HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Jing, H.-b. Articles by Jiang, Y.-q. Search for Related Content PubMed PubMed Citation Articles by Jing, H.-b. Articles by Jiang, Y.-q. Agricola Articles by Jing, H.-b. Articles by Jiang, Y.-q.

Epidemiological analysis of group A streptococci recovered from patients in China

¹ State Key Laboratory of Pathogen and Biosecurity, The Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, no. 20 Dongda Street, Fengtai District, Beijing, 100071, People's Republic of China

² 304 Hospital, Haidian District, Beijing, 100037, People's Republic of China

Correspondence
Yong-qiang Jiang
jiangyq{at}nic.bmi.ac.cn

Received 13 July 2005
Accepted 15 March 2006

Abbreviations: GAS, group A streptococcal; NF, necrotizing fasciitis; STSS, streptococcal toxic shock syndrome.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Group A streptococci are an important group of Gram-positive extracellular bacterial pathogens that cause a variety of diseases, including pharyngitis, scarlet fever, cellulitis, bacteraemia, impetigo, acute rheumatic fever, glomerulonephritis, necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). Since the mid-1980s, an increase in the incidence of invasive infections caused by group A streptococci has been reported in many countries and regions (Banks et al., 2002; Carapetis et al., 1995; Holm et al., 1992; Kaul et al., 1997; Kiska et al., 1997; O'Brien et al., 2002; Chiou et al., 2004; Ho et al., 2003). This dramatic rise may be due to changes in specific virulence factors (Vlaminckx et al., 2003; Chatellier et al., 2000), hence strain characterization has assumed great importance.

Among the many virulence factors produced by group A streptococci, the M protein encoded by the emm gene is considered to be a major factor. About 150 different M protein gene sequence types have been documented (Bisno et al., 2003; Cunningham, 2000). It has been reported that strains of certain M types are epidemiologically associated with particular clinical syndromes. For example, the majority of recently reported invasive group A streptococcal (GAS) infections have been caused by strains of the M1 or M3 type, and infections with these organisms are associated with increased mortality (O'Brien et al., 2002; Chatellier et al., 2000; Musser et al., 1995; Ikebe & Wader, 2002). Moreover, a particularly virulent subclone of M1 associated with invasive infections has been spreading globally for more than 20 years. However, the mechanism(s) by which any M type causes a specific clinical manifestation still remains controversial.

Among the virulence factors that regulate GAS pathogenicity, the known extracellular products, including streptolysin O (SLO), streptococcal pyrogenic exotoxin B (SpeB) and superantigen toxins, are principal contributors. The virulence-related superantigens are the pyrogenic exotoxins SpecA, SpeC, SSA and SMEZ. They are mitogenic for certain T-cell subsets and do not require processing by antigen-presenting cells, and thus can activate a much larger number of T cells than conventional antigens and release excessive amounts of inflammatory cytokines (Bisno et al., 2003; Cunningham, 2000; Kotb, 1995). They are encoded on various phage genomes and are associated with gene variation. SLA is a prophage-encoded secreted extracellular phospholipase A₂ in GAS strains and is associated with host–pathogen interaction (Banks et al., 2003; Beres et al., 2002; Nagiec et al., 2004). The sil gene is the streptococcal invasion locus, which controls GAS spread into deeper tissues and may be involved in DNA transfer (Hidalgo-Grass et al., 2002, 2004).

