Development and application of SYBR Green â real-time quantitative reverse transcription PCR assay for detection of swine Getah virus.

Getah virus (GETV), a mosquito-borne virus belonging to the Alphavirus genus of family Togaviridae, has become increasingly problematic, which poses a huge threat to the safety of animals and public health. In order to detect GETV quickly and accurately, we have developed a SYBR Green I real-time quantitative reverse transcription PCR (RT-qPCR) assay for GETV with the detection limit of 66 copies/µL, excellent correlation coefficient (R2) of 0.9975, and amplification efficiency (E) of 98.90%, the target selected was the non-structural protein 3 of GETV. The sensitivity of it was higher than that of ordinary RT-PCR by 1000 folds, and the inter-assay and intra-assay CV values were all less than 0.99%. The newly developed RT-qPCR assay exhibited good sensitivity and reproducibility, which will provide technical support for the reliable and specific rapid diagnosis, and quantitative analysis of GETV infection.

The role of GtxA during Gallibacterium anatis infection of primary chicken oviduct epithelial cells.

Gallibacterium anatis (G. anatis), one of the major pathogens causing reproductive tract disorders in laying hens, leads to a reduction in egg production and increased mortality, caused by either single or mixed infections with other pathogens. As a specific virulence factor of G. anatis, the role of GtxA in layers' salpingitis remains unclear. In this study, we explored the effect of GtxA on G. anatis infection by comparing wild strain Yu-PDS-RZ-1-SLG (RZ) and its GtxA deleted counterpart RZΔgtxA in primary chicken oviduct epithelial cells (COEC). Their adherence, invasion, cytoxicity, and ability to induce apoptosis and and cytokine secretion were evaluated and the cytotoxicity and cytokine secretion of the recombinant GtxA protein and its N-terminal adenylate cyclase and C-terminal RTX hemolysin domain were also analyzed. We found that the adhesion ability of RZΔgtxA was significantly lower than that of parental strain RZ, and its toxicity to COEC was weakened; Meanwhile, apoptosis was inhibited and the expression of IL-6, IL-2, TNF-α and IFN-γ were dramatically reduced in COEC infected by RZΔgtxA. In contrast, the recombinant protein GtxA inhibited the proliferation of oviduct cells and induced obvious cytotoxicity, and the expression of IL-6, TNF-α and IFN-γ were up-regulated in COEC interacted with recombinant proteins. Our study indicates that GtxA promotes G. anatis adherence to cells, changes cells permeability and expression of inflammatory factors, resulting in cell damage and apoptosis.

Antibiotic Resistance Profiles of Haemophilus influenzae Isolates from Children in 2016: A Multicenter Study in China.

BACKGROUND AND OBJECTIVE: Haemophilus influenzae (HI) is a common cause of community-acquired pneumonia in children. In many countries, HI strains are increasingly resistant to ampicillin and other commonly prescribed antibiotics, posing a challenge for effective clinical treatment. This study was undertaken to determine the antibiotic resistance profiles of HI isolates from Chinese children and to provide guidelines for clinical treatment. METHODS: Our Infectious Disease Surveillance of Pediatrics (ISPED) collaboration group includes six children's hospitals in different regions of China. The same protocols and guidelines were used by all collaborators for the culture and identification of HI. The Kirby-Bauer method was used to test antibiotic susceptibility, and a cefinase disc was used to detect ß-lactamase activity. RESULTS: We isolated 2073 HI strains in 2016: 83.9% from the respiratory tract, 11.1% from vaginal secretions, and 0.5% from blood. Patients with respiratory isolates were significantly younger than nonrespiratory patients (P < 0.001). Of all 2073 strains, 50.3% were positive for ß-lactamase and 58.1% were resistant to ampicillin; 9.3% were ß-lactamase-negative and ampicillin-resistant. The resistance rates of the HI isolates to trimethoprim-sulfamethoxazole, azithromycin, cefuroxime, ampicillin-sulbactam, cefotaxime, and meropenem were 71.1%, 32.0%, 31.2%, 17.6%, 5.9%, and 0.2%, respectively. CONCLUSIONS: More than half of the HI strains isolated from Chinese children were resistant to ampicillin, primarily due to the production of ß-lactamase. Cefotaxime and other third-generation cephalosporins could be the first choice for the treatment of ampicillin-resistant HI infections.

