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1.
Streptococcus uberis, a well‐known bacterial pathogen associated with bovine mastitis, appears to be biochemically and serologically almost indistinguishable from the closely related species Streptococcus parauberis. In the present study, species‐specific oligonucleotide primers were designed using internal parts of the genes sodA, encoding superoxide dismutase A, and cpn60 encoding chaperonin 60 of S. uberis and S. parauberis, respectively. The two oligonucleotide primer pairs allowed a rapid and reliable PCR‐mediated identification and differentiation of both species. These studies, performed with S. uberis and S. parauberis reference cultures and clinical isolates from routine diagnostics, revealed that the occurrence of S. parauberis as causative agent of bovine mastitis appears to be rare. In addition the sodA and cpn60 sequence data confirmed that both species could taxonomically be classified to the pyogenic group of genus Streptococcus.  相似文献   

2.
Species-specific PCR tests, based on the manganese-dependent superoxide dismutase A encoding gene (sodA) and the chaperonin 60 encoding gene (cpn60), were developed for the identification of Streptococcus phocae, a bacterial pathogen of seals. The selection of both oligonucleotide primer pairs was performed after amplification and sequencing of internal parts of both genes using universal oligonucleotide primers. The sequence studies of both genes additionally confirmed that S. phocae could taxonomically be classified to the pyogenic group of the genus Streptococcus.  相似文献   

3.
Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.  相似文献   

4.
The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.  相似文献   

5.
Streptococcus uberis, as one of the principal causes of bovine streptococcal mastitis, has been characterized serologically and biochemically. Serological grouping of S. uberis revealed polysaccharide antigens of groups E, G, P and U. The biochemical properties of S. uberis, determined with the Strep-Zym identification system, differed clearly from those of S. agalactiae and S. dysgalactiae. Some cultures of S. uberis produced the enzymes hyaluronidase and neuraminidase. In addition S. uberis partly demonstrated CAMP-like synergistic hemolytic activities on sheep blood agar, reacted specifically with the lectins from Helix pomatia and Dolichos biflorus and produced bacteriocin-like inhibitors. This reactions, possibly of importance as virulence factors, as well as "DNA-fingerprinting" of S. uberis, might serve as individual markers of the respective cultures in epidemiological studies.  相似文献   

6.
In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.  相似文献   

7.
Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.  相似文献   

8.
Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.  相似文献   

9.
We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.  相似文献   

10.
The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E. coli. Killing of S. uberis in milk by Ply700 (50 microg/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 31% killing (P<0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing (P<0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calcium-dependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis.  相似文献   

11.
Heifers managed under pastoral conditions are at risk from Streptococcus uberis mastitis infections at calving. A total of 397 heifers from six farms around New Zealand were enrolled in a study to identify and enumerate S. uberis on teat-ends of heifers in the peri-partum period, and to understand the effect of teat-spraying in the pre-calving period on the prevalence and incidence of S. uberis mastitis post-calving. Heifers were randomly assigned to Control or Sprayed groups. Sprayed heifers were teat-sprayed once, three times a week (Monday, Wednesday and Friday) with a commercial iodine-based teat sanitizer, starting at 3 weeks prior to calving and ending at day of calving. Across three farms, all glands of cows in both groups were sampled at calving to determine S. uberis intra-mammary infection (IMI) prevalence. For all farms, clinical mastitis (CM) cases detected during the week after calving were sampled and submitted for bacteriological analysis. Swabbing of teat-ends of 54 heifers from one farm showed that heifers had a pre-existing S. uberis contamination averaging 610 colony-forming units per swab (cfu/swab), at 3 weeks prior to calving. At calving, teat-end contamination was 560 cfu/swab for Sprayed heifers and 1775 cfu/swab for Control heifers. Two weeks after calving, teat-end contamination was similar between both groups, at 30 cfu/swab. The prevalence of S. uberis IMI was significantly lower in the Sprayed (3.5% glands) vs. the Control (7.4%) heifers in the first week after calving. There was a trend for Sprayed heifers (3.6% heifers) to have a lower incidence of S. uberis CM compared with Control heifers (7.4% heifers). It is concluded that teat-spraying in the dry period is a management option that could contribute to controlling heifer S. uberis mastitis in the transition period.  相似文献   

