Differences in Pathogenicity and Vaccine Resistance Discovered between Two Epidemic Strains of Marek's Disease Virus in China.

Despite highly effective vaccines, Marek's disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 and DH/18) with clinically different pathotypes. We studied each strain's infection process and pathogenicity and observed differences in immunosuppression and vaccine resistance. Specific pathogen-free chickens, unvaccinated or vaccinated with CVI988, were challenged with AH/1807 or DH/18. Both infections induced MD damage; however, differences were observed in terms of mortality (AH/1807: 77.8%, DH/18: 50%) and tumor rates (AH/1807: 50%, DH/18: 33.3%). The immune protection indices of the vaccine also differed (AH/1807: 94.1, DH/18: 61.1). Additionally, while both strains caused interferon-ß and interferon-γ expression to decline, DH/18 infection caused stronger immunosuppression than AH/1807. This inhibition persisted even after vaccination, leading to increased replication of DH/18 that ultimately broke through vaccine immune protection. These results indicate that both strains have different characteristics, and that strains such as DH/18, which cause weaker pathogenic damage but can break through vaccine immune protection, require further attention. Our findings increase the understanding of the differences between epidemic strains and factors underlying MD vaccination failure in China.

Revascularization versus drug therapy for coronary artery disease in patients aged over 80 years: a real-world study.

BACKGROUND: Revascularization for the treatment of coronary artery disease (CAD) is advancing rapidly and is used increasingly in old patients. This study aimed to compare the efficacy and safety of revascularization with drug therapy in CAD patients aged over 80 years at a real-world clinical setting. METHODS: A total of 501 CAD patients aged over 80 years were consecutively enrolled from January 2011 to January 2016 in Anzhen Hospital (Beijing, China), Capital Medical University. The patients were treated with percutaneous coronary intervention (PCI) (n=283), coronary artery bypass grafting (CABG) (n=106), or drug therapy (n=112). All-cause mortality, cardiovascular-related mortality, readmission rate, and Seattle Angina Questionnaire (SAQ) score were compared between the three treatment methods. RESULTS: A total of 411 patients (82.04%) were followed with a median duration of 25 months. All-cause mortality and cardiovascular-related mortality in the drug therapy group were significantly higher than the PCI and CABG groups (both P<0.05). Readmission rate for cardiovascular events in the CABG group was significantly lower than the PCI and drug therapy groups (both P<0.05). Scores of physical limitation, angina frequency, treatment satisfaction, and disease perception of the SAQ in the PCI and CABG groups were significantly higher than the drug therapy group (both P<0.05). Scores of angina stability did not differ significant between the three groups (P=0.127). CONCLUSIONS: Revascularization is superior to drug therapy in efficacy and safety in the treatment of oldest-old patients with CAD.

An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

Clinical efficacy of different treatments and their impacts on the quality of life of octogenarians with coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) in octogenarians (age of ≥80 years) has a high risk of mortality and high medical expenses. Research shows that the prevalence of CAD is higher among octogenarians than that among younger people, but few such patients undergo percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG). This study aimed to evaluate different treatments with respect to their clinical effects and impacts on quality of life of octogenarians with CAD. METHODS: Data of 519 octogenarians with CAD consecutively treated at Beijing Anzhen Hospital, Capital Medical University (Beijing, China) from January 2010 to January 2016 were collected in this study. The patients were categorized into three groups based on the treatments they received: the PCI group (n = 292), CABG group (n = 110), and medical treatment group (n = 117). The followings were recorded during follow-up: clinical data, death (all-cause and cardiovascular-related), re-hospitalization time, Seattle Angina Questionnaire (SAQ) score, and occurrence of hemorrhagic events (cerebral bleeding, gastrointestinal bleeding, and dermal ecchymosis). RESULTS: The median follow-up duration was 25.0 (25th, 75th percentile: 17.0, 55.5) months among 417 patients. The all-cause death rates (28.2% vs. 12.0% and 14.6%, respectively) and cardiovascular-related death rates (15.4% vs. 3.8% and 6.4%, respectively) were significantly higher in the medical treatment group than those in the PCI group and CABG group (all P < 0.05). The re-hospitalization rate for cardiovascular events was significantly lower in the CABG group than those in the PCI group and medical treatment group (3.8% vs. 12.8% and 14.9%, respectively) (χ = 8.238, P = 0.018). The SAQ scores of physical limitation, angina frequency, treatment satisfaction, and disease perception were significantly higher in the PCI group and CABG group than those in the medical treatment group (all P < 0.05). No significant difference in the angina stability score was observed among the three groups (F = 3.179, P = 0.204). CONCLUSION: PCI and CABG result in reduced mortality and better quality of life in octogenarians with CAD.