[Clinical characteristics and drug sensitivity in children with invasive pneumococcal disease: a multicenter study].

OBJECTIVE: To study the clinical characteristics, drug sensitivity of isolated strains, and risk factors of drug resistance in children with invasive pneumococcal disease (IPD). METHODS: The clinical characteristics and drug sensitivity of the isolated strains of 246 hospitalized children with IPD in nine grade A tertiary children's hospitals from January 2016 to June 2018 were analyzed. RESULTS: Of the 246 children with IPD, there were 122 males and 124 females. Their ages ranged from 1 day to 14 years, and among them, 68 (27.6%) patients were less than 1 year old, 54 (22.0%) patients were 1 to 2 years old, 97 (39.4%) patients were 2 to 5 years old, and 27 (11.0%) patients were 5 to 14 years old. Pneumonia with sepsis was the most common infection type (58.5%, 144/246), followed by bloodstream infection without focus (19.9%, 49/246) and meningitis (15.0%, 37/246). Forty-nine (19.9%) patients had underlying diseases, and 160 (65.0%) had various risk factors for drug resistance. The isolated Streptococcus pneumoniae strains were 100% sensitive to vancomycin, linezolid, moxifloxacin, and levofloxacin, 90% sensitive to ertapenem, ofloxacin, and ceftriaxone, but had a low sensitivity to erythromycin (4.2%), clindamycin (7.9%), and tetracycline (6.3%). CONCLUSIONS: IPD is more common in children under 5 years old, especially in those under 2 years old. Some children with IPD have underlying diseases, and most of the patients have various risk factors for drug resistance. Pneumonia with sepsis is the most common infection type. The isolated Streptococcus pneumoniae strains are highly sensitive to vancomycin, linezolid, moxifloxacin, levofloxacin, ertapenem, and ceftriaxone in children with IPD.

Antibiotic resistance profiles and multidrug resistance patterns of Streptococcus pneumoniae in pediatrics: A multicenter retrospective study in mainland China.

Emergent resistance to antibiotics among Streptococcus pneumoniae isolates is a severe problem worldwide. Antibiotic resistance profiles for S pneumoniae isolates identified from pediatric patients in mainland China remains to be established.The clinical features, antimicrobial resistance, and multidrug resistance patterns of S pneumoniae were retrospectively analyzed at 10 children's hospitals in mainland China in 2016.Among the collected 6132 S pneumoniae isolates, pneumococcal diseases mainly occurred in children younger than 5 years old (85.1%). The resistance rate of S pneumoniae to clindamycin, erythromycin, tetracycline, and trimethoprim/sulfamethoxazole was 95.8%, 95.2%, 93.6%, and 66.7%, respectively. The resistance rates of S pneumoniae to penicillin were 86.9% and 1.4% in non-meningitis and meningitis isolates, while the proportions of ceftriaxone resistance were 8.2% and 18.1%, respectively. Pneumococcal conjugate vaccine was administered to only 4.1% of patients. Penicillin and ceftriaxone resistance, underling diseases, antibiotic resistant risk factors, and poor prognosis appeared more frequently in invasive pneumococcal diseases. The incidence of multidrug resistance (MDR) was 46.1% in patients with invasive pneumococcal disease which was more than in patients with non-invasive pneumococcal disease (18.3%). Patients with invasive pneumococcal disease usually have several MDR coexistence.S pneumoniae isolates showed high resistance to common antibiotics in mainland China. Penicillin and ceftriaxone resistance rate of invasive streptococcal pneumonia patients were significantly higher than that of non-invasive S pneumoniae patients. Alarmingly, 46.1% of invasive clinical isolates were multidrug resistant, so it is important to continued monitor the resistance of S pneumoniae when protein conjugate vaccine (PCV13) is coming in mainland China.

