of BPA in containers and wrappers of daily life. However, their safeties are questioned due to their similar chemical structure and possible physiological effects as BPA. To investigate the neurotoxic effects of BPA, BPS, and BPB as well as their underlying mechanism, IMR-32 cell line from male and SK-N-SH cell line from female were exposed respectively to BPA, BPS and BPB with concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM for 24 h. Additionally, 24 h exposure of BPA combining epigallocatechin gallate (EGCG) (4 μM and 8 μM for IMR-32 and SK-N-SH respectively) were conducted. Results demonstrated that BPs exposure could promote reactive oxygen species production and increase level of malondialdehyde (MDA) while decrease levels of superoxide dismutase (SOD). Intensive study revealed that after exposure to BPA mitochondrial membrane potential (MMP) dropped down and the protein expression levels of Bak-1, Bax, cytochrome c and Caspase-3 were up-regulated but Bcl-2 were down-regulated significantly. Moreover, apoptosis rate was raised and cell activity declined remarkably in the neuroblastoma cells. All the effects induced by BPA could be alleviated by the adding of EGCG, which similar alleviations could be inferred in IMR-32 and SK-N-SH cells induced by BPS and BPB. Furthermore, BPS showed lower neurotoxic effects compared to BPA and BPB. Interestingly, the neurotoxic effects of BPA on IMR-32 cells were significantly higher than those on SK-N-SH cells. In conclusion, the results suggested that BPA, BPS and BPB could induce oxidative stress and apoptosis via mitochondrial pathway in the neuroblastoma cells and male is more susceptible to BPs than female.

Seed traits are agronomically important for Cucurbita breeding, but the genes controlling seed size, seed weight and seed number have not been mapped in Cucurbita maxima (C. maxima). In this study, 100 F2 individual derived from two parental lines, “2013-12” and “9-6”, were applied to construct a 3,376.87-cM genetic map containing 20 linkage groups (LGs) with an average genetic distance of 0.47 cM using a total of 8,406 specific length amplified fragment (SLAF) markers in C. maxima. Ten quantitative trait loci (QTLs) of seed width (SW), seed length (SL) and hundred-seed weight (HSW) were identified using the composite interval mapping (CIM) method. The QTLs affecting SW, SL and HSW explained a maximum of 38.6%, 28.9% and 17.2% of the phenotypic variation and were detected in LG6, LG6 and LG17, respectively. To validate these results, an additional 150 F2 individuals were used for QTL mapping of SW and SL with cleaved amplified polymorphic sequence (CAPS) markers. We found that two major QTLs, SL6-1 and SW6-1, could be detected in both SLAF-seq and CAPS markers in an overlapped region. Based on gene annotation and non-synonymous single-nucleotide polymorphisms (SNPs) in the major SWand SL-associated regions, we found that two genes encoding a VQ motif and an E3 ubiquitin-protein ligase may be candidate genes influencing SL, while an F-box and leucinerich repeat (LRR) domain-containing protein is the potential regulator for SW in C. maxima. This study provides the first high-density linkage map of C. maxima using SNPs developed by SLAF-seq technology, which is a powerful tool for associated mapping of important agronomic traits, map-based gene cloning and marker-assisted selection (MAS)-based breeding in C. maxima.

Abstract Background Eimeria tenella is an obligate intracellular apicomplexan protozoan parasite that has a complex life-cycle. Calcium ions, through various calcium-dependent protein kinases (CDPKs), regulate key events in parasite growth and development, including protein secretion, movement, differentiation, and invasion of and escape from host cells. In this study, we identified proteins that interact with EtCDPK4 to lay a foundation for clarifying the role of CDPKs in calcium channels. Methods Eimeria tenella merozoites were collected to construct a yeast two-hybrid (Y2H) cDNA library. The Y2H system was used to identify proteins that interact with EtCDPK4. One of interacting proteins was confirmed using bimolecular fluorescence complementation and co-immunoprecipitation in vivo. Co-localization of proteins was performed using immunofluorescence assays. Results Eight proteins that interact with EtCDPK4 were identified using the Y2H system. One of the proteins, E. tenella serine protease inhibitor 1 (EtSerpin), was further confirmed. Conclusion In this study, we screened for proteins that interact with EtCDPK4. An interaction between EtSerpin and EtCDPK4 was identified that may contribute to the invasion and development of E. tenella in host cells.

