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1.

Background

Campylobacteriosis is a zoonotic disease, and animals such as poultry, pigs and cattle may act as reservoirs for Campylobacter spp. Cattle shed Campylobacter spp. into the environment and they can act as a reservoir for human infection directly via contact with cattle or their faeces or indirectly by consumption of contaminated food. The aim of this study was to determine the prevalence, the quantitative load and the genetic strain diversity of Campylobacter spp. in dairy cattle of different age groups.

Results

Faecal samples of 200 dairy cattle from three farms in the central part of Lithuania were collected and examined for Campylobacter. Cattle herds of all three farms were Campylobacter spp. positive, with a prevalence ranging from 75% (farm I), 77.5% (farm II) to 83.3% (farm III). Overall, the highest prevalence was detected in calves (86.5%) and heifers (86.2%). In contrast, the lowest Campylobacter prevalence was detectable in dairy cows (60.6%). C. jejuni, C. coli, C. lari and C. fetus subsp. fetus were identified in faecal samples of dairy cattle. C. upsaliensis was not detectable in any sample. The high counts of Campylobacter spp. were observed in faecal material of dairy cattle (average 4.5 log10 cfu/g). The highest numbers of Campylobacter spp. were found in faecal samples from calves (average 5.3 log10 cfu/g), whereas, faecal samples from cows harboured the lowest number of Campylobacter spp. (average 3.7 log10 cfu/g). Genotyping by flaA PCR-RFLP analysis of selected C. jejuni isolates showed that some genotypes were present in all farms and all age groups. However, farm or age specific genotypes were also identified.

Conclusions

Future studies are needed to investigate risk factors related to the degree of colonisation in cattle. Based on that, possible measures to reduce the colonisation and subsequent shedding of Campylobacter in cattle could be established. It is important to further investigate the epidemiology of Campylobacter in the cattle population in order to assess associated risks to public health.  相似文献   

2.
Fecal samples of 2,056 dairy cattle from 14 farms were collected in three geographical regions of China and stained using a modified acid-fast staining technique to identify Cryptosporidium oocysts. A total of 387 (18.82%) positive samples were identified and further analyzed by polymerase chain reaction (PCR) using primers designed to amplify DNA fragments from the small subunit ribosomal RNA. The PCR products were sequenced and the sequences were deposited in the GenBank database under accession numbers EU369377-84 and GU070730-33. Phylogenetic analysis was performed and a distances matrix generated from these sequences confirmed the existence of Cryptosporidium (C.) parvum ''mouse'' genotype, C. bovis, C. andersoni, C. hominis, and C. serpentis in cattle. These results represent the first report on the prevalence and genetic identification of Cryptosporidium species, and may contribute to a better understanding of the epidemiology of Cryptosporidium in cattle in China.  相似文献   

3.

Background

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Prevalence data in ruminant species are important to support risk assessments regarding public and animal health. The aim was to investigate the presence of or exposure to C. burnetii in cattle, sheep, goats and moose, and to compare two enzyme-linked immunosorbent assays (ELISAs). National surveys of antibodies against C. burnetii were performed for dairy cattle (n=1537), dairy goats (n=58) and sheep (n=518). Bovine samples consisted of bulk milk, caprine of pooled milk, and ovine of pooled serum. Antibodies were investigated in moose samples (n=99) from three regions. A one-year regional cattle bulk milk survey was performed on the Isle of Gotland (n=119, four occasions). Cattle, sheep and goat samples were analysed with indirect ELISA and moose samples with complement fixation test. For the sheep, goat, and parts of the cattle survey, samples were run in parallel by ELISAs based on antigens from infected ruminants and ticks. Bulk milk samples from the regional cattle survey and vaginal swabs from a subset of the sheep herds (n=80) were analysed for the agent by polymerase chain reaction. Spatial clustering was investigated in the national cattle survey.

Results

The prevalence of antibodies in dairy herds was 8.2% with large regional differences. High risk clusters were identified in the southern regions. The prevalence among dairy herds on the Isle of Gotland varied from 55.9% to 64.6% and 46.4% to 58.9.0% for antibodies and agent, respectively, overall agreement between agent and antibodies was 85.2%. The prevalence of antibodies in sheep was 0.6%, the agent was not detected the vaginal swabs. Antibodies were not detected in goats or moose, although parts of the moose samples were collected in an area with high prevalence in cattle. The overall agreement between the two ELISAs was 90.4%.

Conclusions

The prevalence of antibodies against C. burnetii in dairy cattle in Sweden shows large regional differences. The results suggest that C. burnetii is a rare pathogen among Swedish moose, dairy goat and sheep. ELISAs based on ruminant and tick antigen performed in a similar manner under Swedish conditions.  相似文献   

4.
This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.  相似文献   

5.
This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.  相似文献   

6.
Bartonella and Babesia infections and the association with cattle breed and age as well as tick species infesting selected cattle herds in Taiwan were investigated. Blood samples were collected from 518 dairy cows and 59 beef cattle on 14 farms and 415 ticks were collected from these animals or in a field. Bartonella and Babesia species were isolated and/or detected in the cattle blood samples and from a selected subset (n = 254) of the ticks either by culture or DNA extraction, PCR testing and DNA sequence analysis. Bartonella bovis was isolated from a dairy cow and was detected in 25 (42.4%) beef cattle and 40 (15.7%) tick DNA samples. This is the first isolation of B. bovis from cattle in Asia and detection of a wide variety of Bartonella species in Rhipicephalus microplus. Babesia spp. were detected only on one farm from dairy cows either infected by Babesia bovis (n = 10, 1.9%) or B. bigemina (n = 3, 0.6%).  相似文献   