Studies of GAS distribution have been performed worldwide, but there are few data from mainland China, where the epidemiology of GAS may be different. In Asia, studies from Malaysia, Thailand and Hong Kong have revealed a different GAS type distribution (Chiou et al., 2004; Ho et al., 2003). In mainland China, a similar GAS infection increase has also been observed in recent years. Severe infections, such as NF and STSS, caused by group A streptococci were earlier almost unknown. To obtain a better understanding of the epidemiology of GAS infections in mainland China, we performed a molecular epidemiology analysis on GAS isolates obtained from patients in China, including GAS emm typing, detection of some virulence factors, and antibiotic-susceptibility tests.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains. A total of 86 strains of group A streptococci were recovered from patients in different regions of mainland China from 2000 to 2004. The sources of the specimens included hospitalized patients, as well as outpatients from emergency rooms and specific outpatient clinics. All GAS strains were grown on blood agar plates in 5 % CO₂ and at 37 °C overnight. All isolates were ß-haemolytic and their identity was confirmed using API strips. Invasive isolates were exclusively from blood (n=10). Non-invasive isolates were obtained from the epipharynx (n=47), pus (n=7), wounds (n=7) and other sources (n=15).

DNA preparation. The DNA preparation of GAS stains was performed as described by the USA Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov/ncidod/biotech/strep/protocols.htm). Briefly, one loopful of bacteria was suspended in 300 µl 0.85 % NaCl and heated for 15 min at 70 °C. The samples were centrifuged and resuspended in 50 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8) supplemented with 10 µl mutanolysin (3000 U ml^–1) and 2 µl hyaluronidase (30 mg ml^–1). The mixture was then incubated for 30 min at 37 °C, heated at 100 °C for 10 min and briefly clarified by centrifugation. The supernatant including genomic DNA was used as the template for PCR.

emm genotyping. The M genotypes of strains were identified as described at the CDC website (http://www.cdc.gov/ncidod/biotech/strep/strepindex.html). Briefly, the emm gene encoding the M protein was amplified with the genomic DNA described above as template and P1 [5'-tatt(c/g)gcttagaaaattaa-3'] and P2 (5'-gcaagttcttcagcttgttt-3') as primers. The cycling parameters were 94 °C for 15 s, 46 °C for 30 s, and 72 °C for 1 min 15 s for the first 10 cycles, and then 94 °C for 15 s, 46 °C for 30 s, 72 °C for 1 min 15 s (with a 10 s increment for each of the subsequent 19 cycles) for the subsequent 20 cycles. The PCR product was sequenced with primer emmseq2 (5'-tattcgcttagaaaattaaaaacagg-3') and the first 160 bp of the 5' emm sequence of every strain was compared with the sequences in the CDC emm database (http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm) to determine emm types. This sequence includes 70 bases encoding the signal peptide and 90 bases encoding the first 30 amino acids of the mature protein. The subtypes were assigned to exact 150 bp sequences encoding the N-terminal 50 residues of the mature M protein.

Detection of virulence genes. Virulence genes, including sepA, sepB, sepC, slo, ssa, smeZ, sla and sil, were detected by PCR with primers as shown in Table 1. Amplification of all genes was performed with an initial 5 min denaturation step at 94 °C followed by 30 cycles of denaturation at 94 °C for 30 s, 30 s of annealing at the appropriate temperature for each gene, as specified in Table 1, and 60 s of extension at 72 °C with a final extension step at 72 °C for 7 min.

Statistical analysis. The chi square test was used for statistical analysis with a P value <0.05 considered significant. The SPSS 10.0 statistical package was used for all analyses.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Distribution of emm types

A total of 21 M types were identified among the 86 clinical isolates. In descending order of frequency, the prevalent M types (with a frequency >3 % in the population) in mainland China were M1 (29.1 %), M12 (23.3 %), M8 (7.0 %), M18 (5.8 %), M80 (4.7 %) and M28 (4.7 %). The other M types (M4, M66, M77, M94, M101, M3, M6, M23, M44, M63, M64, M75, M85, M86 and M88) were detected in only one or two isolates. M1 and M12 together accounted for 60.0 and 51.3 % of the invasive and non-invasive isolates, respectively.

A total of six different M types, including M1, M12, M18, M80, M6 and M86, were found in the 10 invasive isolates. A total of 19 different M types were identified in the 76 non-invasive isolates. Besides the most prevalent M1 and M12 types, other common types in non-invasive isolates (with a frequency >3 %) were M8 (7.9 %), M18 (5.3 %), M28 (5.3 %), M80 (3.9 %) and M101 (3.9 %). M8 and M28 were absent in the 10 invasive isolates and more frequent in the non-invasive isolates (P <0.05).