12.
The ability to attach to the host cell surface has been considered an important virulence strategy in many bovine mammary gland pathogens, including Streptococcus uberis. Research conducted in our laboratory lead to the identification of an S. uberis adhesion molecule (SUAM) with affinity for bovine lactoferrin (LF) and delineation of its role in adherence of S. uberis to bovine mammary epithelial cells. Using a selected bacterial surface protein extraction protocol and affinity chromatography, a 112-kDa protein that had a similar molecular mass and the LF affinity as one of the identified S. uberis LBP described by Fang and Oliver in 1999 was found. To further characterize SUAM, the N-terminal amino acid sequence of this protein was elucidated. A protein query versus translated database TBLASTN search of the National Center for Biotechnology (NCBI), non-redundant database, nr, with the LBP N-terminal amino acid sequence showed no significant similarity with previous entries. Antibodies directed against SUAM and a 17 amino acid long N-terminal sequence (pep-SUAM) inhibited adherence to and internalization of S. uberis UT888 into bovine mammary epithelial cells. Data presented suggests that we have discovered a novel bacterial protein involved in the pathogenesis of this economically important mastitis pathogen.  相似文献   

13.
Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.  相似文献   

14.
OBJECTIVE: To determine whether lactoferrin (LF) or milk influenced adherence of Streptococcus uberis to bovine mammary epithelial cells. SAMPLE POPULATION: Three strains of S uberis from cows with mastitis, pooled milk samples from 3 clinically healthy Jersey cows early in the lactation period, and bovine mammary epithelial cells from a clonal cell line. PROCEDURES: Adherence of S uberis to bovine mammary epithelial cells in the presence of various concentrations of LF or milk and after pretreatment of bacteria with LF or milk was tested. Bacteria were cultured with mammary epithelial cell monolayers for 1 hour. The culture supernatant was removed, and the epithelial cells were lysed. Adherence index was calculated as number of colony-forming units (CFU) in the cell lysate divided by number of CFU in the supernatant times 10,000. RESULTS: All 3 strains of S uberis were found to bind to purified LF and LF in milk. Addition of LF to the culture medium enhanced adherence of all 3 strains to mammary epithelial cells, whereas addition of milk enhanced adherence of 2 strains and decreased adherence of the third. Pretreatment of bacteria with LF or milk increased adherence of 1 of the strains but decreased adherence of the other 2. Increased adherence was antagonized by rabbit antibovine LF antibody. CONCLUSIONS: Results suggest that LF may function as a bridging molecule between S uberis and bovine mammary epithelial cells, facilitating adherence of the bacteria to the cells.  相似文献   

15.
Streptococcus uberis causes a significant proportion of clinical and subclinical intramammary infections (IMI) in lactating and non-lactating dairy cows. In spite of this, its pathogenesis is incompletely understood. A study was conducted to determine leukocyte and cytokine dynamics during experimentally induced S. uberis mastitis. Five Jersey and five Holstein cows were challenged via intramammary inoculation of S. uberis into two uninfected mammary glands. Sixteen of 20 challenged mammary glands developed clinical mastitis with peak clinical signs observed at 144 h. The number of S. uberis in milk increased (P<0.05) 48 h after challenge, in spite of an increase in milk somatic cells that began at 18 h (P<0.001) and remained elevated throughout the study. Increased tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (IL-8) in milk were detected 66 h after challenge (P<0.05). Peak TNF-alpha and IL-8 concentrations occurred 120 h after challenge and preceded peak clinical signs. Experimental S. uberis IMI induced local production of TNF-alpha, IL-1beta and IL-8, which may play a role in the pathogenesis of S. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland, since increases in milk somatic cells occurred earlier than cytokine production.  相似文献   

16.
Streptococcus uberis is the most common environmental mastitis pathogen causing udder inflammations of different severities in dairy cows. The aim of the study was to investigate if the different clinical outcome of mastitis induced by different strains of S. uberis can be reflected in the mammary immune response. Mammary epithelial cells and somatic milk cells were treated with heat inactivated and living S. uberis of strain A and strain B in vitro. Strain A was repeatedly isolated from a chronically infected quarter during 8 months, and persisted in the quarter despite antibiotic treatment. Strain B caused an acute clinical mastitis and was not further isolated after a single antibiotic treatment. Treatment with Strain B induced a more pronounced increase of mRNA-expression of various immune factors (interleukin-8, interleukin-1beta, RANTES, and lactoferrin) in mammary epithelial cells than strain A. In contrast to mammary epithelial cells the response of removed somatic milk cells showed no differences between the stimulation with two S. uberis strains. Tumor necrosis factor-alpha mRNA expression was not differently induced by the two strains. In conclusion, the characteristics of different severities of mastitis that are induced by different S. uberis strains in vivo can also be reflected at the level of the immune response of the mammary gland in vitro.  相似文献   