A multi-center clinical investigation on invasive Streptococcus pyogenes infection in China, 2010-2017.

BACKGROUND: Invasive S. pyogenes diseases are uncommon, serious infections with high case fatality rates (CFR). There are few publications on this subject in the field of pediatrics. This study aimed at characterizing clinical and laboratory aspects of this disease in Chinese children. PATIENTS AND METHODS: A retrospective study was conducted and pediatric in-patients with S. pyogenes infection identified by cultures from normally sterile sites were included, who were diagnosed and treated in 9 tertiary hospitals during 2010-2017. RESULTS: A total of 66 cases were identified, in which 37 (56.1%) were male. The median age of these patients, including 11 neonates, was 3.0 y. Fifty-nine (89.4%) isolates were determined from blood. Fever was the major symptom (60/66, 90.9%) and sepsis was the most frequent presentation (64/66, 97.0%, including 42.4% with skin or soft tissue infections and 25.8% with pneumonia. The mean duration of the chief complaint was (3.8 ± 3.2) d. Only 18 (27.3%) patients had been given antibiotics prior to the hospitalization. Among all patients, 15 (22.7%) developed streptococcal toxin shock syndrome (STSS). No S. pyogenes strain was resistant to penicillin, ceftriaxone, or vancomycin, while 88.9% (56/63) and 81.4% (48/59) of the tested isolates were resistant to clindamycin and erythromycin respectively. Most of the patients were treated with ß-lactams antibiotics and 36.4% had been treated with meropenem or imipenem. Thirteen (19.7%) cases died from infection, in which 9 (13.6%) had complication with STSS. CONCLUSIONS: Invasive S. pyogenes infections often developed from skin or soft tissue infection and STSS was the main cause of death in Chinese children. Ongoing surveillance is required to gain a greater understanding of this disease.

Antibiotic usage in Chinese children: a point prevalence survey.

BACKGROUND: Children as a population have high antimicrobial prescribing rates which may lead to high resistance of bacteria according to data from some single-center surveys of antibiotic prescribing rates in China. The acquirement of baseline data of antibiotic prescribing is the basis of developing intervention strategies on inappropriate antimicrobial prescriptions. Few studies show clearly the pattern and detailed information on classes of antibiotics and distribution of indications of antibiotic prescriptions in children in China. This study aims to assess the antibiotic prescribing patterns among children and neonates hospitalized in 18 hospitals in China. METHODS: A 24-hour point prevalence survey on antimicrobial prescribing was conducted in hospitalized neonates and children in China from December 1st, 2016 to February 28th, 2017. Information on the antibiotic use of patients under 18 years of age who were administered one or more on-going antibiotics in the selected wards over a 24-hour period was collected. These data were submitted to the GARPEC (Global Antimicrobial Resistance, Prescribing and Efficacy in Children and Neonates) web-based application ( https://pidrg-database.sgul.ac.uk/redcap/ ). For statistical analysis, Microsoft Excel 2007 and SPSS 22.0 were used. RESULTS: The antibiotic data were collected in 35 wards in 18 hospitals from 9 provinces. In total, 67.76% (975/1439) of the patients (n = 1439) were given at least one antibiotic, including 58.1% (173/298) of neonates (n = 298) and 70.3% (802/1141) of children (n = 1141). In neonates, the three most frequently prescribed antibiotics were third-generation cephalosporins (41.7%), penicillins plus enzyme inhibitor (23.8%), and carbapenems (11.2%). In children, the three most frequently prescribed antibiotics were third-generation cephalosporins (35.5%), macrolides (23.2%), and penicillins plus enzyme inhibitors (15.9%). The most common indication for antibiotics was proven or probable bacterial lower respiratory tract infection (30.9% in neonates and 66.6% in children). CONCLUSIONS: Antibiotics are commonly prescribed in the Chinese children population. It is likely that the third-generation cephalosporins and macrolides are currently overused in Chinese children. Efforts must be made to ensure safe and appropriate antibiotic prescribing to reduce and prevent the future development of antibiotic resistance.

