In freshwater cages, 13,500 adult female triploid rainbow trout (3.2 ± 0.1 kg) were cultured for 77 days. Triplicate cages (500 fish per cage) were used as repetitions of each experimental diet. The findings revealed that as DP increased to 400 g kg−1 and DL raised to 300 g kg−1, the weight gain ratio (WGR) elevated significantly (P < 0.05). However, when DP ≥ 350 g kg−1, WGR was similar in the DL250 and DL300 groups. As DP raised to 350 g kg−1, the feed conversion ratio (FCR) notably decreased (P < 0.05). In the DP350DL300 group, lipids had a protein-sparing impact. High DP diet (400 g kg−1) generally improved fish health status by increasing antioxidant capacity in the liver and intestine. A high DL diet (300 g kg−1) showed no harmful effect on hepatic health based on plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and antioxidant capacity in the liver. For fillet quality, a high DP diet could increase fillet yield, improve fillet hardness, springiness, and water-holding capacity values, and inhibit the production of off-flavors caused by n-6 fatty acids. A high DL diet could increase odor intensity, and EPA, DHA, and n-3 fatty acid concentrations decrease the thrombogenicity index value. The maximum fillet redness value was discovered in the DP400DL300 group. Overall, for adult triploid rainbow trout (≥3 kg), the minimum recommended DP and DL according to growth performance were 400 and 250 g kg−1, respectively; DP and DL based on feed utilization were 350 and 200 g kg−1, respectively; DP and DL based on fillet quality were 400 and 300 g kg−1, respectively.The research is aimed at investigating the effects of dietary protein and lipid levels on adult triploid rainbow trout growth performance, feed utilization, digestive and metabolic enzyme activities, antioxidative capacity, and fillet quality. Nine diets containing three dietary protein levels (DP) (300, 350, and 400 g kg Oncorhynchus mykiss), belonging to the order Salmoniformes and the family Salmonidae, is an essential cold-water aquaculture fish [Rainbow trout . As a result, this study was conducted using a two-factor design to explore the recommended level of lipid and protein for adult triploid rainbow trout (≥3 kg) based on growth performance, feed utilization, digestion and metabolism, antioxidant response, and fillet quality. It is expected to provide a basis for the establishment of a nutritional database and the development of an efficient and environmentally diet of triploid rainbow trout.Regarding triploid rainbow trout, our earlier research has indicated that the minimum requirement of lipid and protein in fish  was 233 g kg−1 [−1 , respect−1, respectively, and the recommended level of protein in diets was 360 g kg−1 for rainbow trout  [−1) and three protein levels (DP)  was utilized to formulate a total of nine diets labelled DP300DL200, DP300DL250, DP300DL300, DP350DL200, DP350DL250, DP350DL300, DP400DL200, DP400DL250, and DP400DL300, respectively. The major lipid and protein sources were fish oil and fish meal, respectively. Based on the results of our previous studies, the minimum level of lipid and protein in the diet for triploid rainbow trout  was 250 and 400 g kg1,500 g) . A two-fn = 3), which were placed in a reservoir in the Qinghai province of China, were chosen to culture the trout fed one of the nine different experimental diets. Fish were hand-fed at 08 : 30 and 16 : 30 using the standard of apparent satiation (stop feeding when most fish were not actively eating). The number and weight of dead fish and feed consumption were documented throughout this period. The feeding trial of 77 days was carried out in the autumn (from October to December). Dissolved oxygen (7.4 ± 1.6 mg L−1) and water temperature (11.1 ± 3.7°C) were measured daily.A total of 13,500 adult triploid rainbow trout  weighting 3.2 kg were taken from the local fisheries in Qinghai province of China. These trout were then randomly dispersed into 27 net cages of 8 × 8 × 8 meters, with 500 fish in each cage. During the feeding trial, three replicate net cages (n = 3) were created for each diet group, and all samples were frozen in liquid nitrogen and subsequently kept at -80°C for further analysis. The fillet samples were prepared on the basis of the method described in our previous study [After 24 h fasting of all the experimental fish following the feeding experiment, all of the fish in each net cage were gathered, and thirty fish were taken at random and weighted following anesthesia with eugenol at a ratio of one to ten thousand . The aforementioned weighting technique was repeated three times, and the mean value of three weights of 30 fish was used as the average final weight of fish in each cage. One ungutted fish per net cage was chosen at random to analyze the whole body composition. Three fish per net cage were chosen to measure body weight and body length after anesthesia, respectively, and then blood samples were obtained from the caudal vein of each fish with 5 mL syringes. Plasma samples were separated by centrifuging at 4,000 × g for 10 min and combined to form one biological duplicate with three fish from one cage of comparable volume. Three bled fish were then dissected to obtain viscera, abdominal fat, hepatic, intestine, and two fillets. In order to determine the viscerosomatic index (VSI), relative intestinal length (RIL), fillet yield (FY), abdominal fat index (AFI), and hepatosomatic index (HSI), the weights of each fish's viscera, abdominal fat, liver, and fillet, as well as the length of the intestine were measured. Liver and intestinal samples were combined to form one biological duplicate with three fish from one cage of comparable size. Three biological replicates  was measured using a Dumas Nitrogen Analyzer . The results were used to calculate the crude protein contents in the whole body, liver, and fillet. The crude lipid contents in the whole body, liver, and fillet were extracted using the Folch et al. [v/v). A commercial test kit with the anthrone-sulfuric acid colorimetric method  was used to determine the glycogen content in the liver.To assess moisture content, whole body, liver, and fillet samples were freeze-dried to a consistent weight at -45°C. The nitrogen content  in a recognized hospital.L∗), redness (a∗), yellowness (b∗), chroma (C∗ab), and hue (Hab°) was also assessed directly using a colorimeter  on the basis of our previous study [The color card  was used to analyze the redness of the fillet visually. Fillet color described by lightness (us study .Fillet pH and water holding capacity (WHC) was assayed by the methods of Meng et al.  by usingTexture profile analyses (TPA) were performed on the texture of fillets using a texture analyzer  following the methodology given in our earlier work . The insN): the first significant break in the first compression cycle;Fracture (N): peak force in the first compression cycle;Hardness (Adhesiveness (mJ): the negative area for the first bite;Cohesiveness: positive area of the second compression cycle/positive area of the first compression cycle;Springiness (mm): the end of the first bite height—the start of the second height;Chewiness (mJ): hardness × cohesiveness × springiness.The fatty acid analysis was performed on the collected lipid following the fillet lipid assay. The fatty acids were methyl esterified and analyzed using gas chromatography-mass spectrometry , and nutritional value indicators of fatty acids  and thrombogenicity index (TI)) were calculated following the methodology given in our earlier work .The approach reported by Ma et al.  was utilA two-factorial design (3 × 3) was used to assess the interaction between DP and DL. Factorial (two-way) and one-way ANOVA  were conducted for all the data using SPSS 26.0 software following the approach described by Xu et al. . The datP > 0.05). According to two-way ANOVA, DP × DL did not affect FW, WGR, and FCR of triploid rainbow trout (P > 0.05). DP significantly affected WGR and FCR (P < 0.05). WGR elevated considerably with DP increasing and peaked in the DP400 group, while FCR declined as DP increased to 350 g kg−1, then plateaued (350 to 400 g kg−1). DL affects FW and WGR significantly (P < 0.05), but no effect on FCR was observed. FW and WGR elevated considerably as DL increased; the highest value was found in the DL300 group (P < 0.05). When DP ≥ 350 g kg−1, the indicators were similar in the DL250 and DL300 groups.Data on growth performance and feed utilization of triploid rainbow trout are presented in P > 0.05, P < 0.05) influence on FY, AFI, and VSI values, while DP × DL had no effect. VSI and AFI values increased and then decreased (P < 0.05) as DP increased, and the highest values were found in the DP350 group. The DP400 group showed significantly (P < 0.05) higher FY value than the other groups. With DL increasing, the VSI and AFI values raised and peaked in the DL300 group (P < 0.05). The FY value in the DL300 group was substantially lower than that in the other DL group. HSI and RIL values were significantly (P < 0.05) affected by the DP × DL. The highest HSI value was found in the DL300 group when DP ≤ 350 g kg−1, but it was not varied as DL when DP was 400 g kg−1. The DP300DL200 group had the lowest RIL value, while the DP350DL200 group had the highest value.Based on two-way ANOVA, the CF value was not affected by DP, DL, and DP × DL  on hepatic glycogen, protein, or lipid content. DP significantly affected hepatic lipid, protein, and glycogen contents (P < 0.05). The DP400 group had greater protein and lipid contents but lower glycogen content than the DP300 and DP350 groups (P < 0.05). DL only affected hepatic glycogen content. The DL300 group had significantly greater glycogen content than the DL200 and DL250 groups (P < 0.05).Data on fish liver compositions are presented in P < 0.05), but the effect of DP × DL was not significant (P > 0.05). The DP400 group had significantly (P < 0.05) lower protease activity than DP300 and DP350 groups. The DL250 group had significantly greater protease activity than the DL200 and DL300 groups (P < 0.05) with DL rising and peaking in the DL300 group. It also potentiated significantly with DP increasing and peaked in the DP350 group (Data on protease and lipase activities in the digestive tract are shown in 0 groups . Lipase 50 group .P < 0.05) . Furthermore, ALT and AST activities in the liver showed an increasing trend with DL increasing when DP ≤ 350 g kg−1, but the tendency was reversed when DP was 400 g kg−1. When DL was 200 g kg−1, the DP300 group had the greatest activities of HL and total lipase in the liver, whereas the DP400 group had the highest LPL activity. In addition, when DL was 300 g kg−1, the maximum LPL and total lipase activities in the liver were found in the DP350 group.As seen in  < 0.05) . When DLP < 0.05). When DP was 300 and 400 g kg−1, the DL250 and DL300 groups exhibited low plasma T-AOC level  . DP × DL significantly affected a∗ and C∗ab values. The a∗ value notably elevated with DL increasing when DP was 400 g kg−1, but it reduced with DL increasing when DP ≤ 350 g kg−1 (P < 0.05). The highest C∗ab value was found in the DP400DL250 and DP400DL300 groups.DP, DL, and DP × DL fail to affect fillet redness value assessed visually, P > 0.05). DP or DL had the significant influence on fracture, hardness, and adhesiveness values (P < 0.05), while the effect of DP × DL was not significant (P > 0.05). The DL200 group had the greatest fracture, hardness, and adhesiveness values. The values of hardness and adhesiveness enhanced with DP increasing up to 350 g kg−1, and then decreased. DP × DL notably (P < 0.05) affected springiness, chewiness, and WHC values. The DP350 group had a lower springiness value than the other groups. The highest springiness and chewiness values were found in the DP300DL200 group. The DP300 group had a lower WHC value than the DP350 and DP400 groups, and the lowest value was found in the DP300DL300 group.Data on fillet texture are presented in E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal, -2,4-heptadienal, -2,6-nonadienal, and -2,4-nonadienal), and 1 furan (2-ethyl-furan). DP, DL, and DP × DL fail to affect the concentrations of 2,3-octanedione, heptanal, nonanal, (E)-2-nonenal, (E)-2-decenal, and -2,4-nonadienal. The concentrations of 1-heptanol, decanal, undecanal, -2,6-nonadienal, and subtotal concentration of n-9 fatty acids (n-9 derived) increased