Differential expression of type I interferon mRNA and protein levels induced by virulent Marek's disease virus infection in chickens.

Marek's disease virus (MDV), an α-herpesvirus targeting avian species, causes fatal Marek's disease (MD) in chickens. The host interferon (IFN) responses play a key role in resisting viral infection. However, host IFN responses following MDV infection in the chicken central immune organs (thymus and bursa of Fabricius), which contain numerous MDV target cells, is poorly understood. In this study, we performed animal experiments in specific pathogen-free chickens infected with two virulent MDV strains (BS/15 and Md5) or without infection as negative controls. Specifically, the type I IFN (IFN-α and IFN-ß) transcriptional and proteomic expression levels at 7, 10, 14, 17, and 21 days post infection (dpi) were detected and analyzed. Our results indicated that the mRNA and protein expression levels of IFN-α and IFN-ß in the thymus and bursa of Fabricius were mainly downregulated in cytolytic infection (such as 10 dpi) and reactivation (such as 17 dpi) stages, but not the latent (such as 14 dpi) stage of MDV infection, which was determined by comprehensively analyzing the MDV viral load and immune organ damage caused by MDV infection. These data suggest that MDV could inhibit the expression of host type I IFNs, which may be involved in the MDV-induced host immunosuppression and contribute to the immune escape of MDV from host immunity. Furthermore, we found that the downregulated expression of the host type I IFNs induced by BS/15 and Md5 infection was significantly different, which we speculated may be related to the diverse virulence and pathogenicity of MDV strains. In conclusion, our study demonstrated that MDV mostly inhibited the expression of type I IFNs in infected hosts, which may be associated to its pathogenesis.

Unfractionated Heparin with Sequential Enoxaparin in Patients with Complex Coronary Artery Lesions during Percutaneous Coronary Intervention.

BACKGROUND: Unfractionated heparin (UFH), despite its limitations, has been used as the primary anticoagulant alternative during the percutaneous coronary intervention (PCI). Some studies indicated that intravenous enoxaparin could be an effective and safe option. Our team used enoxaparin alone at one time according to the guidelines (Class IIA) and found a little catheter thrombosis during PCI. We recommend a new anticoagulation strategy using enoxaparin in combination with UFH. Enoxaparin has a more predictable anticoagulant response with no need of repeatedly monitoring anticoagulation during PCI. This retrospective study aimed to evaluate the efficacy and safety of using enoxaparin in combination with UFH in PCI patients with complex coronary artery disease. METHODS: Between January 2015 and April 2017, 600 PCI patients who received intravenous UFH at an initial dose of 3000 U plus intravenous enoxaparin at a dose of 0.75 mg/kg (observation group) and 600 PCI patients who received UFH at a dose of 100 U/kg (control group) were consecutively included in this retrospective study. The endpoints were postoperative 48-h thrombolysis in myocardial infarction (TIMI) bleeding and transfusion and 30-day and 1-year major adverse cardio-cerebrovascular events (MACCE). RESULTS: Baseline clinical, angiographic, and procedural characteristics were similar between groups, except there was less stent implantation per patient in the observation group (2.13 vs. 2.25 in the control group, P = 0.002). TIMI bleeding (3.3% vs. 4.7%) showed no significant difference between the observation group and control group. During the 30-day follow-up, the rate of MACCE was 0.9% in the observation group and 1.5% in the control group. There was no significant difference in the rates of MACCE, death, myocardial infarction, target vessel revascularization, cerebrovascular event, and angina within 30 days and 1 year after PCI between groups as well as in the subgroup analysis of transfemoral approach. CONCLUSIONS: UFH with sequential enoxaparin has similar anticoagulant effect and safety as UFH in PCI of complex coronary artery disease.

Co-Infection with Marek's Disease Virus and Reticuloendotheliosis Virus Increases Illness Severity and Reduces Marek's Disease Vaccine Efficacy.

Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) cause Marek's disease (MD) and reticuloendotheliosis (RE), respectively. Co-infection with MDV and REV is common in chickens, causing serious losses to the poultry industry. However, experimental studies of such co-infection are lacking. In this study, Chinese field strains of MDV (ZW/15) and REV (JLR1501) were used as challenge viruses to evaluate the pathogenicity of co-infection and the influence of MD vaccination in chickens. Compared to the MDV-challenged group, the mortality and tumor rates increased significantly by 20.0% (76.7 to 96.7%) and 26.7% (53.3 to 80.0%), in the co-challenged group, respectively. The protective index of the MD vaccines CVI988 and 814 decreased by 33.3 (80.0 to 47.7) and 13.3 (90.0 to 76.7), respectively. These results indicated that MDV and REV co-infection significantly increased disease severity and reduced the vaccine efficacy. The MDV genome load showed no difference in the feather pulps and spleen, and pathogenicity-related MDV gene expression (meq, pp38, vIL-8, and ICP4) in the spleen significantly increased at some time points in the co-challenged group. Clearly, synergistic pathogenicity occurred between MDV and REV, and the protective efficacy of existing MD vaccines was attenuated by co-infection with Chinese field MDV and REV strains.

A Chinese Variant Marek's Disease Virus Strain with Divergence between Virulence and Vaccine Resistance.

Marek's disease (MD) virus (MDV) has been evolving continuously, leading to increasing vaccination failure. Here, the MDV field strain BS/15 was isolated from a severely diseased Chinese chicken flock previously vaccinated with CVI988. To explore the causes of vaccination failure, specific-pathogen free (SPF) chickens vaccinated with CVI988 or 814 and unvaccinated controls were challenged with either BS/15 or the reference strain Md5. Both strains induced MD lesions in unvaccinated chickens with similar mortality rates of 85.7% and 80.0% during the experimental period, respectively. However, unvaccinated chickens inoculated with BS/15 exhibited a higher tumor development rate (64.3% vs. 40.0%), but prolonged survival and diminished immune defects compared to Md5-challenged counterparts. These results suggest that BS/15 and Md5 show a similar virulence but manifest with different pathogenic characteristics. Moreover, the protective indices of CVI988 and 814 were 33.3 and 66.7 for BS/15, and 92.9 and 100 for Md5, respectively, indicating that neither vaccine could provide efficient protection against BS/15. Taken together, these data suggest that MD vaccination failure is probably due to the existence of variant MDV strains with known virulence and unexpected vaccine resistance. Our findings should be helpful for understanding the pathogenicity and evolution of MDV strains prevalent in China.

Characterization of a Gallid herpesvirus 2 strain with novel reticuloendotheliosis virus long terminal repeat inserts.

A bacterial artificial chromosome clone, designated LCY, was constructed from a Gallid herpesvirus 2 (GaHV-2) isolate from a GaHV-2 and reticuloendotheliosis virus co-infected clinical sample. The LCY GaHV-2 insert was sequenced and found to consist of 175,319 nucleotides. LCY GaHV-2 open reading frames (ORFs) had a high sequence identity to those of reference strains. The major difference was that two REV long terminal repeats (LTRs), in the same direction, were inserted at the internal repeat short (IRs)/unique short (Us) and Us/terminal repeat short (TRs) junctions. In addition, the a-like sequence and UL36 were different from other strains. Phylogenetic analysis revealed that LCY was closely related to pandemic strains in China. A pathogenicity study and a vaccination-challenge test were performed on LCY and the reference strain, GA. The results showed that LCY induced gross Marek's disease (MD) lesions and mortality in 71.4 and 7.1% of chickens, respectively, which are lower rates than those observed for the reference strain GA (85.7 and 35.7%). The commercially available CVI988 vaccine provided complete protection against LCY and GA (100%). These results showed that the isolate exhibited lower pathogenicity in SPF chickens. This study revealed that a novel pattern of LTR inserts was found in the strain LCY and that the strain was of low virulence. The present work expands the available genetic information for GaHV-2 and will be useful for the control of MD in China.

Integrin αvß1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or ß1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and ß1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvß1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV.

Molecular and pathogenicity characterization of Gallid herpesvirus 2 newly isolated in China from 2009 to 2013.

