Role of noscomial infection informatics surveillance system in the prevention and control of multidrug-resistant organisms infections

OBJECTIVE: To analyze the role of nosocomial infection informatics surveillance system in the prevention and control of multidrug-resistant organisms(MDROs) infections. METHODS: The First Affiliated Hospital of Guangdong Pharmaceutical University was selected as the study subjects, which had adopted the nosocomial infection informatics surveillance system since Jan.2020. The period of Jan.to Dec.2020 were regarded as the study period, and Jan.to Dec.2019 were regarded as the control period. The situation of nosocomial infection and MDROs infections in the two periods were retrospectively analyzed. RESULTS: The incidence of nosocomial infections and underreporting of nosocomial infection cases in this hospital during the study period were 2.52%(1 325/52 624) and 1.74%(23/1 325), respectively, and the incidences of ventilator associated pneumonia(VAP), catheter related bloodstream infection(CRBSI), catheter related urinary tract infection(CAUTI)were 4.10(31/7 568), 2.11(14/6 634), and 2.50(25/9 993) respectively, which were lower than those during the control period(P< 0.05). The positive rate of pathogenic examination in the hospital during the study period was 77.95%(1 269/1 628), which was higher than that during the control period(P<0.05), the overall detection rate of MDROs was 15.77%(206/1 306), the detection rates of MDROs in Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus epidermidis, Pseudomonas aeruginosa and Staphylococcus aureus were lower than those during the control period(P<0.05). CONCLUSION: The development and application of the informatics technology-based surveillance system of nosocomial infection could effectively reduce the incidence of nosocomial infections and device related infections, decrease the under-reporting of infection cases, and also reduce the detection rate of MDROs as well as the proportion of MDROs detected in common pathogenic species.

Face masks as sources of cross-contamination during food preparation

Amid the COVID-19 pandemic, mask-wearing has become a common practice in the foodservice industry to prevent the spread of respiratory diseases. Like kitchen utensils, a mask may serve as a vehicle for cross-contamination of pathogens during food handling. The objective of this study was to quantify cross-contamination between tasks of handling contaminated chicken and chopping lettuce. Chicken breasts were inoculated with a high or a low level of nonpathogenic Escherichia coli surrogates (ca. 6 or 4 log CFU/ml) and sliced for 1, 5, or 10 min. During slicing, duplicate, single-use medical masks were touched each minute. One mask was immediately sampled, but the second mask was used to contaminate lettuce by touching the mask each minute while chopping the lettuce for 5 min. E. coli were enumerated from the second mask and lettuce. Masks touched while slicing both high- and low-inoculated chicken showed significant contamination (0.8-4.9 log CFU/cm2) after each slicing scenario of 1, 5, or 10 min (P > 0.05). Lettuce was significantly contaminated regardless of inoculation level (1.0-3.2 log CFU/g). Slicing time was a significant factor in some cases (P < 0.05), whereas inoculation level was not (P > 0.05). Data indicate masks can be a source of cross-contamination if not replaced appropriately.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate. Materials and Methods: B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Results: The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E. coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000. Conclusion: According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.

Establishment and applications of a duplex quantitative real-time RT-PCR assay for simultaneous detection of bovine rhinitis A virus and bovine rhinitis B virus

Bovine rhinitis virus (BRV) is an important pathogen responsible for the bovine respiratory disease complex (BRDC) and can be divided into two genotypes (BRAV and BRBV). To establish a duplex quantitative real-time RT-PCR assay for simultaneous detection of BRAV and BRBV, specific primers and TaqMan probes targeting the 5'NTR of BRAV and 3'NTR of BRBV were designed. A duplex quantitative real- time RT- PCR assay for simultaneous detecting BRAV and BRBV was preliminarily established by optimizing reaction conditions for each step. The assay specifically detects BRAV and BRBV, and no crossreaction with other common bovine respiratory pathogens, including IDV, BCoV, BVDV-1, BRSV, BPIV-3, BAdV-3, mycoplasma bovis, Pasteurella multocida, Mannheimia haemolytica, Escherichia coli, and Salmonella, was observed. In addition, the sensitivity test showed that the detection limits of this assay were 3.2x101 copies/L for both BRAV and BRBV plasmid standards. Besides, the repeatability test showed that the variation coefficients of this assay were less than 0.05 from both lot-to-lot and intra-lot. These results showed that the assay has high specificity, extreme sensitivity, and good repeatability. Moreover, a total of 43 nasal swabs of BRDC cattle were tested by our assay and four other quantitative real-time RT-PCR assays, including 3 BRAV assays and 4 BRBV assays. The results showed that the detection rates of our assay were 32.56%(14/43) for BRAV and 30.23%(13/43) for BRBV, and the detection rates of other quantitative real-time RT-PCR assays were 0(0/43), 2.33%(1/43), 23.26%(10/43) for BRAV and 27.91% (12/43), 27.91%(12/43), 27.91%(12/43), 27.91%(12/43) for BRBV, indicating that our assay has a more substantial detection capability than other assays. This study firstly established a duplex quantitative real-time RT-PCR assay for simultaneous detection of BRAV and BRBV, and the assay exhibited high specificity, sensitivity, and stability. Moreover, the study firstly confirmed the existence of BRAV in China, contributing to the prevention and control of BRDC.