as DP increasing (P < 0.05) and peaked in the DP350 group, while the concentrations of 1-octen-3-ol, 2,3-pentanedione, hexanal, octanal, (E)-2-hexenal, (E)-2-heptenal, and total concentration (TC) and n-6 derived tended to increase as DP increasing up to 350 g kg−1, and then decrease. (E)-2-hexenal and -2,4-heptadienal concentrations increased with DL increasing. The concentrations of 1-octen-3-ol, hexanal, (E)-2-octenal, -2,6-nonadienal, 2-ethyl-furan, TC, n-3 derived, and n-6 derived notably increased as DL increasing (P < 0.05) and peaked in the DL250 group. DP × DL had a significant influence on 3,5-octadien-2-one concentration (P < 0.05), it enhanced as DL increasing when DP ≥ 350 g kg−1.Data on volatile compounds with OAVs in the fillet are shown in P > 0.05) by DP × DL, DP, and DL. Fillet protein content was only affected by DP. The DP300 group had considerably lower fillet protein content than the DP350 and DP400 groups (P < 0.05).Data on fillet protein and lipid content are shown in P > 0.05) by DP × DL and DP, but were notably affected by DL (P < 0.05). The DL300 group had higher EPA, DHA, and ∑n-3 contents than the other groups, while the TI value showed the opposite trend (P < 0.05).Data on fatty acid compositions are shown in −1, and there was no negative effect of the high DL diet (300 g kg−1) on fish growth. This is substantiated by Meng et al. [−1) of rainbow trout  [−1, respectively.The current study discovered that the growth of triploid rainbow trout was enhanced when the fish were fed the diet with DL ≥ 250 g kg DL diet 00 g kg−11,500 g) . Triploi1,500 g) . Overall−1, no effect of DL on FI was also found in subadult triploid rainbow trout [−1 improved FI in subadult triploid rainbow trout. The phenomenon was not observed in adult fish, which indicated that large fish fed actively and were not picky eaters. The minimum recommended DP and DL for adult triploid rainbow trout (≥3 kg) based on FCR results was 350 and 200 g kg−1, respectively. Fish feed utilization was related to the processes of nutrient digestion. Digestion is performed by a variety of substrate-specific digestive enzymes, and there is a limit to the digestion and absorption of protein and lipid in diet by fish [−1 on the basis of FCR in the study. In addition, the DL300 group enhanced lipase activity but inhibited protease activity in the present study. Similar findings were reported in subadult fish [Fish growth is also affected by feed utilization; hence, FI and FCR were thoroughly evaluated. DP and DL had no effect on FI in the present study. When DL ≥ 195 g kgow trout . Howeverow trout  discover by fish . The curw trout ≥ kg based−1 was attributed to lipid deposition. Similar findings have been found in other fish species [Based on whole body composition in the study, the growth improvement by the diets with DL ≥ 250 g kg species . It was  species  showed t species . Combine species .−1), which led to more glucose being stored as glycogen. The main reason will require further study. An intriguing finding in the study was that high a DP diet (400 g kg−1) might mitigate the increase in HSI value produced by a high DL diet. It was related to decreasing hepatic glycogen content. As seen in earlier studies [−1. It was agreed that the optimal DP may enhance antioxidative capacity and benefit fish health [−1) or high DL group (300 g kg−1), which were different from the results in subadult fish [The liver is a crucial organ in fish, performing metabolic and detoxifying tasks , 33. Liv studies , 37, HSI studies , a high  studies . In addih health . The exph health . It indiult fish . Adult f DP diet 0 g kg−1 ult fish . Similara∗). Generally, a firm texture is preferred by consumers of salmon [Fillet is the most important product produced by salmonids, which is the majority of the edible component for consumers . Thus, tf salmon . Previouf salmon . The DL2E)-2-hexenal, is generally pleasant, while the volatile compounds derived from n-6 fatty acids, such as 1-octen-3-ol and hexanal, may cause off-flavors [−1 in the study. The results indicated that a diet with DL ≥ 250 g kg−1 could increase odor intensity overall, regardless of good or bad. The study discovered an intriguing result that the concentrations of n-6 derived and n-9 derived were enhanced with DP increasing up to 350 g kg−1, while n-6 derived dropped as DP increased from 350 to 400 g kg−1. It indicated that a high DP diet could inhibit the production of off-flavors.In addition to color and texture, the odor of fish flesh is a significant quality measure . The OAV-flavors . The con−1, respectively.Fish is not only a wonderful cuisine, but it also contributes essential nutrients for human health. In the study, fillet protein content was increased in the DP350 and DP400 groups. A similar pattern has been discovered in another investigation , which i−1, respectively; DP and DL based on feed utilization were 350 and 200 g kg−1, respectively; DP and DL based on fillet quality were 400 and 300 g kg−1, respectively. In addition, a high DP diet (400 g kg−1) was beneficial to fish health, and a high DL diet (300 g kg−1) showed no influence on hepatic health.In the study, for adult triploid rainbow trout (≥3 kg), the minimum recommended DP and DL based on growth performance were 400 and 250 g kg


Spodoptera frugiperda is one of the most harmful pests that attack maize and other major food crops and causes huge economic loss every year in China and other countries and regions. Beauveria bassiana, a kind of entomological fungus that is highly pathogenic to pests, is harmless to the environment and human beings. However, at present, S. frugiperda has gradually developed resistance to many pesticides and microbial insecticides. In this study, transcriptome sequencing was conducted to analyze the differences in gene expression between B. bassiana-infected and -uninfected S. frugiperda. More than 160 Gb of clean data were obtained as 150-bp paired-end reads using the Illumina HiSeq™ 4000 platform, and 2,767 and 2,892 DEGs were identified in LH36vsCK36 and LH144vsCK144, respectively. In order to explore the roles of JAK/STAT, Toll, and Imd signaling pathways in antifungal immune response in S. frugiperda against B. bassiana infection, the expression patterns of those signaling pathway-related genes in B. bassiana-infected S. frugiperda were analyzed by quantitative real-time PCR. In addition, antifungal activity experiments revealed that the suppression of JAK/STAT, Toll, and Imd signaling pathways by inhibitors could inhibit the antifungal activity to a large extent and lead to increased sensitivity of S. frugiperda to B. bassiana infection, indicating that JAK/STAT, Toll, and Imd signaling pathways and their associated genes might be involved in the synthesis and secretion of antifungal substances. This study implied that JAK/STAT, Toll, and Imd signaling pathways played crucial roles in the antifungal immune response of the S. frugiperda larvae, in which the related genes of these signaling pathways could play special regulatory roles in signal transduction. This study would improve our understanding of the molecular mechanisms underlying innate immunity and provide the basis for a wide spectrum of strategies against antifungal resistance of S. frugiperda. Arthropoda (Insecta) mainly relies on humoral and cellular immunity. The humoral immunity of insects is mainly composed of antimicrobial peptides and lysozymes against pathogenic fungi and antiviral factors, lectins, and other immune factors against viruses and other pathogens  signaling pathway, Toll signaling pathway, Imd (immunodeficiency) signaling pathway, and JNK (c-Jun NH (2)-terminal protein kinase) signaling pathway} in insects that mediate the immune response to different pathogens. The Toll signaling pathway and Imd signaling pathway are classical immune signaling pathways that mediate the transcription and synthesis of most antimicrobial peptides (AMPs) and other active substances .The JAK/STAT signaling pathway is guided by a variety of cytokines and involved in the regulation of many important physiological processes, including growth regulation , cell prDrosophila  have been registered and utilized for the control of S. frugiperda both at home and abroad  medium at 27°C ± 1°C for 1 week. Then, 10 ml 0.15 mol/L NaCl solution was added into the tubes which were then oscillated gently. The conidia and hyphae were transferred into a centrifuge tube with 20 μL Tween-80, vortex-shaken for 10 min, and then filtered. The spores were counted using a blood count plate and diluted with 0.15 mol/L NaCl solution to a concentration of 108 spores/ml. Then, 1 mL of the prepared conidia suspension was added into 10 mL potato dextrose (PD) medium and then cultured at 30°C ± 1°C, 180 rpm for 3 days. The mixture of hyphae and blastospores were autoclaved at 121°C for 20 min to generate the heat-inactivated suspension of B. bassiana. Finally, the B. bassiana conidial suspension and inactivated B. bassiana conidial suspension were used for immune induction in S. frugiperda.S. frugiperda were treated with 1 × 108/mL spore suspension of B. bassiana Bb378, while those treated with 0.05% Tween-80 solution were set as controls. The infected larvae were collected at 36 and 144 h after treatment, then flash-frozen with liquid nitrogen, and stored at −80°C. Each treatment was performed using three biological replicates. For every sample, the total RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. Contaminating genomic DNA was removed using RNase-free DNase I , and then, the quantity and quality of RNA were assessed. RNA with high concentration, integrity, and purity was chosen for cDNA library construction and final Illumina sequencing at Novogene Bioinformatics Technology Co., Ltd. . The obtained cDNA was then tested and sequenced on the Illumina HiSeq™ 4000 platform as 150-bp paired-end reads.The larvae of de novo contigs using Trinity software  sequences were removed from the raw data to obtain clean reads. Then, the quality of the clean reads was assessed by Q30 , the GC content, and sequence duplication level. The clean data were assembled into software . Subsequsoftware .−5. The annotation of unigenes was obtained using HMMER software , and then, Gene Ontology (GO) annotations were performed on all unigenes using Blast2GO. Subsequently, WEGO was used to determine GO functional classification and evaluate the distribution of gene functions at the macro-level  protein, UniProt, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) with an E-value <10ro-level . Metabolro-level .S. frugiperda at 12, 24, 36, 48, 60, 72, and 144 h after treatment with three biological replicates. A total of 10 annotated unigenes were selected randomly and quantified by real-time quantitative PCR (qRT-PCR) with specific primers . To obtain the first-strand cDNAs, 1 μg of total RNA from the transcriptome samples was reverse-transcribed in a 20-μL reaction system, according to the manufacturer’s instruction . qRT-PCR was performed using LightCycler®480 SYBR Green I Master  and run on the LightCycler®480 Real-time PCR system (Roche Diagnostics Ltd.) according to the manufacturer’s instructions. Each reaction was conducted in a 10-μL reaction system with 1 μL cDNA (2 ng/μL), 5 μL SYBR Green I Master , 0.5 μL/primer, and 3 μL ddH2O. The amplification conditions for qRT-PCR were as follows: denaturation at 95°C for 5 min, followed by 40 cycles for 5 s at 95°C, 20 s at 60°C, and 20 s at 72°C. gapdh was used as the internal reference gene, and each gene was tested in triplicate. The relative expression levels of the candidate chemosensory genes normalized to the internal control gene were calculated using the 2−ΔΔCT method  signaling pathway, and JAK/STAT  signaling pathway was verified by real-time quantitative PCR (qRT-PCR) with specific primers . Tissue  primers  to verifT method . AnalysiB. bassiana conidial suspension, and 0.15 mol/L NaCl solution (as the control), with a volume of 1 μL. A total of 60 larvae were injected in each treatment and performed in triplicate. After 24 h, the injected S. frugiperda larvae were submerged in B. bassiana conidial suspension  for 5 s and then reared with fresh corn at 70% humidity and 26°C ± 2°C. The number of dead larvae was recorded every 12 h. A lethal time of 50% (LT50) was used to show the resistance of S. frugiperda larvae against B. bassiana. LT50 and