During the course of our continuous surveillance of Gallid herpesvirus 2 (GaHV-2), 44 isolates were obtained from GaHV-2-positive chickens of different flocks in China from 2009 to 2013. The meq gene, considered as a major GaHV-2 oncogene, was sequenced and was found to contain an open reading frame of 1020 nucleotides encoding a 339 amino acid (aa) polypeptide in all isolates. Compared with the GaHV-2 GA strain, the meq genes in 15.9 % (7/44) of the isolates analyzed in this study contained an aa substitution mutation at position 88 (A to T) of which is the first report. The main characteristics of Chinese GaHV-2 isolates meq genes included the substitutions K77E, D80Y, V115A, T139A, P176R, and P217A, and the aa substitution frequency at positions 139 and 176 showed an increase. To test the pathogenicity of the isolates, a pathogenicity study and a vaccination-challenge test were performed on three selected isolates (ZY/1203, WC/1203, and WC/1110) and reference strain GA. The results showed that the three isolates induced gross Marek's disease (MD) lesions in 95.0-100 % cases, which was a higher rate than that obtained for strain GA (82.4 %). Three isolates induced mortality in 10-21.1 % of specific-pathogen-free chickens, which was similar to results with strain GA (23.5 %). The commercially available CVI988 vaccine induced lower protective indices (PIs) against ZY/1203 (82.4) and WC/1110 (83.3) as compared to those against WC/1203 (100) and GA (100). These results showed an evolving trend in the meq genes of the isolates; three isolates exhibited higher morbidity as compared to the reference strain and the vaccine induced lower PIs against two isolates as compared to that against the reference strain.

Isolation and full-genome sequence of two reticuloendotheliosis virus strains from mixed infections with Marek's disease virus in China.

Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China.

Pathogenic characteristics of Marek's disease virus field strains prevalent in China and the effectiveness of existing vaccines against them.

The virulence of Marek's disease virus (MDV) is continuously evolving, and more virulent MDV pathotypes are emerging, thereby reducing the effectiveness of the existing vaccines. In this study, feather pulps were collected from diseased chickens in commercial chicken flocks in China that presented significant MD visceral tumors in 2011 and were inoculated into a monolayer of duck embryo fibroblasts (DEFs). Three field isolates of MDV were obtained by plaque cloning and identified as MDV via PCR and designated strains LCC, LLY, and LTS. Unvaccinated and CVI988 vaccine-vaccinated specific pathogen-free chickens were challenged at 7 days post vaccination (dpv) with 1000 plaque forming units of each of the respective MDV isolates. These strains induced gross MD lesions in all (100%) of the unvaccinated chickens, and the mortality rates of the unvaccinated chickens were 42.9%, 46.7%, and 23.1% by 60 days post challenge (dpc), respectively. The CVI988 vaccine induced protective indices (PIs) of 85.7, 92.3, and 66.7, respectively. These results showed that the pathogenic characteristics of the Chinese isolates were diverse and that vaccine CVI988 provided different levels of protection against them. These data indicated that the existence of variant MDV strains was a possible reason of immunity failure in China.

Antigenic analysis of monoclonal antibodies against different epitopes of σB protein of avian reovirus.

BACKGROUND: Avian reovirus (ARV) causes arthritis, tenosynovitis, runting-stunting syndrome (RSS), malabsorption syndrome (MAS) and immunosuppression in chickens. σB is one of the major structural proteins of ARV, which is able to induce group-specific antibodies against the virus. METHODS AND RESULTS: The present study described the identification of two linear B-cell epitopes in ARV σB through expressing a set of partially overlapping and consecutive truncated peptides spanning σB screened with two monoclonal antibodies (mAbs) 1F4 and 1H3-1.The data indicated that (21)KTPACW(26) (epitope A) and (32)WDTVTFH(38) (epitope B) were minimal determinants of the linear B cell epitopes. Antibodies present in the serum of ARV-positive chickens recognized the minimal linear epitopes in Western blot analyses. By sequence alignment analysis, we determined that the epitopes A and B were not conserved among ARV, duck reovirus (DRV) and turkey reovirus (TRV) strains. Western blot assays, confirmed that epitopes A and B were ARV-specific epitopes, and they could not react with the corresponding peptides of DRV and TRV. CONCLUSIONS AND SIGNIFICANCE: We identified (21)KTPACW(26) and (32)WDTVTFH(38) as σB -specific epitopes recognized by mAbs 1F4 and 1H3-1, respectively. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against ARV groups.

