Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

Quantification of Cell Proliferation and Alpha-Toxin Gene Expression of Clostridium perfringens in the Development of Necrotic Enteritis in Broiler Chickens

Cell proliferation and alpha-toxin gene expression of Clostridium perfringens in relation to the development of necrotic enteritis (NE) were investigated. Unlike bacitracin-treated chickens, non-bacitracin-treated birds exhibited typical NE symptoms and reduced growth performance. They also demonstrated increased C. perfringens proliferation and alpha-toxin gene expression that were positively correlated and progressed according to the regression model y = b₀ + b₁X − b₂X². The average C. perfringens count of 5 log₁₀ CFU/g in the ileal digesta appears to be a threshold for developing NE with a lesion score of 2.     

Clostridium perfringens in retail chicken

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% “atypical” genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.     

Real-time PCR assay for Clostridium perfringens in broiler chickens in a challenge model of necrotic enteritis

We compared ileal Clostridium perfringens quantification results produced by real-time PCR and culture-based methods in broiler chickens in a challenge model of necrotic enteritis. Assessment of the relative standard deviations (RSDs) revealed that the real-time PCR assay generated a smaller standard deviation and thus was more precise than the culture-based method. Linear regression analysis indicated that the bacterial counts of these two methods were highly correlated (R(2) = 0.845). We suggest that real-time PCR could be a replacement of the culture method for quantifying C. perfringens in the intestinal tracts of broiler chickens.     

Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract

The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as dietary fat type significantly affects microbiota activity in the broiler chicken gastrointestinal tract.     

Differential Responses of Cecal Microbiota to Fishmeal,Eimeria and Clostridium perfringens in a Necrotic Enteritis Challenge Model in Chickens

Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for progressing towards clinical symptoms of necrotic enteritis.     

The ability of disease and non-disease producing strains of Clostridium perfringens from chickens to adhere to extracellular matrix molecules and Caco-2 cells

Clostridium perfringens is a major enteric pathogen that is responsible for causing necrotic enteritis of poultry. The ability to adhere to the host’s intestinal epithelium and to extracellular matrix molecules (ECMM) in the gut, are strategies used by numerous bacterial enteropathogens, however, C. perfringens has received comparatively little attention in this respect. The present study investigated sixteen type A C. perfringens isolates from chickens, with varying disease producing ability with respect to necrotic enteritis in chickens, for their ability to adhere to nine different extracellular matrix molecules (ECMM) and to the intestinal epithelial cell line Caco-2. C. perfringens strains were able to bind to ECMMs and there was strain variation. Strains of C. perfringens that produced severe disease, were capable of binding to collagen type III, IV and V, fibrinogen, laminin and vitronectin at higher levels than less severe disease producing strains, suggesting that the ability to adhere to ECMMs might enhance virulence with respect to induction of necrotic enteritis. In addition, severe disease producing strains also bound better to collagen type III and IV and fibrinogen, than non-disease producing strains. The present study also showed that some strains of C. perfringens possessed the ability to adhere to Caco-2 cells; however no relationship was found between the ability to adhere to Caco-2 cells and disease producing ability.     

Dissemination of Fluoroquinolone-Resistant Campylobacter spp. within an Integrated Commercial Poultry Production System

While characterizing the intestinal bacterial community of broiler chickens, we detected -proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.     

Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log₁₀ CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log₁₀ were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log₁₀ CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP⁻/rtPCR⁺ colonies were identified as C. perfringens, whereas 3 mCP⁺/rtPCR⁻ colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.     