confidence intervals of 95% of each treatment were analyzed and calculated using SPSS21.0.The third-instar larvae were injected with the Toll inhibitor (BAY 11-7082), Imd inhibitor (parthenolide), JAK/STAT inhibitor mix , heat-inactivated S. frugiperda were submitted to the NCBI Short Archive (SRA) with BioProject PRJNA987447. Their accession numbers are SAMN35982286, SAMN35982287, SAMN35982288, and SAMN35982289.The Illumina reads of After removing the low-quality reads, trimming off the adapter sequences, sequencing, and a subsequent quality control process, a total of 1,155,833,496 raw reads and 1,111,415,334 clean reads were obtained from those 12 libraries. Furthermore, more than 160 Gb of clean data were obtained. The Q20, Q30, and GC content of each library were over 97.30%, 92.52%, and 43.95%, respectively . UnigeneAccording to the statistical analysis, 2,767 and 2,892 DEGs were identified in LH36vsCK36 and LH144vsCK144, respectively. In LH36vsCK36, 1,541 DEGs were upregulated and 1,226 DEGs were downregulated, while in LH144vsCK144, 1,261 DEGs were upregulated and 1,631 DEGs were downregulated .p-value <1 was used to screen the significantly enriched GO terms, and the up- and downregulated DEGs in significantly enriched GO terms in LH36vsCK36 and LH144vsCK144 were statistically analyzed.GO enrichment analyses for the DEGs were processed in LH36vsCK36 and LH144vsCK144. A total of 623 and 670 DEGs of LH36vsCK36 and LH144vsCK144 were enriched in GO annotation under three main terms, respectively. In LH36vsCK36, 338, 63, and 222 DEGs were enriched in “biological process,” “cellular component,” and “molecular function,” while in LH144vsCK144, 365, 63, and 241 DEGs were enriched, respectively . A correFrom the KEGG enrichment results, the most significant 20 KEGG pathways were selected to draw a scatter plot. In LH36vsCK36, the DEGs were mainly enriched in the “biosynthesis of cofactors” and “carbon metabolism,” followed by “fatty acid metabolism,” “metabolism of xenobiotics by cytochrome P450,” and “drug metabolism—cytochrome P450,” while in LH144vsCK144, the DEGs were mainly enriched in the “biosynthesis of cofactors,” “lysosome,” and “drug metabolism—other enzymes,” followed by “drug metabolism—cytochrome P450,” “metabolism of xenobiotics by cytochrome P450” and “retinol metabolism” .2 values of fold changes in gene expression, normalized to gapdh relative to each sample. In LH36vsCK36, the qRT-PCR results supported the data obtained by DEG analysis . The qPCR result revealed that after being infected with nfection . The exp to 60 h .Sfrutube, Sfrupell, Sfrumyd88, Sfrucactus, and Sfrudif, have become the core signal transfer factors in the Toll signaling pathway. The relative expression of Sfrutube was significantly upregulated within 48–60 h after infection; the relative expression of Sfrucactus was upregulated and peaked within 60–72 h after infection; the relative expression of Sfrupell and Sfrumyd88 peaked at 36 and 48 h, respectively  and lead to the subsequent immune response. When nd 144 h ; Sfrusocand 48 h .S. frugiperda against B. bassiana infection, the effect of signaling pathway inhibitors on the survival rate of S. frugiperda infected by B. bassiana was tested. The results showed that dead larvae could be found earliest at 48 h and overgrown by the fungus approximately 144 h after injection. In 50 analysis confirmed this observation. As shown in the tables, LT50 of the group injected with the Toll inhibitor was 20 h less than that of the control group , five cytoplasmic signal transduction factors , and Sfruspatzle in the Toll signaling pathway in S. frugiperda against B. bassiana infection. The Toll pathway is activated by the activation of Spätzle (Spz), the Toll receptor ligand  and Sfruspatzle, Sfrumyd88, Sfrutube, and Sfrupell all showed a significant upregulation at 36 or 48 h after infection, indicating that the Toll signaling pathway of S. frugiperda was activated after B. bassiana infection and the relative high expression of their corresponding proteins. We speculated that this might be related to the formation of a receptor-proximal oligomeric complex assembled by proteins Spz, Toll receptors and MyD88, Tube, and Pelle, and this complex will further trigger the phosphorylation and regulation of expression of different antimicrobial peptides (AMPs)  . The relbyx mori , while tB. bassiana in S. frugiperda, the antifungal activity in vivo was conducted by inhibition assay. The result showed that BAY 11-7082 increased the sensitivity of S. frugiperda to B. bassiana infection and decreased the antifungal activity of the hemolymph of S. frugiperda, suggesting that the Toll signaling pathway plays a crucial role in the synthesis of antifungal substances against B. bassiana infection.In order to further verify the role of the Toll signaling pathway in defense against Drosophila, the Imd pathway is activated by meso-diaminopimelic acid (DAP)-type PG of Gram-negative bacteria and some bacilli species   had a lower survival rate and a 20 h reduction in LT50. So the JAK/STAT signaling pathway might regulate the synthesis of antifungal substances in S. frugiperda, which further indicates that the JAK/STAT signaling pathway plays an important role in the resistance of S. frugiperda to B. bassiana infection. However, there are also complex regulatory mechanisms in the JAK/STAT signaling pathway, which need to be further studied.The JAK/STAT signaling pathway, which is involved in the regulation of a variety of important physiological processes, is the key signal pathway of immune regulation . STAT isresponse . At the as STAT) , and Kenas STAT) . In this Sfruken . The resSfrulysozyme showed a high level from 24 to 72 h after infection by B. bassiana (G. mellonella larvae (S. frugiperda. The relative expression of Sfrucecropin, Sfruattacin, Sfrugloverin, and Sfrulebocin all showed upregulated expression patterns to a varying degree after S. frugiperda was infected with B. bassiana, indicating that these antimicrobial peptides were involved in the immune response of S. frugiperda to B. bassiana infection.AMPs play an irreplaceable role in the process of innate immunity in insects, and a wide range of antimicrobial peptides display both antibacterial and antifungal functions . In thisbassiana ; a simila larvae , suggestS. frugiperda. Further studies are needed to investigate the resistance mechanism of S. frugiperda to pathogenic microorganisms. This study not only lays a solid foundation for the research and development of new fungal agents to control S. frugiperda and other pests but also provides an important scientific basis for the development and use of new biocontrol agents.In summary, the different expression patterns of Toll, Imd, and JAK/STAT signaling pathway-related genes revealed their different and specific functions in immune signal transduction in


Extreme environments at high altitudes pose a significant physiological challenge to animals. We evaluated the gut microbiome and fecal metabolism in Sanhe heifers from different altitudes.Twenty Sanhe heifers  selected from two regions of China: the Xiertala Cattle Breeding Farm in Hulunbeier, Inner Mongolia  and Zhizhao Dairy Cow Farm in Lhasa, Tibet , were used in this study. Fecal samples were collected and differences in the gut microbiota and metabolomics of Sanhe heifers were determined using 16S rRNA gene sequencing and metabolome analysis.p > 0.05). However, 16S rRNA gene sequencing showed that altitude significantly affected gut microbial composition. Principal coordinate analysis based on Bray–Curtis dissimilarity analysis revealed a significant difference between the two groups (p = 0.001). At the family level, the relative abundances of Peptostreptococcaceae, Christensenellaceae, Erysipelotrichaceae, and Family_XIII were significantly lower (p < 0.05) in LA heifers than in HA heifers. In addition, the relative abundances of Lachnospiraceae, Domibacillus, Bacteroidales_S24-7_group, Bacteroidales_RF16_group, Porphyromonadaceae, and Spirochaetaceae were significantly higher in HA heifers than in LA heifers (p < 0.05). Metabolomic analysis revealed the enrichment of 10 metabolic pathways, including organismal systems, metabolism, environmental information processing, genetic information processing, and disease induction. The genera Romboutsia, Paeniclostridium, and g_unclassified_f_Lachnospiraceae were strongly associated with the 28 differential metabolites. This study is the first to analyze the differences in the gut microbiome and metabolome of Sanhe heifers reared at different altitudes and provides insights into the adaptation mechanism of Sanhe heifers to high-altitude areas.The results showed that altitude did not significantly affect the concentrations of fecal volatile fatty acids, including acetate, propionate, butyrate, and total volatile fatty acids ( Using high-throughput sequencing and LS-MS-based untargeted metabolome analyzes, we aimed to reveal the effect of altitude on the gut microbiome of Sanhe cattle and to improve the understanding of the role of the gut microbiome in high-altitude adaptability. In addition, the gut microbiome and metabolomics of Sanhe heifers from low-and high-altitude regions were compared to provide insights into the high-altitude adaptability of ruminants.2.The study protocol was approved by the Ethical Committee of the College of Animal Science and Technology of China Agricultural University (project number AW22121202-1-2).2.1.Twenty Sanhe heifers  fed in two different altitude regions of China were selected for the experiments. Ten Sanhe heifers were selected from each trial site.2.2.Cattle from two regions were analyzed: those from the origin of Sanhe cattle, including Hulunbuir, Inner Mongolia Autonomous Region , and Lhasa, Tibet Autonomous Region . Lhasa has an average annual temperature of 8.6 Â °C and annual precipitation of 472.5 mm , whereas2.3.g at 4°C for 10 min, and the supernatant was collected and evaluated using gas chromatography , and those used to analyze volatile fatty acids (VFAs) were stored at −20°C. For VFA analysis, the fecal sample from each animal was thawed, diluted, and centrifuged at 8,000 × tography .2.4.Total microbial genomic DNA was extracted from 1 g of fecal samples using an OMEGA kit , following the manufacturer’s instructions. A Nanodrop 2000 spectrophotometer  was used to confirm the purity and concentration of the extracted DNA. The V3–V4 region of the gut bacterial 16S rRNA gene was amplified using the forward primer 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer 806R (3′-GGACTACNNGGGTATCTAAT-5′). The PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 28 cycles at 95°C for 45 s, 55°C for 50 s, and 72°C for 45 s, with a final extension at 72°C for 10 min. Amplified fragments were visualized using 2% agarose gel electrophoresis, and the respective bands were purified using an Agencourt AMPure XP kit  according to the manufacturer’s instructions and quantified using QuantiFluor-ST . Purified PCR products were sequenced on an Illumina MiSeq , reads <200 bp, and reads containing ambiguous bases or unmatched primer sequences were filtered out using QIIME 1.8 , and barThe resulting sequences were clustered into OTUs based on a 97% sequence similarity threshold using the Ribosomal Database Project classifier  with a cThe OTU level alpha diversity of the bacterial communities was determined using the Chao1, Shannon, and Simpson indices and procedures within QIIME 1.8, and visualized using the “ggplot2” package of R  was added to an 80 mg fecal sample and vortexed for 60 s to extract metabolites. The samples were incubated on ice for 20 min and centrifuged at 14,000 × g for 20 min at 4°C. The supernatant was collected for liquid chromatography (LC)-MS analysis. The samples were dissolved in 100 μl of acetonitrile/water  and transferred to LC vials.The cold extraction solvent methanol/acetonitrile/H2.7.Gut microbiota metabolites were separated using an ultra-high-performance liquid chromatography system  coupled to a quadrupole time-of-flight . The fecal samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 μm column . In both the positive and negative electrospray ionization modes, the mobile phase contained 25 mM ammonium acetate and 25 mM ammonium hydroxide in water and acetonitrile, respectively. The gradient was 85% acetonitrile for 1 min, which was linearly reduced to 65% in 