Identification of a linear B-Cell epitope on avian reovirus protein sigmaC.

SigmaC (σC) protein, which mediates virus attachment to target cells, is the most variable proteins of avian reovirus (ARV). It is responsible for inducing protective antibody immune responses in animals. To understand the antigenic determinants of σC protein, a set of partially overlapping and consecutive peptides spanning σC were expressed and then screened with the monoclonal antibody (mAb) 2B5 directed against σC. The mAb 2B5 recognized peptides with the σC motif (45)ELLHRSISDISTTV(58). Further identification of the displayed B-cell epitope was conducted with a set of truncated peptides expressed as GST fusion proteins. The Western blot and ELISA results indicated that (45)ELLHRSISDI(54) was the minimal determinant of the linear B-cell epitope. Using sequences analysis, we found that this epitope was not a common motif shared among the other members of the ARV and DRV groups. Furthermore, cross reactivity analysis showed that the associated coding motif of other ARV and DRV groups was not recognized by 2B5. These data suggested that (45)ELLHRSISDI(54) was a type-specific linear B-cell epitope of avian reovirus. The results in this study may have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against ARV, which is prevalent in China.

[Clinical and coronary angiographic features of young women with acute myocardial infarction].

OBJECTIVE: To observe the clinical and coronary angiographic features of young women (≤ 44 years) with acute myocardial infarction (AMI). METHODS: Clinic and coronary angiographic features were compared among 57 young women with AMI and 60 non-CHD young women, 78 young men with AMI and 80 elderly women with AMI were included, all patients were admitted to hospital from June 2003 to December 2010 and underwent coronary angiography. Body mass index (BMI), smoking history, familial history of early coronary artery disease, essential hypertension, type 2 diabetes mellitus, hyperlipidemia, serum uric acid (UA), menopause, lipids, total bilirubin (TBil), direct bilirubin (DBil), hemoglobin (Hb), red cell distribution width (RDW), fasting blood glucose (FBG) were measured. RESULTS: (1) Prevalence of essential hypertension, hypertriglyceridemia and levels of fasting blood glucose, triglyceride, serum uric acid were significantly higher while high density lipoprotein cholesterol, and hemoglobin were significantly lower in young women with AMI group than in age-matched non-CHD control group (all P < 0.05). (2) Logistic regression analysis showed that essential hypertension (OR = 23.187), lower hemoglobin level (OR = 1.010) and uric acid (OR = 1.040) were independent risk factors for young women with AMI (all P < 0.05). (3) Coronary angiography revealed normal coronary finding in 6 cases, single-vessel disease in 35 cases (26 cases with left anterior descending artery disease) and two-vessel disease in 12 patients. The ratio of single vessel disease involved left anterior descending artery in young women was higher than that of young men and old women with AMI (all P < 0.05). CONCLUSION: Essential hypertension, lower hemoglobin and uric acid are risk factors of young women with AMI. Single vessel coronary disease is the most common coronary angiographic feature of young women with AMI.

Tacrolimus-related hypertrophic cardiomyopathy in an adult cardiac transplant patient.

Left ventricular hypertrophy associated with the use of tacrolimus is a rare complication of solid organ transplantation in adult recipients. We present a cardiac transplant recipient who developed severe concentric left ventricular hypertrophy with congestive heart failure related to myocardial hypertrophy on tacrolimus. Hypertrophy improved when the drug was discontinued and replaced with sirolimus.

[Expression and identification of sigmaC gene of avian reovirus by baculovirus expression system].

Recombinant baculovirus containing sigmaC gene of Avian reovirus was constructed using Bac-To-Bac Baculovirus expression system, and recombinant sigmaC protein was expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, sigmaC gene of Avian reovirus was cloned and inserted into donor plasmid pFastBacHTA to obtain recombinant donor plasmid pFsigmaC. Plasmid pFsigmaC was transformed into E. coli DH10Bac for integration into bacmid vector and the recombinant bacmid plasmid BacmidsigmaC was obtained. Recombinant baculovirus rBacsigmaC was obtained by transfection of the sf9 cells with BacmidsigmaC. Western blot and indirect immunofluorescence assay (IFA) were carried and the results showed that the recombinant sigmaC protein with 37 kDa molecular weight was expressed successfully.