Only few emm sequence variations were found in our study. In nine of 86 isolates, types emm1.22, emm12.13, emm3.1, emm6.10, emm18.12 and emm86.1 were found to contain one to six base substitutions relative to M type reference strains, while no deletions and insertions were found in other isolates. Most of these subtype variants occurred in one or two isolates (Table 2). The remaining 77 isolates all had DNA sequences identical to the corresponding reference emm sequences.

The association of virulence genes and source is shown in Table 4. The speA gene was found in 70 % of isolates from blood and 51 % of isolates from the epipharynx, while speC was found in 40 % of isolates from blood and 62 % of isolates from the epipharynx. The speC gene was significantly over-represented among isolates from the epipharynx. The occurrence rates of speA, speC and ssa in wound isolates were much lower than in isolates from other sources. This could be explained by the reduced need for GAS virulence factors to establish infection after the destruction of the skin barrier. Because two out of four strains containing the sla gene were obtained from blood, the sla gene was more frequent in invasive isolates than non-invasive isolates overall (2/10 versus 2/64; P <0.01). Consistent with our expectation, none of six M8 isolates lacking speA, speC and ssa caused invasive infection in blood.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Only minor emm gene sequence alterations were noted for six emm types in our study. Allelic variation in the emm genes of our isolates, compared to reference sequences, was predominantly due to single-base substitutions. This study also showed that the GAS emm type distribution in mainland China differs from those in Thailand and Hong Kong (Chiou et al., 2004; Ho et al., 2003).

In order to study the factors contributing to the aggressive clinical picture seen with some GAS infections, the isolates were tested for the presence of different toxin genes. The streptococcal pyrogenic exotoxin B (SepB) is a highly conserved extracellular cysteine protease that is associated with pyrogenicity, T-lymphocyte mitogenicity, and the ability to increase susceptibility to endotoxic shock in individuals infected with group A streptococci (Collin & Olsen, 2001; Watanabe et al., 2002). Louie & Simor (1988) reported that speB was positive in all GAS isolates by PCR assay, but the assay was negative in all patients without culture or serological evidence of streptococcal infection. Our results were the same, indicating that PCR detection of the speB gene in GAS strains may be a useful method to confirm GAS infection.

The isolates were tested for the presence of the phage-encoded superantigens. It has been reported that speA is located in phages T12, 270 and 49 (Yu & Ferretti, 1991) and speC in phage CS112 (Goshorn & Schlievert, 1989). We showed that these toxin genes were exclusively associated with particular M types. Some of these observed associations between M types and toxin genes have been reported by others. For instance, Schmitz et al. (2003) have found that all but one of 239 M1 and M3 strains carry a speA allele and lack a speC gene, suggesting that the presence of speA affects the acquisition of a speC gene in M1 and M3 types. Vlaminckx et al. (2003) speculated that the exclusive combination of speA and smeZ in M1, which encode proteins known to be immunoactive, might contribute to intrinsic virulence. This specific set of virulence genes might make them more efficient as pathogens, findings in accordance with our study.

The presence of speA, as opposed to speC, was positively correlated with invasive infection in blood. This finding was consistent with the notion that speA is an important virulence factor required to cause invasive infection. The speC gene occurred more frequently in strains obtained from the epipharynx than in those obtained from other sources, a finding supportive of the conclusion that speC and bacteriophage induction are pharyngeal-cell dependent. Broudy et al. (2001) have reported that speC is induced along with phage CS112 when group A streptococci are co-cultured with human pharyngeal cells.