11 min, reduced to 40% in 0.1 min and maintained for 4 min, and increased to 85% in 0.1 min, with a 5 min re-equilibration period. The electrospray ionization source conditions were as follows: ion source Gas1 as 60, ion source Gas2 as 60, curtain gas as 30, source temperature, 600°C; and ion spray voltage floating ±5,500 V. During MS acquisition, the instrument was set to acquire signals over an m/z range of 60–1,000 Da, and the accumulation time for the time-of-flight MS scan was set to 0.20 s/spectra. In the auto-MS/MS acquisition mode, the instrument was set to acquire signals over an m/z range of 25–1,000 Da, and the accumulation time for the production scan was set to 0.05 s/spectra. The production scan was acquired using information-dependent acquisition in high-sensitivity mode. The collision energy was fixed at 35 ± 15 eV. The declustering potential was set at ±60 V.Raw MS data (Wiff. scan files) were converted to MzXML files using ProteoWizard MSConvert, and processed using XCMS for feature detection, retention time correction, and alignment. The metabolites were identified using accuracy mass spectrometry (<25 ppm) and MS/MS data, which were matched with the standard database.t-test  on the raw data. The metabolites were considered significant when they had VIP values >1.0, VIP values <0.05, and p-values less than 0.05. Differential metabolites were identified using three databases, including the Kyoto Encyclopedia of Genes and Genomes (KEGG)For the extracted ion features, only variables with >50% of the nonzero measurement values in at least one group were retained. The MetaboAnalyst2.8.t-test in the SPSS software . Alpha diversity indices, which reflect the significance between the LA and HA groups, were analyzed using the Wilcoxon rank test with the “dplyr” package  in R. PCoA was performed based on the Bray–Curtis dissimilarity matrices in R, and “ggplot2” package in R was used to visualize the results. The differences in the relative abundance of organisms at the phylum, family, and genus levels and microbiota function between the two groups were tested using the Wilcoxon method in R (version 4.0.5). Spearman’s rank correlation was used to identify the relationship between the relative abundance of the core OTUs, altitude, fecal fermentation parameters, and serum antioxidant indices using the “Psych” package  and visualized using the “corrplot” package  in R. All data were reported as the mean, and differences with p < 0.05 were considered as significant.Fecal fermentation parameters were analyzed using the 3.3.1.p > 0.05) in the concentrations of acetate, propionate, butyrate, and total VFAs between fecal samples of Sanhe cattle reared at different altitudes. Compared with the LA group, the acetate-to-propionate ratio (A/P) increased significantly (p < 0.05) in the HA group.As shown in 3.2.3.2.1.A total of 868,445 raw sequences were generated with an average of 43,422 ± 4,392.71 (mean ± SD) per sample. An average of 2,054 ± 133.75 OTUs across all samples were identified at 3% sequence dissimilarity. Rarefaction curves showed that the number of new OTUs decreased as the number of sequences per sample increased , Bacteroidetes (32.33%), Tenericutes 0.93%; ,B. Among3.2.2.>10% total relative abundance: Ruminococcaceae (26.10%), Rikenellaceae , Rikenellaceae_RC9_gut_group . However, we found no significant difference (p < 0.05) in Chao 1 richness, Shannon diversity index, and Simpson’s diversity index between the groups  plot, as shown in p > 0.05) between the LA and HA groups. In contrast, compared with the LA group, the relative abundances of the phyla Proteobacteria and Actinobacteria were significantly (p < 0.05) lower (p < 0.05) higher in the HA group. At the family level (family of relative abundance >0.01%), lower relative abundances of Peptostreptococcaceae, Christensenellaceae, Erysipelotrichaceae, Family_XIII, Acidaminococcaceae, Peptococcaceae, Enterobacteriaceae, Spirochaetaceae and Coriobacteriaceae were observed in the HA group than in the LA group (p < 0.05) higher, and those of 15 genera were significantly lower in the HA group , Eubacterium_xylanophilum_group , Corynebacterium , Escherichia-Shigella , and Domibacillus  lower , whereasLA group , and theLA group . At the HA group ; of thes3.2.4.p < 0.05) correlated with altitude; of these, 20 OTUs were negatively correlated with altitude, eight of which were in the family Ruminococcaceae (p < 0.05), four in the family Rikenellaceae (p < 0.05), and two in the family Lachnospiraceae (p < 0.05). Additionally, OTUs within unidentified_o_Clostridiales, Clostridiales_vadinBB60_group, Clostridiaceae_1, Bacteroidales_BS11_gut_group, Bacteroidales_RF16_group, and Spirochaetaceae were significantly negatively (p < 0.05) correlated with altitude. Thirty-eight OTUs were positively correlated with altitude, among which 14 were in the family Ruminococcaceae (p < 0.05), 11 in the family Christensenellaceae (p < 0.05), three in the family Family_XIII (p < 0.05), three in the family Lachnospiraceae (p < 0.05), and three in the family Peptostreptococcaceae (p < 0.05). In addition, OTUs within the families Erysipelotrichaceae, Acidaminococcaceae, Bacteroidaceae, and unidentified_o_Gastranaerophilales were significantly and positively correlated with altitude (p < 0.05).To explore the role of gut bacteria in production and fermentation of VFAs, we analyzed the relationship between fecal VFA concentration  and the relative abundance of OTUs using Spearman’s rank correlations, as shown in p < 0.05) correlated with the acetate-to-propionate ratio (AP), among which six and three were in the families Ruminococcaceae and Rikenellaceae, respectively. In addition, 23 OTUs were positively correlated (p < 0.05) with AP, of which eight were in the family Ruminococcaceae, six in the family Christensenellaceae, three in the family Peptostreptococcaceae, and two in the family Lachnospiraceae. Furthermore, OTUs within the families Family_XIII, Erysipelotrichaceae, unidentified_o_Gastranaerophilales, and Acidaminococcaceae were significantly and positively correlated with AP (p < 0.05).Fourteen OTUs were negatively (Eubacterium coprostanoligenes group. Sixteen OTUs were significantly (p < 0.05) correlated with propionate concentration, among which 10 OTUs were negatively correlated with propionate concentration, three OTUs were in the family Christensenellaceae, three OTUs were in the family Peptostreptococcaceae, two OTUs were in the family Lachnospiraceae, and one OTU was in the family Erysipelotrichaceae. Six OTUs were significantly and positively (p < 0.05) correlated with propionate concentration; of these, four OTUs belonged to the family Ruminococcaceae, one OTU belonged to the family Bacteroidales_RF16_group, and one OTU belonged to the family Lachnospiraceae. The total VFA concentration was negatively (p < 0.05) correlated with the relative abundance within the family Ruminococcaceae.Analysis of VFAs showed that acetate concentration was negatively correlated with the relative abundance of OTUs in the 3.3.3.3.1.p < 0.05) for differential metabolites, 368 metabolites differed significantly between the LA and HA groups, of which 231 and 137 were detected in the positive and negative ion modes, respectively.A total of 1,727 differential metabolites were identified in the gut metabolome; of these, 1,101 and 626 metabolites were detected in positive and negative ion modes, respectively. To compare the metabolome compositions of the gut samples in the two groups, the datasets obtained from LC–MS in the positive and negative ion modes were evaluated using PCA ,B. The m3.3.2.Metabolic pathway analysis based on the significantly different gut metabolites revealed the enrichment of 10 metabolic pathways , with “n3.4.r > |0.8|, p < 0.05; Romboutsia were significantly negatively and positively correlated with seven and two metabolites. Respectively. OTUs belonging to the genus Paeniclostridium were significantly negatively and positively correlated with 13 and five metabolites, respectively. OTUs belonging to the genus unclassified_f_Lachnospiraceae were significantly positively correlated with a single metabolite.Spearman’s correlation network between the core gut microbiota and gut metabolites was analyzed, that revealed 28 significant correlations . A previous study showed that Romboutsia encodes a versatile array of metabolic capabilities involved in carbohydrate utilization, fermentation of single amino acids, anaerobic respiration, and metabolic end-products -4-methyl thiazole, oxeladin, hypoxanthine, 2-aminoadipic acid, melatonin, 1,2-dimyristoyl-sn-glycero-3-phosphate, N-omega-propyl-l-arginine, artemisinin, heliotrine N-oxide, ondansetron, Cer 20:1-d7 (d18:1-d7/20:1), deoxyinosine, and d-fructose). Paeniclostridium is an anaerobic pathogen in animals  can be found in the article/The animal study was reviewed and approved by the study protocol was approved by the Ethical Committee of the College of Animal Science and Technology of China Agricultural University (project number AW22121202-1-2). Written informed consent was obtained from the owners for the participation of their animals in this study.XZ performed experiments and wrote the manuscript. WW, ZC, HY, YW, and SL reviewed and provided guidance for the manuscript and experiment. All authors contributed to the article and approved the submitted version.The services used in this study were purchased by the Ministry of Agriculture and Rural Affairs of China: Experiment and Demonstration of Adaptive Production Technology for Dairy Cows in High Altitude Regions (no.16190319) and China Agriculture Research System of MOF and MARA (CARS36).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates.Shiga toxin-producing E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates  were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance  against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates , both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively.There was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia—Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples  were identified as suspicious E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains.The results of this study provides evidence for the occurrence of  Escherichia coli (STEC) is considered as an important foodborne zoonotic pathogen worldwide that causes gastrointestinal complications in humans ), and stx2 (encodes variant 2 [Stx2]).PCR analysis was performed to confirm the presence of the main virulence factor in STEC O157:H7; Shiga toxins (stx1 (encodes variant 1 [Stx1]), and stx2 (encodes variant 2 [Stx2])). Figure stx2 gene while isolate CF13 had stx1 and stx2 genes.PCR analysis was performed to confirm the presence of main virulence factor in both STEC O157:H7 isolates; Shiga toxins genes  was similar 98% to strain CP024273,CP024269 and CP02422 that were enterotoxigenic Escherichia coli Strain E.coli O169:H41 [Escherichia coli Stx2 producing strain serotype O100:H19 [Escherichia coli. On the other hand, the sequence of di toxin E.coli O157:H7 isolate No (CF13) was similar 99% to Escherichia coli that cause diarrhea and gastrointestinal disease [In Table O169:H41 , 26, and disease .Table 4IE. coli O157:H7 causing environmental hazards and human diseases, and transmission may occur through a variety of routes[E. coli O157 by using EMB, and CT-SMAC media. However, E.coli ferment lactose and sucrose appear as metallic green sheen colony on EMB [E.coli O157 [E.coliO157 isolation [E.coli O157.Cattle are considered to be the principal reservoirs of pathogenic of routes. For exaof routes–32. Addiof routes, 34. Oury on EMB . While, oli O157 , and supsolation . SMAC meE. coli O157 isolates (ranged between 0.15 and 0.62). These values are higher than the accepted international standard for MAR index [E.coli O157 isolates, that showed resistance against two or more antibiotics having different mode of actions, and can be classified as multidrug-resistant (MDR) isolates. This percentage of resistance among our local suspected E.coli O157 isolates were significantly higher than the isolates of the E. coli O157:H7 from cattle (53%) and beef (57%) reported by [E.coli O157 isolated from food and animal sources [E.coli O157 collected from food products and cattle faeces. Katsuda et al., 2009 [Our results revealed high values of multiple antibiotic resistance (MAR) index for all