The emergence of helicity from the densest possible packings of equal-sized hard spheres in narrow cylindrical confinement can be understood in terms of a density maximization of repeating microconfigurations. At any cylinder-to-sphere diameter ratio D∈(1+3/2,2), a sphere can only be in contact with its nearest and second nearest neighbors along the vertical z-axis, and the densest possible helical structures are results of a minimized vertical separation between the first sphere and the third sphere for every consecutive triplet of spheres. By considering a density maximization of all microscopic triplets of mutually touching spheres, we show, by both analytical and numerical means, that the single helix at D∈(1+3/2,1+43/7) corresponds to a repetition of the same triplet configuration and that the double helix at D∈(1+43/7,2) corresponds to an alternation between two triplet configurations. The resulting analytic expressions for the positions of spheres in these helical structures could serve as a theoretical basis for developing novel chiral materials.

The pedunculopontine nucleus (PPN) is composed of a morphologically and neurochemically heterogeneous population of neurons, which is severely affected by Parkinson’s disease (PD). However, the role of each subtype of neurons within the PPN in the pathophysiology of PD has not been completely elucidated. In this study, we present the discharge profiles of three classified subtypes of PPN neurons and their alterations after 6-hydroxydopamine (6-OHDA) lesion. Following 6-OHDA lesion, the spike timing of the Type II (GABAergic) and Type III (glutamatergic) neurons had phase-lock with the oscillations in the delta and beta band frequency range in the PPN, respectively. Morphological evidence has shown distinct alteration in three kinds of neurons after 6-OHDA lesion. These findings revealed that the changes in the firing characteristics of neurons in PPN in hemi-parkinsonism rats are closely associated with damaged neuronal morphology, which would make contributions to the divergence of dysfunctions in Parkinsonism.

Reversible data hiding (RDH) has become a hot spot in recent years as it allows both the secret data and the raw host to be perfectly reconstructed, which is quite desirable in sensitive applications requiring no degradation of the host. A lot of RDH algorithms have been designed by a sophisticated empirical way. It is not easy to extend them to a general case, which, to a certain extent, may have limited their wide-range applicability. Therefore, it motivates us to revisit the conventional RDH algorithms and present a general framework of RDH in this paper. The proposed framework divides the system design of RDH at the data hider side into four important parts, i.e., binary-map generation, content prediction, content selection, and data embedding, so that the data hider can easily design and implement, as well as improve, an RDH system. For each part, we introduce content-adaptive techniques that can benefit the subsequent data-embedding procedure. We also analyze the relationships between these four parts and present different perspectives. In addition, we introduce a fast histogram shifting optimization (FastHiSO) algorithm for data embedding to keep the payload-distortion performance sufficient while reducing the computational complexity. Two RDH algorithms are presented to show the efficiency and applicability of the proposed framework. It is expected that the proposed framework can benefit the design of an RDH system, and the introduced techniques can be incorporated into the design of advanced RDH algorithms.

In order to investigate the influence of ammonia on broiler intestinal microflora and growth performance of broiler chickens, 288 21-day-old male Arbor Acres broilers with a similar weight were randomly divided into four groups with different NH3 levels: 0 ppm, 15 ppm, 25 ppm, and 35 ppm. The growth performance of each group was recorded and analyzed. Additionally, 16s rRNA sequencing was performed on the cecal contents of the 0 ppm group and the 35 ppm group broilers. The results showed the following: a decrease in growth performance in broilers was observed after 35 ppm ammonia exposure for 7 days and 25 ppm ammonia exposure for 14 days. At phylum level, the relative abundance of Proteobacteria phylum was increased after 35 ppm ammonia exposure. At genus level, ammonia increased the relative abundance of Escherichia–Shigella and decreased the relative abundance of Butyricicoccus, Parasutterella, Lachnospiraceae_UCG-010, Ruminococcaceae_UCG-013 and Ruminococcaceae_UCG-004. Negative correlation between Escherichia–Shigella and growth performance, and positive correlation between bacteria genera (including Butyricicoccus, Parasutterella, Lachnospiraceae_UCG-010, Ruminococcaceae_UCG-013 and Ruminococcaceae_UCG-004) and growth performance was observed. In conclusion, ammonia exposure caused changes in the structure of cecal microflora, and several species were either positively or negatively correlated with growth performance. These findings will help enhance our understanding of the possible mechanism by which ammonia affect the growth of broilers.