Increased intracellular calcium level and impaired nutrient absorption are important pathogenicity traits in the chicken intestinal epithelium during <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> colonization

Although a high number of chickens carry Campylobacter jejuni, the mechanistic action of colonization in the intestine is still poorly understood. The current study was therefore designed to investigate the effects of C. jejuni on glucose uptake, amino acids availability in digesta, and intracellular calcium [Ca²⁺]_i signaling in the intestines of broiler chickens. For this, we compared: control birds (n?=?60) and C. jejuni-infected birds (n?=?60; infected orally with 1?×?10⁸ CFU of C. jejuni NCTC 12744 at 14 days of age). Our results showed that glucose uptake was reduced due to C. jejuni infection in isolated jejunal, but not in cecal mucosa at 14 days postinfection (dpi). The decrease in intestinal glucose absorption coincided with a decrease in body weight gain during the 2-week post-infectious period. A reduction in the amount of the amino acids (serine, proline, valine, leucine, phenylalanine, arginine, histidine, and lysine) in ileal digesta of the infected birds at 2 and/or 7 dpi was found, indicating that Campylobacter utilizes amino acids as a carbon source for their multiplication. Applying the cell-permeable Ca²⁺ indicator Fluo-4 and two-photon microscopy, we revealed that [Ca²⁺]_i was increased in the jejunal and cecal mucosa of infected birds. The muscarinic agonist carbachol induced an increase in [Ca²⁺]_i in jejunum and cecum mucosa of control chickens, a response absent in the mucosa of infected chickens, demonstrating that the modulation of [Ca²⁺]_i by Campylobacter might be involved in facilitating the necessary cytoskeletal rearrangements that occur during the bacterial invasion of epithelial cells. In conclusion, this study demonstrates the multifaceted interactions of C. jejuni with the gastrointestinal mucosa of broiler chickens. For the first time, it could be shown that a Campylobacter infection could interfere with intracellular Ca²⁺ signaling and nutrient absorption in the small intestine with consequences on intestinal function, performance, and Campylobacter colonization. Altogether, these findings indicate that Campylobacter is not entirely a commensal and can be recognized as an important factor contributing to an impaired chicken gut health.     

Acidified Litter Benefits the Intestinal Flora Balance of Broiler Chickens

The alterations in the balance of the normal intestinal bacterial flora of chickens exposed to acidified wood-derived litter were analyzed and compared to those of a control group exposed to nonacidified litter. A total of 1,728 broilers were divided into two groups, with six replicates in each. One group was exposed to dry wood-derived litter, and the other was exposed to dry wood-derived litter sprayed with a mixture of sodium lignosulfonate, formic acid, and propionic acid. At five different times, five chickens from each pen were killed and the intestinal contents from ileum and caeca were collected. The samples were diluted and plated onto selective media to identify coliforms, Lactobacillus spp., Clostridium perfringens, and Enterococcus spp. Covariance analysis of bacterial counts showed significantly lower counts for C. perfringens in the caeca and the ileum and for Enterococcus spp. and Lactobacillus spp. in the ileum in chickens exposed to the acidified litter. Lactobacillus spp. showed significantly higher counts in the caeca in chickens exposed to acidified litter. There was no difference between the two litters with regard to coliforms in the ileum and the caeca or to Enterococcus spp. in the caeca. The study shows that exposing the chickens to acidified litter lowers the intestinal bacterial number, especially in the ileum, without negative consequences for the chicken's health or performance. Of special interest are the lower counts of C. perfringens and Enterococcus spp. that might reduce the risk of developing clinical or subclinical necrotic enteritis and growth depression.     

Clinicopathological and immunological studies on toxoids vaccine as a successful alternative in controlling clostridial infection in broilers

The present study was conducted to evaluate the efficacy and safety of three vaccination regimes of Clostridium perfringens (C. perfringens) type A, C and combined A&C toxoids based on their clinical signs and immunological effects. The vaccines were administered two times at two weeks interval (7 & 21 days old), then the birds were challenged (35 days old) with virulent strains of C. perfringens type A, C and combined A&C. Blood samples were taken one week after the first and second vaccination as well as after challenge. The evaluated parameters in this study included: clinical signs, gross intestinal lesions, complete blood count (CBC), serum protein, liver profiles, and enzyme-linked immunosorbent assay (ELISA) test for detecting serum antibody titers. The results revealed that immunization of broilers with C. perfringens type A, C and combined A&C toxoids resulted in a significant decrease in numbers of chickens with intestinal lesions particularly with the A&C toxoids vaccine. Results of the CBC values were significantly increased in all treated groups and challenged groups. Total leukocytic count decreased in challenged non vaccinated group while increased in challenged vaccinated birds. Results of biochemical assays implicated that there were a significant increase in serum protein and liver profiles. ELISA results explored a significant increase in antibody titers after immunization of broilers with C. perfringens type A, C and combined A&C toxoids particularly after the second dose of vaccination. We concluded that immunization of broilers with toxoid vaccines particularly the combined type A & C is safe, well-tolerated and can protect broiler chickens against necrotic enteritis particularly after the second booster dose of the vaccine.     