It has been reported that the prophage-encoding sla is not present in M3 GAS strains recovered before 1987, and that strains containing an sla gene are more likely to come from a severe infection such as NF. When the resurgence of severe invasive disease occurred in North America, Europe and Japan (Banks et al., 2003; O'Brien et al., 2002), Nagiec et al. (2004) found that the sla gene was more widely distributed in GAS than just serotype M3 strains. The sla gene was present in 10.8 % of 1189 GAS strains tested, including emm1, emm2, emm3, emm4, emm22, emm28 and emm7 (Nagiec et al., 2004). In our studies, we found the sla gene occurred at a lower rate (4.65 %) than those in other countries, and only four strains were sla positive, including emm18, emm101 and emm86, suggesting a horizontal transmission of the prophage-encoded gene in mainland China. Moreover, two of the four sla gene-positive strains could cause severe infection and death in mice (data not shown). Therefore, the sla gene is also an important virulence gene and plays a major role in invasive infection in M types other than M3.

The sil gene is the streptococcal invasion locus. Within this locus, a gene, silCR, is predicted to encode a 41 amino acid bacterial pheromone peptide that abrogates chemokine proteolysis. Hidalgo-Grass et al. (2004) showed that the M14 GAS invasive strains have a missense mutation (ATGATA) in the start codon of silCR, suggesting that the peptide might not be produced. We found that 11 strains carried the sil gene (Table 4), of which only one was from invasive infection. Sequence analysis of sil showed that these strains did not contain a missense mutation in the start codon of silCR, suggesting that the pheromone peptide might be produced in these strains and decrease host damage.

The overall rate of erythromycin resistance in our GAS isolates was high (91.8 %) compared to reported rates of 2.6 % in the USA, 2.1–4.6 % in Canada, 8.6 % in Finland and 32 % in Hong Kong (Ho et al., 2003). We also found a high rate of resistance to tetracycline (93.4 %) compared to 4 % in the USA and 53 % in Hong Kong. The high rates of resistance that we found in mainland China are probably a reflection of the high level of antibiotic usage in China. Fortunately, the GAS strains in this study were still highly susceptible to penicillin, ceftriaxone, vancomycin, ciprofloxacin and chloramphenicol. Resistance to penicillin and ceftriaxone has not emerged in GAS, despite extensive use of ß-lactam drugs in China. In general, the observations of GAS resistance to erythromycin, tetracycline and clindamycin are consistent with previous reports, despite a much higher drug-resistance rate (Ho et al., 2003; Bemer-Melchior & Juvin, 2000; Deazavedo et al., 1999; Palavecino et al., 2001).

This epidemiological study has demonstrated different emm types and virulence factors in mainland China, and such data may help understand both pathogenesis and vaccine development. Fewer M3 and M14 types, lower carriage of the sla and sil genes, lack of the missense mutation in the start codon of silCR, and the continued susceptibility of GAS to antibiotics, may explain fewer severe clinical infections, such as NF and STSS, in mainland China.

	ACKNOWLEDGEMENTS

 
We thank Dr Li Cheng for critical reading of and helpful suggestions for this manuscript. This work was supported by key programmes of the National Science and Technology Foundation of China (no. 2003BA712A04-06).

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Banks, D. J., Beres, S. B. & Musser, J. M. (2002). The fundamental contribution of phages to GAS evolution, genome diversification and strain emergence. Trends Microbiol 10, 515–521.[CrossRef][Medline]

Banks, D. J., Lei, B. & Musser, J. M. (2003). Prophage induction and expression of prophage-encoded virulence factors in group A streptococcus serotype M3 strain MGAS315. Infect Immun 71, 7079–7086.[Abstract/Free Full Text]

Bemer-Melchior, P. & Juvin, M.-E. (2000). In vitro activity of the new ketolide telithromycin compared with those of macrolides against Streptococcus pyogenes: influences of resistance mechanisms and methodological factors. Antimicrob Agents Chemother 44, 2999–3002.[Abstract/Free Full Text]

Beres, S. B. G. L., Sylva, K. D. & Lei, B. B. (2002). Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype and clone emergence. Proc Natl Acad Sci U S A 99, 10078–10083.[Abstract/Free Full Text]