suspected AR index . Whereasorted by  from anoorted by . Severalorted by , 42. Mea sources . The sam sources  and from sources . All ant sources  Neverthe sources . The sam sources . In Egyp sources . This mal., 2009  reportedE.coli O157 in Al-Sharkia, Egypt that may cause environment and public health risk, was the CF samples. Where, eight out of the nine (89%) CF samples that were suspect to have E.coli O157 isolates had the highest MAR index (ranged between 0.23- 0.62), and were considered as MDR. Our local results suggest that CF samples are the leading cause of pathogenic E. coli O157 contamination. Wang et al. [E. coli O157 and non-O157 in faeces of dairy and beef cattle [It is noteworthy in this study that the most frequent source for MDR g et al.  pointed f cattle .E. coli O157, were both obtained from CF samples , and confirmed as E.coli O157:H7 by serological test, as they gave strong agglutination with O157 antigen and H7 antigen. The agglutination test appeared specific for the diagnosis of E. coli O157:H7, these results are in agreement with, [E. coli O157:H7. It is a rapid, reliable test that is easy to perform.Two isolates out of our local suspected nt with, , 52, whoE.coli O157:H7 in Al-Sharkia, Egypt (two out of 30 samples) is equal to 6.7%. This ratio is similar to results that were obtained from sheep faeces in another Egypt governorate (Giza) [E. coli O157 in sheep faeces at Pastures in Scotland (6.5%) [E.coli O157 in Al-sharkia, Egypt is clearly higher than that recorded in South Yorkshire, UK (1–2%) and Spain (2.6%) by [Our results revealed that, the prevalence of e (Giza) , which id (6.5%) , and 7.1d (6.5%) . Nonethe2.6%) by , 56, res2.6%) by , respectE.coli O157:H7 is associated with different virulence factors, production of Shiga toxins (stx1 and stx2) being the most potent one [E.coli O157:H7 isolates, CF8 was found to carry stx2 gene, and CF13 carry stx1 and stx2 genes; this is according to molecular analysis by multiplex PCR.Pathogenicity of tent one , 61 . Lotent one  reportedE. coli O157 can survive in the environment for more than 10 months, as reported by [Particularly, as orted by , humans orted by ; whom haorted by . OccurreE. coli (STEC) has been deeply involved in foodborne, waterborne and airborne outbreaks in several studies all over the world [E. coli O157:H7, caused the infection for 20 campers out of 337 at scout camp on May 2000, it was due to an environmental source. Investigations do not suggest that it was a food vehicle, as they did not find any E.coli isolates in food or drinking water. However, investigations supported that the transmission of E. coli O157:H7 was due to exposure to the contaminated environment by hands, either straightly from hand to mouth or via food. They found that the food in studied cases worked as an inert vehicle for E. coli O157:H7 transmission, not as a growth medium (source of infection) for the bacterial pathogen. The contamination of the environment may be due to the fact that the camp took place on an agricultural showground used for sheep grazing. Additionally, the location of the camp had been fully contaminated with sheep faeces.Shiga toxin-producing he world , and havE.coli strains carrying stx1 and stx2 genes mainly showed MDR towards numerous antibiotics that are medically important, as they commonly used in human and animal treatment. There is a high possibility that these resistant strains can endure the environment for a long time and, then, eventually transmit these resistance determinants to other environmental bacteria [The isolated toxigenic O157 bacteria .Such transfer of resistance determinants could fuel the spread of MDR bacteria, this could have grave implications on the health of humans and animals; thus, increasing the burden of disease in the community. Therefore, there is a great need for urgent policy formulations on the prudent use of antimicrobials in both human and veterinary medicine, because failure in this regard could spell doom in the near future .There are high recommendations from different authorities worldwide for testing all ground meat/poultry samples for STEC O-serotypes, as these organisms are shed in faeces, and will lead to environmental contamination for an extended period of time as the USDA Food Safety and Inspection Service states.E.coli O157 in Zagazig City et al.-Sharkia, Egypt by 15.4% in grounding beef, as hamburger and cattle faeces samples are of epidemiological significance. Furthermore, there is a high prevalence of MDR (80%) determinants in suspected E.coli O157. Accordingly, this will lead to the emergence of superbugs of clinical relevance due to the ease of transfer of resistance genes to other pathogens. In addition, two isolates (6.7%) recovered from cattle faeces were carried the Shiga toxin genes stx1 and/or stx2, which suggests that the farm environment may serve as a channel for the dissemination of antimicrobial-resistant STEC strains to other niches, including the food chain. Risk assessment is needed to protect human, animal, and environmental health. Furthermore, animal faeces should be treated before discharge, continuous monitoring should be considered, and surveillance for environment, livestock, and food product should be strengthened.The importance of recent study is to monitor the prevalence of suspected A total of sixty-five samples were collected in the period between Jan. 2021 to March 2021. Out of this, 35 samples  were collected from diverse markets and butcher’s shops in Zagazig City, AL-Sharkia Governorate- Egypt. While, the remaining 30 samples, were cattle’s faecal samples collected from different house farms, and the Veterinary Medicine in Zagazig University at Zagazig City, AL-Sharkia Governorate- Egypt.Following the same procedure as , all samAt the laboratory, each sample underwent the following process. Twenty-five grams of the sample was transferred to a sterile flask using sterile spatula, and 225 ml of Tryptone Soya Broth (TSB) was added. Homogenization took place in a clean blender at 2000 r.p.m for 1–2 min. Then, the homogenized sample was transferred into a sterilized flask and incubated at 37 ± 2 °C for 18-24 h.E. coli) in each sample alone, the three-tubes most probable number (MPN) technique was performed as in the U.S. Food and Drug Administration Bacteriological Analytical Manual, which is well described by [To enumerate the low concentration of the total fecal coliform bacteria FCB  . After an incubation period 18-24 h at 37 ± 2 °C, colonies with metallic sheen appearance were picked up and purified as E. coli isolates for a completed test.Then, the confirmatory test was performed by picking up pure colonies of FCB isolates and streaked on eosin methylene blue (EMB) agar medium , in order to isolate and differentiate E. coli colonies obtained from previous assays were separately streaked onto selective differential sorbitol MacConkey agar media with cefixime- telurite supplement (CT-SMAC) , which has a selective supplement (cefixime- telurite) for specific E. coli O157 isolation. After the incubation period, E. coli serogroup O157 colonies had a pale (colourless) appearance due to their inability to ferment the sorbitol [Purified sorbitol . These cE. coli O157:H7 were tested for their susceptibilities to the selected antibiotics by standard Kirby-Bauer disc diffusion method [E. coli isolates. The 13 chosen antibiotics include the following; cefoxitin , cefepime , ceftazidime , ceftriaxon , amoxicillin/clavulanic acid , and imipenem  belonging to β-lactam antibiotics, piperacillin , gentamicin , amikacin ,trimethoprim-sulfamethoxazole , ciprofloxacin , erythromycin , and polymixin (Pb30 µg). The test was performed as illustrated by [All presumptive purified n method . Thirteen method , 73. Addrated by  and the Isolates with high MAR index were recorded as multidrug-resistant (MDR) isolates, especially if they showed resistance to more than two antibiotics from different groups .E. coli O157:H7, and they were selected to approve their carrying of Shiga toxin genes.According to the antibiogram pattern, the multidrug- resistant (MDR) isolates were serologically identified by slide agglutination according to  by usingE. coli O157:H7 isolates gave positive reaction for agglutination test with both anti-O157 and anti-H7 antibodies, and was extracted by using QIAamp DNA Mini kit . Next, the isolates were subjected to molecular analysis for the presence of Shiga toxins (stx1 and stx2) as a potential virulence gene in E. coli O157:H7, by using multiplex PCR. The primer sequences were designed by [DNA from igned by , as illuPCR was carried out in a 50 µL reaction containing 25 µL of EmeraldAmp GT PCR mastermix (2 × premix), 1 µL of each primer (20 pmol), 13 µL of water, and 8 µL of DNA template. The reactions were performed in an Applied Biosystems 2720 thermal cycler . The products of PCR were separated by electrophoresis on 1.5% (Wt/Vol) agarose gel  in 1 × TBE buffer  at room temperature using gradients of 5 V/cm. For gel analysis, 20 µL of the products were loaded in each gel slot. A Gelpilot 100 bp Ladder  was used to determine the fragment sizes. The gel was photographed by a gel documentation system  and the data was analyzed using associated software .E. coli O157:H7 using universal 1492r primer [PCR amplification was performed to confirm the identity of r primer , as in TThe extracted bacterial genome must be diluted before PCR performance by adding 495 μl of nuclease free water into 1.5 ml micro centrifuge tube and 5 μl of the supernatant from the PrepMan ultra extracted sample was added to get 1: 100 dilution; then, vortexed and mixed well. Thermo-scientific was used to amplify the first 500 base pairs of the 16S rRNA bacterial gene in the sample. PCR set up was performed by adding the following components: 12.5 μl of Dream Taq Green PCR Master Mix (2X) with Cat. No. K1081, 0.5 μl of Forward Primer (10 pmol), 0.5 μl of Reverse Primer (10 pmol), 10.5 μl Nuclease Free Water and 1.0 μl DNA Template; the tube was closed tightly and placed in the thermal cycler. PCR reaction conditions were initial denaturation at 95 °C for 4 min, 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 40 s. Final extension at 72 °C for 10 min was done. The PCR product was run and visualized by loading 10 μl of the PCR product per lane on 1.8% agarose gel that stained by ethidium bromide against 100 bp DNA ladder as a marker using 1X TAE as a running buffer, and photographed by gel documentation system to identify its size.PCR products were purified according to the manufacturing procedure of Gene JET PCR Purification Kit  as per the following steps: five microliters of the PCR reaction product was mixed with 2 μl of GeneJET reagent for a total of 7 μl of the reaction volume, and incubated at 37 °C for 15 min to degrade the remaining primers and nucleotides. Then, the sample was incubated at 80 °C for 15 min to inhibit the reagent reaction. The purified PCR product stored at -20 °C for sequencing.PCR products were sequenced using the standard Sanger method on ABI 3730XL DNA Sequencer at Macrogen sequencing services  [Pair wise and multiple DNA sequence alignment were carried out using CLUSTALW multiple sequence alignment programme version (7.0.9)  . Bootstrlustalw)  from CLUThe Gene-Bank nucleotide sequence accession numbers for partial sequences of 16S rRNA gene were generated in this study using DDBJ Center .All statistics with a probability value of ≤ 0.05 were considered significant. The bacterial counts were normalized by transforming the data using log10. The MPN estimated by selecting the dilutions of positive tubes, and all the dilutions between them by using  formula;ethod of .3\documeE. coli. The slope of the line was calculated to obtain a growth rate for the organism (the unit for growth rate is log10 CFU).The mean value (from the three replicates) was plotted against time by linear regression analysis using cultural detection of