In vitro adherence and invasion of primary chicken oviduct epithelial cells by Gallibacterium anatis.

Gallibacterium anatis (G. anatis) has been suggested to have a causal role in salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production and increased mortality worldwide. Adherence and invasion of epithelial cells are thought to play a role in the pathogenesis of G. anatis infection. The purpose of this article was to study adherence and invasion of G. anatis using two G. anatis strains of different virulence (Yu-PDS-RZ-1-SLG strain, highly virulent and F149T strain, non-virulent) via infection of the primary chicken oviduct epithelial cells (PCOECs).The results showed that Yu-PDS-RZ-1 -SLG strain was able to attach to PCOECs at higher levels than that of F149T strain, but no invasion was observed with either strain. However, cell debris and cell apoptosis were observed after being exposed to G. anatis Yu-PDS-RZ-1-SLG for 90min, whereas G. anatis F149T did not cause cell damage, and adherence was prevented by trypsin treatment of bacterial cells. Cytokines were detected by ELISA after infection, and the results showed that the expression of IL-6, TNF-α, and IFN-γ levels was higher in virulent strain infection than that of the avirulent group. Results also indicated that the highly virulent strain G. anatis displayed an increased level of adherence. Changes in cytokine profiles in this study suggested that the production of cytokines might influence the microenvironment of oviduct and promote adherence, serving as a possible mechanism inducing cell damage.

A case report of hepatic abscesses with soft tissue infection caused by methicillin resistant Staphylococcus aureus in a young child.

RATIONALE: Pyogenic hepatic abscess in children is a rare clinical condition. Hepatic abscesses caused by methicillin resistant Staphylococcus aureus are extremely rare. PATIENT CONCERNS: A 6-year-old boy was referred to a tertiary children's hospital for a 6-day history of right lower abdominal pain and fever. Radiographic findings showed hepatic abscesses and soft tissue abscesses around the left femur. DIAGNOSES: Bacteriology of blood, hepatic abscesses, and soft tissue abscesses showed methicillin resistant Staphylococcus aureus. INTERVENTIONS: Our patient received adequate drainage of MRSA abscesses and a complete course of antibiotics. OUTCOMES: The hepatic abscesses were healed and no recurrence has been founded until now. LESSONS: This report describes an extremely rare case of hepatic abscesses with soft tissue infection caused by MRSA. Adequate drainage and appropriate systemic antibiotics should be considered as a standard treatment of MRSA abscesses in order to reduce the mortality rate and improve the quality of life.

Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry.

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

Complete Genome Sequence of a Novel Porcine Reproductive and Respiratory Syndrome Virus That Emerged in China.

A novel porcine reproductive and respiratory syndrome virus (PRRSV) strain with 393 nucleotide deletions in the nonstructural protein 2 (Nsp2) region was examined in this study. Results will help improve our understanding of the epidemiology and genetic diversity of the North American-type PRRSV in China.

Successful reduction in central line-associated bloodstream infections in a Chinese neonatal intensive care unit.

BACKGROUND: Few data are available on central-line associated bloodstream infections (CLABSIs) in Chinese neonatal intensive care units (NICUs). The aims of this study were to characterize CLABSIs among neonates in a Chinese NICU and evaluate the impact of a multifaceted evidence-based practice for improving quality program to decrease CLABSI. METHODS: We conducted a prospective before-after intervention study with a 1-year follow-up among patients with central lines at the NICU of the Children's Hospital of Fudan University between January 2008 and December 2010. The study was conducted in 3 phases: before, during, and after the intervention. A multifaceted infection control program was introduced in phase 2 with successive surveillance. CLABSIs were prospectively monitored and compared. RESULTS: A total of 171 patients with central lines (CLs) were observed; 29 of them developed CLABSI corresponding to 7.35 per 1,000 catheter days, with a CL utilization ratio of 37.9%. Overall CLABSI rate decreased gradually from 16.7 per 1,000 CL days in phase 1 to 7.6 per 1,000 CL days in phase 2 (P = .08) to 5.2 per 1,000 CL days in phase 3 (P < .01). Gram-negative bacterium (54.5%) was the predominant pathogen in CLABSIs. CONCLUSION: A multifaceted infection control program is effective in reducing the CLABSI rate among neonates. Such interventions could be extended to other resource-limited countries.