Bisno, A. L., Brito, M. O. & Collins, C. M. (2003). Molecular basis of group A streptococcal virulence. Lancet Infect Dis 3, 191–200.[CrossRef][Medline]

Broudy, T. B., Pancholi, V. & Fischetti, V. A. (2001). Induction of lysogenic bacteriophage and phage-associated toxin from group A streptococci during coculture with human pharyngeal cells. Infect Immun 69, 1440–1443.[Abstract/Free Full Text]

Carapetis, J., Robins-Browne, R., Martin, D., Shelby-James, T. & Hogg, G. (1995). Increasing severity of invasive group A streptococcal disease in Australia: clinical and molecular epidemiological features and identification of a new virulent M-non-typeable clone. Clin Infect Dis 21, 1220–1227.[Medline]

Chatellier, S., Ihendyane, N. & Kansal, R. G. (2000). Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and non-severe invasive diseases. Infect Immun 68, 3523–3534.[Abstract/Free Full Text]

Chiou, C.-S., Liao, T.-L., Wang, T.-H., Chang, H.-L., Liao, J.-C. & Li, C.-C. (2004). Epidemiology and molecular characterization of Streptococcus pyogenes recovered from scarlet fever patients in Central Taiwan from 1996 to 1999. J Clin Microbiol 42, 3998–4006.[Abstract/Free Full Text]

Collin, M. & Olsen, A. (2001). Effect of speB and EndoS from Streptococcus pyogenes on human immunoglobulins. Infect Immun 69, 7187–7189.[Abstract/Free Full Text]

Cunningham, M. W. (2000). Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13, 470–511.[Abstract/Free Full Text]

Deazavedo, J. C. S., Yeung, R. H., Bast, D. J., Duncan, C. L., Borgia, S. B. & Low, D. E. (1999). Prevalence and mechanisms of macrolide resistance in clinical isolates of group A streptococci from Ontario, Canada. Antimicrob Agents Chemother 43, 2144–2147.[Abstract/Free Full Text]

Goshorn, S. C. & Schlievert, P. M. (1989). Bacteriophage association of streptococcal pyrogenic exotoxin type C. J Bacteriol 171, 3068–3073.[Abstract/Free Full Text]

Hidalgo-Grass, C., Ravins, M., Dan-Goor, M., Jaffe, J. & Moses, A. E. (2002). A locus of group A streptococcus involved in invasive disease and DNA transfer. Mol Microbiol 46, 87–99.[CrossRef][Medline]

Hidalgo-Grass, C., Dan-Goor, M. & Maly, A. (2004). Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotizing soft-tissue infections. Lancet 363, 696–703.[CrossRef][Medline]

Ho, P. L., Johnson, D. R., Yue, A. W. Y., Tsang, D. N. C., Que, T. L., Beall, B. & Kaplan, E. L. (2003). Epidemiologic analysis of invasive and noninvasive group A streptococcal isolates in Hong Kong. J Clin Microbiol 41, 937–942.[Abstract/Free Full Text]

Holm, S. E., Norrby, A., Bergholm, A. M. & Norgren, M. (1992). Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 1988–1989. J Infect Dis 166, 31–37.[Medline]

Ikebe, T. & Wada, A. (2002). Dissemination of the phage-associated novel superantigen gene sepL in recent invasive and noninvasive Streptococcus pyogenes M3/T3 isolates in Japan. Infect Immun 70, 3227–3233.[Abstract/Free Full Text]

Kaul, R. A., McGeer, A., Low, D. E., Green, K., Schwartz, B. & Simor, A. E. (1997). Population-based surveillance for group A Streptococcal necrotizing fasciitis, clinical features, prognostic indicators, and microbiologic analysis of seventy-seven cases. Am J Med 103, 18–24.[CrossRef][Medline]