S. aureus population and the enterotoxin gene content in wild boar. In 1025 nasal swabs from wild boars, 121 S. aureus isolates were identified. Staphylococcal enterotoxin (SE) genes were identified in 18 isolates (14.9%). The seb gene was found in 2 S. aureus isolates, sec in 2 isolates, the see and seh genes were found in 4 and 11 isolates, respectively. The production of SEs was evaluated in bacteria grown in microbial broth. Concentration of SEB reached 2.70 µg/ml after 24 h and 4.46 µg/ml at 48 h. SEC was produced at 952.6 ng/ml after 24 h and 7.2 µg/ml at 48 h. SEE reached 124.1 ng/ml after 24 h and 191.6 ng/ml at 48 h of culture. SEH production reached 4.36 µg/ml at 24 h and 5.42 µg/ml at 48 h of culture. Thirty-nine spa types were identified among S. aureus isolates. The most prevalent spa types were t091 and t1181, followed by t4735 and t742, t3380 and t127. Twelve new spa types, i.e., t20572‒t20583 were identified. The wild boar S. aureus population was shown to contain previously identified animal/human-associated spa types and spa types not identified in humans or animals. We also indicate that wildlife animals can be a significant reservoir of see-positive S. aureus.Little is known about the structure of The online version contains supplementary material available at 10.1007/s00248-023-02236-4. Staphylococcus aureus is considered one of the most common opportunistic pathogens of humans and animals. This bacterial species is responsible for a wide range of infections, from superficial skin and tissue infections to serious, life-threatening diseases such as pneumonia and sepsis [S. aureus an important foodborne pathogen [d sepsis . Furtherpathogen .S. aureus can colonise both animals and humans. Due to changes in the natural environment, previously isolated ecological niches are now frequently overlapping [S. aureus that affect animals. S. aureus belonging to clonal complex 5 (CC5) was shown to pass from human to poultry [S. aureus to a new niche changes many of its properties, including those involved in pathogenesis. For a number of clonal lineages host specificity is less pronounced and therefore adaptation to new hosts is more frequent  [rlapping . It was  poultry  and CC39 poultry , 7. The S. aureus in the wild boar population. Meemken et al. [S. aureus is a rare coloniser of wild boars, as this pathogen was isolated from 6.8% of the investigated animals. Among these isolates, spa types t1181, t6782, and t6384‒t6386 were identified. According to Porrero et al. [S. aureus. The isolates belonged to 36 spa types, the most common of which was t3750. In these studies, no CC398 S. aureus isolates were found, suggesting a low frequency of this clonal complex in wild boars. However, in 2017 the first isolation of CC398 from a wild boar was reported [Very little is known about the occurrence and genetic diversity of n et al.  concludereported .S. aureus from 177 nasal swabs from wild boars from Portugal. The most prevalent spa type was t11502 with 37 isolates, and the presence of t011 and t034 isolates that are typically associated with livestock was found in 9 and 8 isolates, respectively.Ramos et al.  obtainedS. aureus. In a study by Seinige et al. [S. aureus cultures were found in 111 wild boar nasal samples. The researchers screened the isolates for the presence of staphylococcal enterotoxin genes (sea‒see and seh). The only enterotoxin gene found was seh. It was present in 11 isolates, 5 of which belonged to the t127 spa type.Even less is known about the enterotoxigenic potential of wild animal–derived e et al. , 36.9% Sspa typing was used to investigate the genetic diversity of S. aureus isolates obtained from wild boars in Poland. The enterotoxigenic potential of wild boar S. aureus isolates was investigated. S. aureus was tested for the sea‒see and seh genes encoding SEs, which are considered the most important food safety hazards.In this study, Sus scrofa) at a game collection point in north-west Poland during the years 2014‒2017. The swabs were taken by placing a sterile cotton swab approximately 10 cm into the nares. Individual swabs were placed in 20 ml of Giolitti-Cantoni broth  and cultured statically for 24‒48 h at 37 °C. Subsequently, cultures were streaked on Baird-Parker agar  for further selection of staphylococcal isolates and incubated for 24‒48 h at 37 °C. Single colonies displaying the characteristic morphology of S. aureus were transferred to 5 ml of BHI broth  and cultured at 37 °C with agitation for 18‒24 h. Species identification was confirmed by PCR, using primers for the nuc and clf genes, encoding thermonuclease and clumping factor, respectively, as previously described [S. aureus isolate per sample was taken for further analysis. The isolates were stored in BHI medium with 15% glycerol.One thousand twenty-five nasal swabs were taken from wild boars , and incubated at 37 °C for 30 min. Then 15 µl of 10% SDS was added and incubated at 37 °C for 10 min, followed by the addition of 200 µl of 5 M guanidine hydrochloride solution and incubation for 10 min at room temperature. DNA was extracted by phenol and chloroform, precipitated by ethanol, dissolved in 50 μl of UltraPure™ Distilled Water  and stored at − 20 °C.Two-millilitre aliquots of an overnight bacterial culture in BHI broth  were centrifuged at 12,000 × S. aureus genomic DNA was screened for the sea–see and seh genes encoding the SEs being the most important food safety hazards. The detection of staphylococcal enterotoxin genes sea–see was performed according to Sharma et al. [seh gene was performed as described previously [a et al. . Detectieviously .S. aureus isolates, spa types were determined using the method described by Harmsen et al. [spa typing and cluster analysis of spa types with the minimum spanning tree algorithm, Ridom SeqSphere + software was used [spa types shorter than 5 repeats were excluded from the analysis. Where possible, isolates were assigned to MLST clonal complexes (CCs), based on information available in the PubMLST database (https://pubmlst.org/) and literature resources.For all n et al. . PCR ampwas used . spa typspa types, a growth curve and enterotoxins production using ELISA were determined. Isolates in which enterotoxin genes were identified were subtyped using RAPD-PCR according to Louws et al. [spa type, all isolates with different RAPD profiles were selected for ELISA. A single colony was inoculated in 5 ml of BHI and incubated overnight at 37 °C. Subsequently, the cultures were diluted to OD600 = 0.03 in 5 ml of BHI broth and incubated for 48 h with cell count and ELISA assay after 24 and 48 h. Bacterial cells were quantified by plating serial dilutions of the culture on BHI agar. Sandwich ELISA for SEC and SEH was performed as previously described [For selected enterotoxigenic isolates representing different s et al. , then wiescribed , and recescribed  served aescribed . PurifieS. aureus isolates were identified. Genes of at least one SE were found in 16 isolates (13.2%). The seb gene was found in 2 S. aureus isolates (1.7%), sec in 2 isolates (1.7%), see and seh gene were found in 4 (3.3%) and 11 (9%) isolates, respectively. One S. aureus isolate was found to carry both see and seh genes (isolate 35WB), and two isolates carried both sec and see genes   was tested using ELISA at 24 and 48 h of culture. The numbers of bacteria ranged from 9.5 ± 0.2 to 9.9 ± 0.2 log CFU/ml after 24 h of culture and from 9.8 ± 0.4 to 10.4 ± 0.3 log CFU/ml after 48 h. Production of SEB was determined in 2 spa types were identified among 121 S. aureus isolates . Based on spa type similarity, the isolates were assigned to 5 spa clusters  or sequence types (ST) based on literature and PubMLST database. Largest spa cluster 1 was formed by 44 S. aureus isolates. Thirty-four of them were assigned to clonal complex CC133  mostly contained already described spa types, and in cluster 2, many new spa types were included (6 identified in this study out of 9 spa types in this cluster). Some of the spa types identified could be assigned to known MLST clonal complexes (CCs). The most common CC identified in wild boar S. aureus was CC133 (n = 36). This clonal complex is considered animal-associated since it was recovered from various animal sources, but not from humans. It has been described as associated with intramammary infections in ruminants, mainly cows [S. aureus isolates from goats and sheep [spa type identified in this study was t091 (n = 18), which belongs to CC7. This lineage, mainly associated with humans [To determine the genotype diversity of wild boar nly cows , 21. It nd sheep , 23, as nd sheep . The sech humans , was alrh humans , 27.S. aureus strains from the CC30 and CC398 clonal complexes are prevalent in pigs [Isolates belonging to CC30 and CC398 can colonize multiple hosts. The CC398 clonal complex was indicated to have emerged in humans and spread to livestock . In Euro in pigs , 29. CC3 in pigs  reportedspa type t742. All of these isolates were included in spa cluster 2. The t742 genotype, assigned to CC425 was identified in wild boars [S. aureus clonal complexes were divided into host generalist and host specialist [S. aureus CC425 [Nine of the bacterial isolates studied here belonged to ld boars  and receld boars . Based oecialist . Accordius CC425 .S. aureus isolates belonging to CC1. The occurrence of this clonal complex in livestock was already observed [S. aureus isolates were assigned to CC5. Previous observations by Lowder et al. [S. aureus isolates from diseased and healthy poultry from Belgium, China, Denmark, Japan, the UK, and the USA belonged to the CC5 complex [S. aureus was assigned to t056 belonging to ST101. This clonal complex is mostly associated with humans [S. aureus isolates as members of CC97. This complex is believed to be associated with human and livestock, and S. aureus belonging to CC97 was identified in cattle and pigs [S. aureus population contains previously identified animal/human-associated genotypes, and genotypes not identified in humans or animals.We identified six wild boar observed , 32-34, observed , 35. Thr complex , 29, 36.h humans , but it h humans , non-humh humans , and rabh humans . We idenand pigs . The wilS. aureus was focused on SEA-SEE and SEH, enterotoxins whose involvement in SFP was widely confirmed [see gene was identified in an S. aureus isolate belonging to spa cluster 4 and assigned to CC1, in one isolate belonging to spa cluster 2 and in two isolates closely related to this cluster. Two S. aureus isolates harbouring the see gene were assigned to new spa type, i.e., t20575. The see-positive S. aureus isolate t4279 was assigned to spa cluster 2 in which we identified a spa type (t742) belonging to CC425, and two isolates of t20575 are closely related to spa cluster 2. In turn, one see-positive isolate was assigned to t922 spa type, which is closely related to t127. t922 was already identified as human, community-associated S. aureus [see in S. aureus. S. aureus carrying this gene was already detected in cows [S. aureus, mainly from human clinical cases and livestock animals, no see gene was identified [see gene was identified in S. aureus from a wildlife isolate, i.e., European badger (GenBank: CP097571.1). Based on the CP097571.1 genome analysis, we identified the ST of this isolate as closely related to ST425. As mentioned above, this ST belongs to CC425, a host specialist complex that mainly includes animal S. aureus isolates. Evidence of an SFP outbreak due to SEE comes from France, 2009 [Most of the enterotoxins identified so far have emetic activity, as demonstrated in experiments conducted in animal models . Howeveronfirmed , 43. The. aureus . So far, in cows , and was in cows . Recent entified . Howeverce, 2009 . In thisS. aureus from wild boar include SEB, SEC, and SEH. The seb gene was identified in two S. aureus isolates assigned to the spa type t160. This spa has already been found in human clinical cases [sec gene was identified in two S. aureus isolates simultaneously carrying the see gene. The seh gene was found in isolates belonging to CC1 and CC133. Some of the SEs investigated were produced at concentrations exceeding a few micrograms per millilitre of medium. In particular, SEB production reached 2.6 and 4.5 µg/ml at 48 h of culture.Other SEs produced by al cases , but alsal cases , 50. TheS. aureus isolates was not assessed previously. We identified wild boar S. aureus isolates which carried and expressed the enterotoxin genes seb, sec, see, and seh. The see gene rarely found in already characterised populations of S. aureus was identified in three isolates displaying unrelated spa types. Our results suggest that wild boar can be a significant reservoir of see-positive S. aureus.Production of enterotoxins by wild boar Supplementary file1 (XLSX 16 KB)Below is the link to the electronic supplementary material.