Inhibition of Clostridium perfringens by a Novel Strain of Bacillus subtilis Isolated from the Gastrointestinal Tracts of Healthy Chickens

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121°C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37°C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.     

Cecal microbiome divergence of broiler chickens by sex and body weight

The divergence of gut bacterial community on broiler chickens has been reported as potentially possible keys to enhancing nutrient absorption, immune systems, and increasing poultry health and performance. Thus, we compared cecal bacterial communities and functional predictions by sex and body weight regarding the association between cecal microbiota and chicken growth performance. In this study, a total of 12 male and 12 female 1-day-old broiler chickens were raised for 35 days in 2 separate cages. Chickens were divided into 3 subgroups depending on body weight (low, medium, and high) by each sex. We compared chicken cecal microbiota compositions and its predictive functions by sex and body weight difference. We found that bacterial 16S rRNA genes were classified as 3 major phyla (Bacteroidetes, Firmicutes, and Proteobacteria), accounting for > 98% of the total bacterial community. The profiling of different bacterial taxa and predictive metagenome functions derived from 16S rRNA genes were performed over chicken sex and bodyweight. Male chickens were related to the enrichment of Bacteroides while female chickens were to the enrichment of Clostridium and Shigella. Male chickens with high body weight were associated with the enrichment of Faecalibacterium and Shuttleworthia. Carbohydrate and lipid metabolisms were suggested as candidate functions for weight gain in the males. This suggests that the variation of cecal bacterial communities and their functions by sex and body weight may be associated with the differences in the growth potentials of broiler chickens.     

Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log₁₀ CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

利用PCR-DGGE方法分析不同鸡群的盲肠微生物菌群结构变化

研究不同品种、饲养阶段的健康和不良鸡群对盲肠细菌种群结构和多样性的影响。使用基于16S rDNA的PCR-DGGE技术,结合割胶回收DNA进行克隆和测序,分别以4、6、10、16、20、40周龄蛋鸡及1、2、4、6、7、8周龄肉鸡健康、不良鸡群盲肠内容物为样本,研究其中特定细菌类群的16S rDNA序列片段指纹图谱,并进行聚类分析,鉴定特异性和共性种群。在两品种健康鸡群盲肠内容物的细菌群落中,Lactobacillus属菌株的相似性均高于不良鸡群,并且在不同饲养阶段的健康、不良鸡群间指纹图谱平均条带数差异显著(P<0.05);而Bacteroides属菌株在健康鸡群盲肠内容物细菌的相似性与不良鸡群较为相近,健康、不良鸡群间平均条带数差异显著(P<0.05);Clostridium属菌株在蛋鸡20、40周龄的平均条带数差异不显著(P>0.05),但肉鸡各周龄健康、不良鸡群间的平均条带数差异显著(P<0.05)。序列测序结果,在蛋鸡产蛋期健康、不良鸡群样本中均检测到能动乳杆菌(Lactobacillus agilis),而育雏和育成期中均检测到鸟乳杆菌(Lactobacillus aviaries)和不可培养细菌;两品种的健康、不良鸡群样本中均检测到Bacteroides属的生酸拟杆菌(Bacteroides acidifaciens)、不可培养物细菌(Uncultured bacterium);而健康、不良蛋鸡群样本中均检测到Clostridium属不可培养的变形菌(Uncultured proteobacterium),健康肉鸡群中检测到索氏志贺菌(Shigella sonnei),而两品种不良鸡群中均缺乏此类菌种。结果显示,不同品种、饲养阶段的鸡群,其盲肠细菌群落的组成差异显著,并且细菌种群结构对鸡群的生长发育影响较大。