Due to the good filter performance for the non-Gaussian noise, the adaptive filters with error nonlinearities have received increasing attention recently. From the viewpoint of the weighted function, in this paper, the existing least mean square (LMS)-based adaptive algorithms with error nonlinearities are divided into three types, i.e., V-shaped, A-shaped, and M-shaped algorithms. Then, to obtain the merits of the V-shaped and A-shaped algorithms, a new family of robust M-shaped error weighted LMS algorithms is proposed. Their steady-state mean square deviation (MSD) analyses are made, which reveal the learning abilities of error nonlinearities: 1) for the V-shaped algorithm, it can achieve smaller steady state MSD for sub-Gaussian noise than that for super-Gaussian noise; 2) the A-shaped algorithm can be used more effectively for super-Gaussian noise than that for sub-Gaussian noise; and 3) the M-shaped algorithm combines the characteristics of the V-shaped and A-shaped algorithms. Furthermore, based on the proposed robust M-shaped function, a proportionate normalized robust M-shaped algorithm is presented for echo cancellation application. Finally, Monte Carlo simulations are conducted to verify the theoretical results and to demonstrate the efficiency of the proposed algorithms in different environments.

Abstract Background Silent information regulator 2 (SIR2) proteins are a family of NAD + -dependent protein deacetylases that are considered potential targets for anti-parasitic agents. In this study, we cloned and characterized SIR2A of the protozoan parasite Eimeria tenella (EtSIR2A) and investigated its protective efficacy as a DNA vaccine. Methods The EtSIR2A gene encoding 33.37 kDa protein from E. tenella second-generation merozoites was cloned, and recombinant EtSIR2A protein (rEtSIR2A) was produced in an Escherichia coli expression system. The rEtSIR2A was used to immunize rabbits. Anti-rEtSIR2A antibodies were used to determine the immunolocolization of EtSIR2A in the parasite by immunofluorescence assay (IFA). Transcript and protein expression of EtSIR2A in different development stages of E. tenella were observed by quantitative real-time PCR (qPCR) and western blot (WB) analysis, respectively. The recombinant plasmid pCAGGS-EtSIR2A was constructed and its efficacy against E. tenella infection in chickens was evaluated. Results qPCR and WB analysis revealed EtSIR2A expression was developmentally regulated at both the mRNA and protein levels. EtSIR2A mRNA levels were higher in unsporulated oocysts than at other developmental stages, including sporulated oocysts, sporozoites and second-generation merozoites. In contrast, EtSIR2A protein expression levels were highest in second-generation merozoites, moderate in unsporulated oocysts and sporulated oocysts and lowest in sporozoites. Immunostaining with anti-rEtSIR2A antibody indicated that EtSIR2A was mainly located in the cytoplasm of sporozoites and second-generation merozoites, and was strongly expressed during first stage schizogony. Animal-challenge experiments demonstrated that immunization with pCAGGS-EtSIR2A significantly increased average body-weight gain, and decreased mean lesion score and oocyst output in chickens. Conclusions These results suggest that EtSIR2A may play an important role in parasite cell survival and may be an effective candidate for the development of new vaccines against E. tenella infection in chickens.

Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. cinerea inoculation on medium and planta based on our study using the tomato-B. cinerea system.

Sentiment analysis is widely studied to extract opinions from user generated content (UGC), and various methods have been proposed in recent literature. However, these methods are likely to introduce sentiment bias, and the classification results tend to be positive or negative, especially for the lexicon-based sentiment classification methods. The existence of sentiment bias leads to poor performance of sentiment analysis. To deal with this problem, we propose a novel sentiment bias processing strategy which can be applied to the lexicon-based sentiment analysis method. Weight and threshold parameters learned from a small training set are introduced into the lexicon-based sentiment scoring formula, and then the formula is used to classify the reviews. In this paper, a completed sentiment classification framework is proposed. SentiWordNet (SWN) is used as the experimental sentiment lexicon, and review data of four products collected from Amazon are used as the experimental datasets. Experimental results show that the bias processing strategy reduces polarity bias rate (PBR) and improves performance of the lexicon-based sentiment analysis method.

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results suggested that EtPDIL might be involved in sporulation in external environments and in host cell adhesion, invasion and development of E. tenella.

Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites). The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1) and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.