7.
Chlamydia pecorum (designated 22–58) was isolated in 2010 in HmLu-1 cells from the jejunum of a calf which died of necrotizing enterocolitis in Yamaguchi Prefecture, Japan. Immunohistochemical staining identified C. pecorum positive reactions in the jejunal villi. C. pecorum, designated 24–100, was isolated from the feces of a calf with diarrhea in another farm in Yamaguchi Prefecture in 2012. A significant increase in neutralizing antibody titers against C. pecorum was confirmed in paired sera. Nucleotide sequence identities of omp1 genes of the 2 isolates were 100%. The isolates were genetically and antigenically more closely related to C. pecorum Bo/Yokohama strain isolated from cattle with enteritis in Japan than to the other prototype strains, Bo/Maeda isolated from cattle with pneumonia and Ov/IPA isolated from sheep with polyarthritis. These results indicate that C. pecorum strains similar to 22–58 and 24–100 might be endemic in Yamaguchi Prefecture and cause enteric disease in cattle.  相似文献   

8.
Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.  相似文献   

9.
This study was carried out to investigate fifteen cases of acute lethal infection of calves (≤ 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79αcy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.  相似文献   

10.
The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.  相似文献   

11.
Bovine tuberculosis (TB) is a major zoonosis that''s caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.  相似文献   

12.
MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.  相似文献   

13.

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.  相似文献   

14.
A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on Tm values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.  相似文献   

15.
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16.
New insights into the epidemiology of bovine piroplasmoses in Italy   总被引：1，自引：0，他引：1  
Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques.Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively.Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.  相似文献   

17.
A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.  相似文献   

18.

Background

Identification of Staphylococci to species level in veterinary microbiology is important to inform therapeutic intervention and management. We report on the efficacy of three routinely used commercial phenotypic methods for staphylococcal species identification, namely API Staph 32 (bioMérieux), RapID (Remel) and Staph-Zym (Rosco Diagnostica) compared to genotyping as a reference method to identify 52 staphylococcal clinical isolates (23 coagulase positive; 29 coagulase negative) from companion animals in Irish veterinary hospitals.

Results

Genotyping of a 412 bp fragment of the staphylococcal tuf gene and coagulase testing were carried out on all 52 veterinary samples along with 7 reference strains. In addition, genotyping of the staphylococcal rpoB gene, as well as PCR-RFLP of the pta gene, were performed to definitively identify members of the Staphylococcus intermedius group (SIG). The API Staph 32 correctly identified all S. aureus isolates (11/11), 83% (10/12) of the SIG species, and 66% (19/29) of the coagulase negative species. RapID and Staph-Zym correctly identified 61% (14/23) and 0% (0/23) respectively of the coagulase-positives, and 10% (3/29) and 3% (1/29) respectively of the coagulase-negative species.

Conclusions

Commercially available phenotypic species identification tests are inadequate for the correct identification of both coagulase negative and coagulase positive staphylococcal species from companion animals. Genotyping using the tuf gene sequence is superior to phenotyping for identification of staphylococcal species of animal origin. However, use of PCR-RFLP of pta gene or rpoB sequencing is recommended as a confirmatory method for discriminating between SIG isolates.  相似文献   

19.

Background

The aims of this study were to investigate the prevalence of Campylobacter species in Swedish dogs, to identify the species of the Campylobacter isolates and to genotype the C. jejuni isolates. Young and healthy dogs were targeted and the sampling was performed at 11 veterinary clinics throughout Sweden from October 2011 to October 2012. Faecal swab samples were collected and sent to the laboratory at the National Veterinary Institute (SVA) for isolation of Campylobacter, speciation and genotyping.

Results

Campylobacter spp. were isolated from 67 of the 180 sampled dogs which yields an overall prevalence of 37%. The most prevalent species of Campylobacter among the participating dogs was C. upsaliensis with 52 of the 67 identified isolates. A lower prevalence was observed for C. jejuni with seven identified isolates and one isolate was identified as C. helveticus. Multi-locus sequence typing (MLST) was carried out on the seven C. jejuni isolates and all sequence types that were found are also commonly found in humans. The dogs were divided into three age groups; 1) under 12 months, 2) 12 to 23 months and 3) 24 months and older. The highest prevalence was found in the two younger age groups. Dogs shedding C. jejuni were between 3–12 months of age while dogs shedding C. upsaliensis were found in all ages.

Conclusions

The present investigation finds that Campylobacter spp. known to cause campylobacteriosis in humans are present in Swedish dogs. The results suggest an age predisposition where dogs under 2 years of age are more likely to shed Campylobacter spp. than older dogs. The most commonly isolated species was C. upsaliensis followed by C. jejuni, which was only detected in dogs up to 12 months of age. All C. jejuni isolates identified in the present study were of the same MLST types that have previously been described both in humans and in animals. The awareness of the Campylobacter risk of healthy young dogs may be an important way to reduce the transmission from dogs to infants, young children and immunocompromised adults.  相似文献   

20.
Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.  相似文献   

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