Kiska, D. L., Thiede, B., Caracciolo, J. & 7 other authors (1997). Invasive group A streptococcal infections in North Carolina: epidemiology, clinical features, and genetic and serotype analysis of causative organisms. J Infect Dis 176, 992–1000.[Medline]

Kotb, M. (1995). Bacterial pyrogenic exotoxins as superantigens. Clin Microbiol Rev 8, 411–426.[Abstract]

Louie, L. & Simor, A. E. (1998). Diagnosis of group A streptococcal necrotizing fasciitis by using PCR to amplify the streptococcal pyrogenic exotoxin B gene. J Clin Microbiol 36, 1769–1771.[Abstract/Free Full Text]

Musser, J. M., Kupur, V. & Szeto, J. (1995). Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype M1 protein: recent international spread of a subclone causing episodes of invasive disease. Infect Immun 63, 994–1003.[Abstract]

Nagiec, M. J., Lei, B., Parker, S. K. & Vasil, M. L. (2004). Analysis of a novel prophage-encoded group A streptococcus extracellular phospholipase A₂. J Biol Chem 279, 45909–45918.[Abstract/Free Full Text]

O'Brien, K. L., Beall, B., Barrett, N. L. & 8 other authors (2002). Epidemiology of invasive group A streptococcus disease in the United States, 1995–1999. Clin Infect Dis 35, 268–276.[CrossRef][Medline]

Palavecino, E. L., Riedel, I., Berrios, X., Bajaksouzian, S., Johnson, D., Kaplan, E. & Jacobs, M. J. (2001). Prevalence and mechanisms of macrolide resistance in Streptococcus pyogenes in Santiago, Chile. Antimicrob Agents Chemother 45, 339–341.[Abstract/Free Full Text]

Schmitz, F.-J., Beyer, A. & Charpentier, E. (2003). Toxin-gene profile heterogeneity among endemic invasive European group A streptococcal isolates. J Infect Dis 188, 1578–1586.[CrossRef][Medline]

Vlaminckx, B. J. M., Mascini, E. M., Schellekens, J., Schouls, L. M., Pauw, A., Fluit, A. C., Novak, R., Verhoef, J. & Schmitz, F. J. (2003). Site-specific manifestations of invasive group A streptococcal disease: type distribution and corresponding patterns of virulence determinants. J Clin Microbiol 41, 4941–4949.[Abstract/Free Full Text]

Watanabe, Y., Todome, Y., Ohkuni, H., Sakurada, S., Ishikawa, T., Yutsudo, T., Fischetti, V. A. & Zabriskie, J. B. (2002). Cysteine protease activity and histamine release from the human mast cell line HMC-1 stimulated by recombinant streptococcal pyrogenic exotoxin B/streptococcal cysteine protease. Infect Immun 70, 3944–3947.[Abstract/Free Full Text]

Yu, C. E. & Ferretti, J. J. (1991). Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (sepA). Mol Gen Genet 231, 161–168.[CrossRef][Medline]

This article has been cited by other articles:

				R. Meisal, E. A. Hoiby, I. S. Aaberge, and D. A. Caugant Sequence Type and emm Type Diversity in Streptococcus pyogenes Isolates Causing Invasive Disease in Norway between 1988 and 2003 J. Clin. Microbiol., June 1, 2008; 46(6): 2102 - 2105. [Abstract] [Full Text] [PDF]

				B. Luca-Harari, K. Ekelund, M. van der Linden, M. Staum-Kaltoft, A. M. Hammerum, and A. Jasir Clinical and Epidemiological Aspects of Invasive Streptococcus pyogenes Infections in Denmark during 2003 and 2004 J. Clin. Microbiol., January 1, 2008; 46(1): 79 - 86. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Jing, H.-b. Articles by Jiang, Y.-q. Search for Related Content PubMed PubMed Citation Articles by Jing, H.-b. Articles by Jiang, Y.-q. Agricola Articles by Jing, H.-b. Articles by Jiang, Y.-q.