In an in vivo pharmacokinetic study, the Cmax and Tmax of the active metabolized sulfoxide were changed from 2.81 µg/mL at 3 h to 10.2 µg/mL at 6 h and the AUC0–48 was increased from 50.72 h⁎μg/mL to 119.95 h⁎μg/mL, indicating that the inclusion complex obtained can be used as a new oral therapeutic anti-anthelmintic and anti-tumor agent formulation.Albendazole is the preferred deworming drug and has strong insecticidal effects on human and animal helminth parasites, showing remarkable activity against hepatocellular carcinoma and colorectal cancer cells. However, it is classified as being in class II in the Biopharmaceutics Classification System due to its poor water solubility (0.2 mg/L) and high permeability, which make the clinical application of albendazole impractical. Through complexation with methyl- Albendazole has a broad anthelmintic spectrum, strong insecticidal effect on human and animal helminth parasites, low toxicity, and good safety, and was recommended by the World Health Organization as the preferred deworming drug . FurtherAlbendazole belongs to class II in the Biopharmaceutics Classification System due to its poor water solubility (0.2 mg/L) and high permeability . AlbendaSince 1991, numerous attempts have been made to improve albendazole’s solubility and thereby enhance its efficacy, such as the use of soybean oil emulsion , surfactβ-Cyclodextrin inclusion complex has been reported to stabilize albendazole and, consequently, improve its water solubility and dissolution rate. However, this complexation only increased the solubility 53.4 times due to β-cyclodextrin’s low aqueous solubility (18 mg/mL). The solid product of the complexation was analyzed using solid NMR spectroscopy, however, signals from albendazole in the complex were not observed in the spectrum [A spectrum ,15.β-cyclodextrin was increased from 2 to 10,000 fold to reach a solubility as high as 1.9 mg/mL [β-cyclodextrin could increase the solubility of albendazole from 0.2 µm/mL to 1.5 mg/mL [β-cyclodextrin in water at 25 °C with a pH of 2.3 for 3 days, its water solubility reached 8 mg/mL. Under the same conditions, HP-β-cyclodextrin could make its water solubility reach 6.4 mg/mL [The solubility of albendazole in a complex with HP-.9 mg/mL ,19,20,21.5 mg/mL ,23,24. W.4 mg/mL .2O.A variety of techniques such as FTIR, DSC, TAG, X-ray, and SEM have been used to confirm the formation of complexes in the solid state . Howeverβ-cyclodextrin, sulfobutylether-β-cyclodextrin, or methyl-β-cyclodextrin in water, its aqueous solubility was remarkably increased from 0.2 μg/mL to 0.79 mg/mL, 1.17 mg/mL, and 1.52 mg/mL, respectively, and these increased water solubilities were confirmed by proton NMR spectra in D2O [When albendazole was treated with 40% of HP-a in D2O ,29,30.β-cyclodextrin, itaconyl-β-cyclodextrin, and succinyl-β-cyclodextrin were synthesized and complexed with albendazole via spray drying. The drug dissolution rate and water solubility were improved slightly, and their proton NMR spectra in 0.1N DCl in D2O were recorded. Since albendazole is soluble in a 0.1 HCl solution (0.4 mg/mL), the chemical shifts from albendazole in the complex in the NMR spectrum could not fully support the drug’s water solubility [Citrate-lubility ,32.A trace amount of water-soluble polymer such as polyvinylpyrrolidone K30 was added to the cyclodextrin complex to increase the drug’s solubility ,34,35.β-cyclodextrin and albendazole using a supercritical antisolvent technique in a miscible solution of DMSO, acetone, and CO2 produced the precipitate as microparticles. After characterization using spectroscopic, thermal, and crystallographic analyses, this new solid form was found to contain tautomer II of albendazole. The dissolution rate was increased about four times in comparison to that of albendazole alone due to the formation of the inclusion complex and a synergetic effect between both components. However, the water solubility increase was not measured and confirmed [Recently, the co-precipitation of onfirmed . It was onfirmed .β-cyclodextrin, where the water solubility of albendazole was increased from 0.2 μg/mL to 30 mg/mL.Overall, even with great efforts towards preparing albendazole/cyclodextrin complexes, the water solubility has still not been significantly improved and is far away from the 5–10 mg/mL that is required for a cancer therapeutic dose. In the continuation of an interest in drug inclusion complexes ,45,46,472 = 0.9991, Y = concentration, and X = peak area).UV spectra of the solutions of albendazole in the mixture of acetic acid and ethanol  and cyclodextrins in water (1 mg/mL) were recorded from 200 nm to 400 nm, and the maximum absorption of albendazole at 295 nm was observed. Cyclodextrins have no UV absorption around this wavelength, and 295 nm was selected for a HPLC analysis of albendazole. A series of albendazole solutions in methanol with concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg/mL were analyzed using HPLC, and, based on the concentrations and peak areas, the regression equation was obtained as Y = 15598X + 10.71 . After stirring at room temperature for 48 h, each mixture was filtered, diluted with water, and checked using HPLC; the results are summarized in β-cyclodextrin had the best solubilization effect on albendazole, and the linear relationship between albendazole and methyl-β-cyclodextrin was classified as AL type according to Higuchi and Connors’s description [Extra albendazole was added to the solutions of 2 supercritical method [β-cyclodextrin was complexed with albendazole. The resulting solutions were analyzed using HPLC, and it was found that the ultrasonic method could provide the complex with the largest increase in water solubility.By using the ultrasonic method, aqueous solution stirring method, hydrothermal reactor, microwave method, and COl method , methyl-β-cyclodextrin, and ultrasonic power affected the complexation. Through single-factor and orthogonal strategies based on the Design of Experiments approach [β-cyclodextrin based on changing the ratio, ultrasonic power, reaction temperature, and reaction time. After the HPLC analysis, the best condition for increased solubility was found as follows: the ratio should be 1:3, the reaction time should be 45 min , the ultrasonic power should be 80%, and the reaction temperature should be 25 °C with occasional ice cooling. The best solubility was found as high as 27 mg/mL.The reaction temperature, reaction time, ratio of albendazole and methyl-approach , many coThrough self-association, cyclodextrins and their complexes can form aggregates or micelle-like structures to solubilize water-insoluble drugs by non-inclusion complexation , and watOnly a trace amount of sodium dodecyl sulfate  existed in the complex; the FTIR, DSC, and NMR spectra of the complex could not exhibit its characteristic signals.β-cyclodextrin. The FTIR spectra of methyl-β-cyclodextrin, albendazole, the physical mixture of albendazole and methyl-β-cyclodextrin, and their inclusion complex are shown in β-cyclodextrin, the characteristic peaks of albendazole appeared at 3463 cm−1 (N-H stretching in carbamate), 2963 cm−1 , 2647 cm−1 (-NH intramolecular in imidazole), 1730 cm−1 (bending vibration of C=O bond in carbamate), 1600 cm−1 (C=C aromatic and N-H out of the plane bending in benzimidazole), 1581 cm−1 (stretching vibration of C=N group), 1491 cm−1 and 1373 cm−1 (C-N and C-O stretching vibrations), 1248 cm−1, 1123 cm−1, and 1029 cm−1 (CH and NH in plane bending vibrations and CH deformation), and 1000–600 cm−1 , and they are obviously seen in the physical mixture of albendazole and methyl-β-cyclodextrin, but are slightly reduced in the inclusion complex. These changes in the characteristic peaks of albendazole confirmed that the albendazole was complexed with cyclodextrin.FTIR spectra were used to confirm the complex of albendazole with random methyl-β-cyclodextrin, their physical mixture, and inclusion complex were recorded and are shown in β-cyclodextrin corresponding to dehydration from the cavity was seen in the range of 50–125 °C, in curve for albendazole, the endothermic event at 225 °C was seen due to its melting effect, in curve for the physical mixture of methyl-β-cyclodextrin and albendazole, the endothermic event in the range of 50–125 °C was seen for the dehydration of methyl-β-cyclodextrin, and the absence of the endothermic event at 225 °C corresponding to albendazole’s melting and the presence of an endothermic event at 205 °C suggested an interaction between albendazole and methyl-β-cyclodextrin in the physical mixture. In the curve of the inclusion complex, the same thermal profile as that of methyl-β-cyclodextrin was presented, probably due to the extra methyl-β-cyclodextrin in the complex. The characteristic endothermic peak of albendazole at 225 °C disappeared due to the loss of the crystalline structure caused by encapsulation, which confirmed the interaction between albendazole and methyl-β-cyclodextrin and the formation of a new phase.The differential scanning calorimetry curves of albendazole, methyl-6, methyl-β-cyclodextrin in D2O, and the inclusion complex in D2O and DMSOd6 are shown in 2O exhibited the signals from albendazole at 7.61 , 7.56 , 7.33 , 2.91 , 1.54 , and 1.06 . The signals of OCH3 overlapped with the signals from methyl-β-cyclodextrin in the range from 4.00 ppm to 3.50 ppm. The proton NMR spectrum of albendazole in DMSOd6 exhibited signals at 7.46 , 7.34 , 7.11 , 3.76 , 2.85 , 1.53 , and 0.95 ; the proton NMR data of albendazole in the complex in DMSOd6 exhibited signals at 7.42 , 7.34 , 7.10 , 2.65 , 1.54 , and 0.95 . The chemical shift difference in the signals of albendazole in DMSOd6 between the complex and the drug alone could not support the formation of the inclusion complex. The Roesy spectrum of the inclusion complex in D2O was recorded , for albendazole sulfoxide as: Y = 40.464X − 0.7299 , and for albendazole sulfone as: Y = 38.613X − 0.6925 . Their standard curves are shown in A series of solutions of albendazole, albendazole sulfone, and albendazole sulfoxide in blank plasma with different concentrations were analyzed using HPLC in the range from 0.1 μg/mL to 5 μg/mL. The peak areas in HPLC and the concentrations of albendazole in the plasma solutions had a linear relationship, and the standard curve equations were obtained for albendazole as: Y = 35.96X − 0.9571  and the AUC0–48 of albendazole was decreased from 9.92 h⁎μg/mL to 3.92 h⁎μg/mL; and the Cmax of albendazole sulfone was