17.
Streptococcus uberis is an important environmental mastitis pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows and heifers throughout the world. Previous work from our laboratory suggests that S. uberis adhesion molecule (SUAM) is involved in S. uberis pathogenesis and may be an excellent target for vaccine development. The objective of this study was to evaluate the antibody response of cattle vaccinated with recombinant SUAM (rSUAM). Uninfected primiparous dairy cows (n=30) in late lactation were divided randomly into three groups of 10 cows each: control, 200 μg rSUAM, and 400 μg rSUAM. Cows in groups vaccinated with 200 μg and 400 μg rSUAM received an emulsion containing adjuvant, phosphate-buffered saline (PBS) and affinity purified rSUAM. Cows in the control group received an emulsion containing adjuvant and PBS. Cows were vaccinated subcutaneously in the neck region at drying off (D-0), 28 d after drying off (D+28) and within 7 d after calving. Serum was collected at D-0, D+28, at calving (C-0), calving vaccination (CV), and during early lactation (CV+14). Serum antibody responses were measured by an ELISA against rSUAM. Following the first vaccination a significant increase in anti-rSUAM antibodies was detected at D+28 in cows from groups vaccinated with 200 μg and 400 μg rSUAM when compared to the control group. This increase in anti-rSUAM antibodies continued following the second immunization at D+28; reaching the highest levels in the post-parturient sampling period (C0), after which antibodies appeared to plateau. S. uberis UT888 pretreated with several dilutions of heat-inactivated serum from cows vaccinated with rSUAM, affinity purified antibodies against rSUAM, and to a 17 amino acid long peptide from the N terminus of SUAM (pep-SUAM) were co-cultured with bovine mammary epithelial cells and adherence to and internalization of S. uberis into epithelial cells was measured. Compared to untreated controls, opsonization of two strains of S. uberis with sera from cows vaccinated with rSUAM, with affinity purified rSUAM antibodies, or with affinity purified pep-SUAM antibodies significantly reduced adherence to and internalization of this pathogen into bovine mammary epithelial cells. In conclusion, subcutaneous vaccination of dairy cows with rSUAM during physiological transitions of the mammary gland either from or to a state of active milk synthesis induced antibodies in serum and milk and these antibodies reduced adherence to and internalization of S. uberis into mammary epithelial cells under in vitro conditions. SUAM appears to be an excellent candidate for vaccine development.  相似文献   

18.
利用编码3-磷酸甘油醛脱氢酶的gapC基因具有高度特异性的特点,建立PCR方法鉴定与奶牛乳房炎相关的链球菌。根据已有gapC基因序列设计1对引物,以分离自患乳房炎奶牛乳样的10株革兰阳性球菌、10株革兰阳性杆菌、10株革兰阴性杆菌作为待检菌株,进行PCR扩增。结果表明,在10株革兰阳性球菌中,有8株球菌可以扩增出约1011bp的目的条带,而其他2株革兰阳性球菌及20株杆菌均无相应PCR产物出现。通过传统的生化鉴定与16S rDNA序列分析相结合证实,能扩出gapC基因的8株革兰阳性球菌分别为乳房链球菌(Streptococcus uberis)、牛链球菌(S.bovis)与猪链球菌(S.suis)。说明基于gapC基因的PCR方法,用于鉴定奶牛乳房炎相关链球菌具有较强的特异性。  相似文献   

19.
Twenty-seven quarters of 18 lactating dairy cows were inoculated intramammarily with 3.6 X 10(4) colony-forming units (CFU) of a strain of Streptococcus uberis isolated from a cow with clinical mastitis. Before quarters were inoculated, 22 were considered as naturally colonized with Corynebacterium bovis, and 5 were considered bacteriologically negative. Streptococcus uberis was isolated from all quarters within 2 days after inoculation, and all quarters developed clinical mastitis by 3 days after inoculation. Mastitis was acute, and most cows had increased rectal temperatures. The number of somatic cells increased significantly (P less than 0.05), and milk production decreased significantly. In many cows, rectal temperatures remained increased, and Str uberis was isolated from infected glands after intramammary and systemic antimicrobial treatments were given. A decreased number (110 CFU) of the same strain of Str uberis caused equally severe mastitis in 3 quarters colonized with C bovis and in 1 bacteriologically negative quarter in 2 cows. Streptococcus uberis was isolated from all inoculated quarters, and all quarters developed clinical mastitis by 2 days after inoculation. Two quarters colonized with C bovis and 2 bacteriologically negative quarters were inoculated once with 25 CFU and once with 240 CFU of a different strain of Str uberis (ATCC 27958). Streptococcus uberis was never isolated from inoculated quarters, and changes in milk yield or number of somatic cells were not observed.  相似文献   

20.
Host-response patterns of intramammary infections in dairy cows   总被引:2,自引:0,他引:2  
Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.  相似文献   

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