[Dynamic distribution of the avian infectious bronchitis virus isolate strain Jin-13 in SPF chickens].

This study aimed to understand the dynamic distribution of infectious bronchitis virus (IBV) Jin-13 strain in SPF chickens. Ninety-day-old SPF chickens were inoculated with Jin-13, a virulent strain, and dissected at day 1, 4, 7, 10, 14, 21, 28 or 35 post-inoculation (dpi). Samples of heart, liver, spleen, lung, trachea, kidney and duodenum were collected and the N gene was detected by Sybr Green I real-time quantitative RT-PCR assays. The established method had a good linear correlation from 7.77 x 10(8) to 10(0) copies/microL. SPF chickens developed typical clinical signs of IBV at the 4th dpi, and the IBV viral concentration of tissues and organs gradually increased with a peak of up to 7.13 x 10(4) copies/microL. The viral concentration of most organs decreased by the 10th dpi, but those of the kidney, trachea and lung remained positive for IBV at 28 dpi and the heart was still positive for IBV at > 35 dpi. The results of this study, showed that the Jin-13 strain can cause prolonged virus excertion in chickens with severe renal damage.

[Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark.

[Investigation of etiology of massive infection with porcine pseudorabies virus in Henan and neighboring Provinces].

In early 2011, the serious outbreak of porcine pseudorabies virus (PRV) infection suddenly recurred in Henan and neighboring Provinces. To investigate the etiology of massive infection with PRV, 16 800 serum samples, 905 porcine epidemic diarrhea virus (PEDV) back-feeding tissues, and 56 PR gene deleted live vaccines were colleted from January 2011 to May 2013 to detect PRV field infection using a PRV gE antibody test kit. The gE and TK genes of 11 new epidemic PRV strains were sequenced by PCR, and their molecular characteristics were analyzed. Moreover, virus titer determination, protective test against PRV, and vaccine potency testing were performed. The results showed that the detection rate of PRV field infection-positive pig farms was 68.06%, and the overall positive rate of PRV field infection in serum was 38.47%; the positive rates in breeding sows, breeding boars, reserve pigs, and commercial pigs were 40.12%, 30.88%, 54.67%, and 26.52%, respectively. The new epidemic strains were in the same evolutionary branch and belonged to the virulent strain group. Compared with the classical PRV strain, the virulence of new epidemic strains changed a little. The length of gE gene was 1 787 bp, and the length of TK gene was 963 bp. The nucleotide homologies of gE and TK genes to Chinese reference strains were 98.2%-99.8% and 98.90%-99.6%, respectively, and the amino acid homologies were 97.1%-99.8% and 97.5%-99.4%, respectively. Commercial vaccine had a 100% protective effect against the new epidemic strains. The positive rate of PRV field infection was 0% in vaccine and 40.44% in back-feeding tissues. The results confirmed that PRV field infection rates were rising sharply among pigs in Henan and neighboring Provinces after 2011. The main virulence genes of new epidemic PRV strains did not change significantly over the years. PR gene deleted live vaccines had no PRV field infection and could completely resist the attack of new strains. The virus carriage of breeding boars and reserve pigs and the serious PRV field infection in PEDV back-feeding tissues were the main causative factors for massive infection with PRV and epidemic outbreak in Henan and neighboring Provinces from 2011 to 2013.