decreased from 1.63 μg/mL at 4 h to 1.16 μg/mL at 4 h and the AUC0–48 of albendazole sulfone was decreased from 34.66 h⁎μg/mL to 22.50 h⁎μg/mL.After oral administration, the albendazole was partially metabolized to sulfoxide and albendazole sulfone, and the albendazole sulfoxide was the anthelmintic active substance. The β-cyclodextrin, the AUC0–48 of the active substance albendazole sulfoxide in vivo was doubled, the Cmax was more than tripled, and the Tmax was doubled, indicating that the inclusion complex of albendazole with methyl-β-cyclodextrin is the best formulation so far of albendazole as a potential anti-anthelmintic and anti-tumor agent.Through complexation with methyl-β-cyclodextrin (average molecular weight: 1303), hydroxypropyl-γ-cyclodextrin, albendazole, albendazole sulfoxide standard (98%), and albendazole sulfone (98%) were obtained from Shanghai Macklin, Shanghai, P. R. China; citric acid, HP-β-cyclodextrin, and β-cyclodextrin were obtained from Sa’en Chemistry Technology, Guangzhou, Guangdong, P. R. China; anhydrous sodium acetate was purchased from Xilong Science Co., Ltd., Shantou, Guangdong, P. R. China; dogs were obtained from Guangdong Medical Laboratory Animal Center, Guangzhou, Guangdong, P. R. China; blank dog plasma was obtained from Guangzhou Rui-Te Co., Ltd., Guangzhou, Guangdong, P. R. China; low-fat dog foods were obtained from Shanghai Jibai Chong Industrial Co., Ltd., Shanghai, P. R. China.Methyl-The solution of albendazole (10 mg) in acetic acid (5 mL) was diluted with ethanol , 5 mL of the solution was diluted with ethanol , and the resulting solution was used for UV  scanning in the range of 200–400 nm.v/v) as a mobile phase with a 1.0 mL/min flow rate. Based on the peak areas in the HPLC peaks and the concentrations, the linear regression equation and HPLC standard curve were obtained.A series of methanolic solutions of albendazole at concentrations of 0.01 mg/mL, 0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, and 0.1 mg/mL were analyzed using HPLC  with a UV detector (295 nm) on a Shim-pack VP-ODS C18 column (250 mm × 4.6 mm) using acetonitrile/water  in acetic acid (5 mL) was added to the solution of methyl-β-cyclodextrin (750 mg) in water (10 mL) with stirring uing a magnetic stirrer . The resulting solution was ultrasonicated in an ultrasonic reactor  at 25 °C and 520 W for 1 h with occasional iced water cooling, refrigerated at 4 °C for a few hours, evaporated, dissolved in water (15 mL), filtered through a 0.22 µm filter membrane. and freeze-dried at around −45 °C under a pressure of 32 pa for 48–72 h to give a clathrate solid powder.Solution method: the solution of albendazole (150 mg) in acetic acid (5 mL) was added to the solution of methyl-β-cyclodextrin (750 mg) in water (10 mL). The mixture was stirred at 50 °C and 800 rpm for 6 h, refrigerated at 4 °C for several hours, evaporated, dissolved in water (15 mL), filtered through a membrane (0.22 µm), and freeze-dried at around −45 °C under a pressure of 32 pa for 48–72 h to give the clathrate solid powder.Using an autoclave: the mixture of albendazole (150 mg) and methyl-β-cyclodextrin (750 mg) in water and acetic acid  was stirred at 120 °C and 800 rpm for 8 h in a high-temperature and pressure reaction kettle , evaporated, added to water, filtered, and freeze-dried at around −45 °C under a pressure of 32 pa for 48–72 h to give the product.Using a microwave reactor: the solution of albendazole (150 mg) in acetic acid (5 mL) was mixed with the solution of methyl-β-cyclodextrin (750 mg) in water (10 mL), and the resulting mixture was stirred at 300 rpm in a microcomputer microwave chemical reactor  for 1 h. During the reaction, every 10 min of heating was followed by 5 min. of no heating. After refrigeration at 4 °C for several hours and evaporation, the residue was dissolved in water (15 mL), filtered (0.22 µm), and freeze-dried at around −45 °C under a pressure of 32 pa for 48–72 h to provide the solid powder.2Using supercritical CO: the solution of albendazole (150 mg) in glacial acetic acid (5 mL) was mixed with the solution of methyl-β-cyclodextrin (750 mg) in water (10 mL), pumped with liquid CO2 in the reactor , and stirred at 25 °C with a pressure of 6.5 Mpa for 12 h. After the pressure was slowly released, the solution was kept in a refrigerator at 4 °C for a few hours, the solvent was removed via rotary evaporation, the residue was dissolved in water (15 mL), and, after filtration through a 0.22 μm filter membrane and freeze-drying at around −45 °C under a pressure of 32 pa for 48–72 h, the product was obtained as a solid powder.β-cyclodextrin were mixed (1:3 molar ratio) and passed through the 80-mesh sieve to provide the physical mixture.The grounded albendazole and methyl-β-cyclodextrin (750 mg) in water (10 mL), 5.6 mg of water-soluble polymer or organic salts  was added, and after stirring for 30 min, the resulting solution was ultrasonicated at 25 °C and 520 W for 40 min with occasional ice cooling, refrigerated at 4 °C for a few hours, evaporated, dissolved in water (15 mL), filtered through a 0.22 µm filter membrane, and freeze-dried at around −45 °C under a pressure of 32 pa for 48–72 h to give a clathrate solid powder. The inclusion complex from the ultrasonic method with the addition of a trace amount of sodium dodecyl sulfate was used for the FTIR, DSC, NMR, and in vitro and in vivo PK study.The solution of albendazole (150 mg) in acetic acid (5 mL) was added to the solution of methyl-β-cyclodextrin, their physical mixture, and inclusion complex were ground in an agate mortar, respectively, on a VERTEX 70 Fourier transform infrared spectrometer . Each of them was mixed with spectrally pure potassium bromide under an infrared lamp, poured into a compression mold, and pressed to provide the KBr pellets for the Fourier-transform infrared spectroscopy analysis in the range of 400–4000 cm−1.The dried samples of albendazole, methyl-β-cyclodextrin, their mixture, and inclusion compound were recorded on a DSC 214 differential scanning calorimeter  by placing them in an aluminum crucible, heating at a rate of 10 °C/min in the scan range from 25 to 300 °C under a nitrogen atmosphere (150 mL/min). Al2O3 was used as a reference.The differential scanning calorimetry curves of albendazole, methyl-β-cyclodextrin, its inclusion complex with albendazole in D2O, the solutions of albendazole, and its inclusion complex in DMSOd6 were recorded on a Bruker spectrometer (400 MHz). The Roesy spectrum of the complex recorded in D2O was recorded on a Bruker spectrometer (600 MHz).The proton NMR spectra of the solutions of methyl-The inclusion complex (100 mg) was dissolved in water (1 mL) with stirring for 2 h, and the solution was diluted with water to the range of 10 μg/mL–100 μg/mL . Based on the HPLC analysis, the albendazole content in the complex was calculated using the regression equation.An excess amount of the inclusion complex was dissolved in water (1 mL) for 2 h to obtain a saturated aqueous solution; after filtration, the sample was ready for HPLC analysis.The inclusion rate and yield of the inclusion compound were calculated by using the methods shown below:β-cyclodextrin  in degassed 0.1 mol/L hydrochloric acid (900 mL) were stirred at 50 r/min at 37 ± 0.5 °C, respectively, on an RC-3 dissolution meter . In total, 5 mL was taken from each of the mixtures at 1, 3, 5, 10, 15, 30, 45, 60, and 75 min, followed by the addition of 5 mL of the same dissolution medium at the same times. After filtration through a 0.22 μm microporous membrane for 30 s, the sample was analyzed using HPLC at a wavelength of 295 nm, and, based on the HPLC data, the dissolution rate curves were obtained.The paddle method in Chinese Veterinary Pharmacopoeia 2010 Edition I was adopted, albendazole (50 mg), the albendazole inclusion compound , and the physical mixture of albendazole and methyl-Wenzhou-kean university approved the study and the ID of the ethics approval is issued as WKULAEC2023-00.Twelve healthy adult dogs  were grouped as the albendazole group and the complex group, weighed, numbered, fed with low-fat food and normal drinking water in a warm and ventilated environment for 1 week, fasted for one day, and orally dosed with albendazole or albendazole complex in water , respectively.Blood samples (2 mL) were collected through the forearm vein after the administration of the drugs at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, and 48 h, transferred immediately to a heparinized test tube, centrifuged at 2000 rpm for 10 min, and stored at −20 °C in a freezer.The thawed plasma sample (0.5 mL) was pipetted into a centrifuge tube (2 mL), added with sodium thiosulfate (200 μg) and ethyl acetate (1 mL), vortexed for 5 min, centrifuged  for 10 min, and transferred to a test tube. After repeated vortexing and centrifugation, the supernatant in the test tube was blown at 40 °C in a nitrogen blower to dryness, added with methanol (0.5 mL), vortexed for 5 min, centrifuged  for 10 min, and filtered with a 0.22 μm microporous membrane for HPLC analysis.The blank plasma (0.5 mL) in a plastic centrifuge tube was added to the solutions of albendazole, albendazole sulfoxide, or albendazole sulfone at concentrations of 5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.1 μg/mL, 0.05 μg/mL, and 0.025 μg/mL, vortexed and vibrated, extracted with ethyl acetate or dichloromethane, blown with nitrogen, dissolved in methanol (1 mL), and analyzed using high-performance liquid chromatography at 295 nm using a Shim-packVP-ODS C18 chromatographic column (250 L × 4.6 mm) to provide the standard curves.p < 0.05.Each experiment was performed in triplicate, and the results were expressed as the mean ± standard deviation. The statistical analysis was performed through a one-way analysis of variance followed by Tukey’s honestly significant difference test using DPS software. Statistical significance was defined as β-cyclodextrin prepared by using the ultrasonic method with the addition of a trace amount of sodium dodecyl sulfate could increase the water solubility of albendazole by around 150,000 times compared to albendazole alone. In its in vivo pk study, the Cmax and Tmax of the active metabolized substance sulfoxide was changed from 2.81 μg/mL at 3 h to 10.2 μg/mL at 6 h, and the AUC0–48 was increased from 50.72 h⁎μg/mL to 119.95 h⁎μg/mL, indicating that this inclusion complex can be used as a new potent oral therapeutic anti-anthelmintic and anti-tumor agent.The inclusion complex of albendazole with methyl-