Distribution and drug resistance analysis of pathogenic bacteria in a hospital blood culture, 2018-2022

Objective: To analyze the distribution and drug resistance of pathogenic bacteria in blood culture specimens of patients with bloodstream infections before and after COVID-19 (2018-2019 and 2020-2021), and to provide scientific basis and reference for rational treatment and effective control of bloodstream infections in the post-epidemic period. Methods: Blood culture specimens were collected from patients in Zhongnan Hospital of Wuhan University in the two years before and after the COVID-19 outbreak (2018-2021). The Automated Blood Culture Systems were used to perform blood culture on blood specimens sent for clinical inspection, and the Vitek MS automatic bacterial identification mass spectrometer was used for strain identification and the Vitek 2 automatic bacterial drug susceptibility analyzer was used for drug susceptibility testing and drug resistance analysis. Results: Blood culture specimens were performed on 28 736 patients with suspected bloodstream infection submitted for inspection from January 2018 to December 2019, and a total of 2 181 strains of pathogenic bacteria were detected after removing duplicate strains, with a positive rate of 7.69%, including 1 046 strains of Gram-negative bacteria, accounting for 47.96%. From January 2020 to December 2021, blood culture specimens from 26 083 patients with suspected bloodstream infection were submitted for inspection, and a total of 2 111 strains of pathogenic bacteria were detected after excluding duplicate strains, with a positive rate of 8.09%, including 1 000 strains of Gram-negative bacteria accounted for 47.37%. The drug resistance of Klebsiella pneumoniae was relatively serious, and the sensitivity rate to ertapenem, polymyxin B and tigecycline was more than 90%. The main non-fermentative bacteria Acinetobacter baumannii was more than 50% sensitive to piperacillin/tazobactam, amikacin and polymyxin B. The sensitivity rates of Pseudomonas aeruginosa to piperacillin/tazobactam, ceftazidime, cefepime, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, piperacillin and meropenem were more than 50%. Conclusions: In the two years before and after COVID-19, there are many types of pathogenic bacteria in bloodstream infection, but the distribution do not differ significantly. The pathogens of bloodstream infection are mainly distributed in ICU, hepatobiliary research institute, and nephrology department. Among them, Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii are the main ones, and different pathogens showed great differences in drug resistance.

Molecular Detection and Genetic Characterization of Bovine Kobuvirus (BKV) in Diarrhoeic Calves in a Central Italy Herd

Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.

In silico identification and validation of phenolic lipids as potential inhibitor against bacterial and viral strains.

The recurrence of coronavirus disease and bacterial resistant strains has drawn attention to naturally occurring bioactive molecules that can demonstrate broad-spectrum efficacy against bacteria as well as viral strains. The drug-like abilities of naturally available "anacardic acids" (AA) and their derivatives against different bacterial and viral protein targets through in-silico tools were explored. Three viral protein targets [P DB: 6Y2E (SARS-CoV-2), 1AT3 (Herpes) and 2VSM (Nipah)] and four bacterial protein targets [P DB: 2VF5 (Escherichia coli), 2VEG (Streptococcus pneumoniae), 1JIJ (Staphylococcus aureus) and 1KZN (E. coli)] were selected to evaluate the activity of bioactive AA molecules. The potential ability to inhibit the progression of microbes has been discussed based on the structure, functionality and interaction ability of these molecules on the selected protein targets for multi-disease remediation. The number of interactions, full-fitness value and energy of the ligand-target system were determined from the docked structure in SwissDock and Autodock Vina. In order to compare the efficacy of these active derivatives to that of commonly used drugs against bacteria and viruses, a few of the selected molecules were subjected to 100 ns long MD simulations. It was found that the phenolic groups and alkyl chains of AA derivatives are more likely to bind with microbial targets, that could be responsible for the improved activity against these targets. The results suggest that the proposed AA derivatives have demonstrated potential to become active drug ingredients against microbial protein targets. Further, experimental investigations are essential for clinical verification of the drug-like abilities of AA derivatives.Communicated by Ramaswamy H. Sarma.

Single center retrospective study on the distribution of pathogens in elderly patients with acute exacerbation of chronic obstructive pulmonary disease

Objective: To explore the distribution and correlation of pathogens in the elderly patients with AECOPD, so as to guide the rational use of antibiotics and hormones in clinic. Methods: A total of 111 patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) admitted to Nanjing First Hospital from January 2019 to January 2020 were retrospectively analyzed. The basic data such as eosinophil, neutrophil and lymphocyte count, the levels of C-reactive protein(CRP) and erythrocyte sedimentation rate (ESR)in blood routine examination were collected. Further, the pathogens were qualified by sputum fluorescence quantitative polymerase chain reaction, and the pathogens distribution was analyzed. Results: The level of ESR and the ratio of cardiovascular diseases showed significant differences between the pathogen-positive group and pathogen-negative group. In this study, the top five pathogens in AECOPD patients were EB virus (21.6%), Haemophilus influenzae (19.8%), Streptococcus pneumoniae (17.1%), herpes simplex virus(14.4%), influenza A virus(14.4%). The detection rate of influenza A virus was correlated with influenza B virus and Aspergillus (P < 0.05);The detection rate of respiratory syncytial virus was correlated with Candida, Moraxella catarrholis, Streptococcus pneumoniae and Haemophilus influenzae (P < 0.05);The detection rate of Escherichia coli was correlated with rhinovirus, adenovirus, Klebsiella pneumoniae and Acinetobacter baumannii (P < 0.05);The detection rate of Candida was correlated with that of Moraxella catarrholis and Pseudomonas aeruginosa(P<0.05);The detection rate of human coronavirus was correlated with Haemophilus influenzae, herpes simplex virus and Streptococcus pneumoniae(P < 0.05). Conclusions: AECOPD are mostly induced by different pathogens, especially mixed infection of bacteria and virus. It is helpful to guide the rational use of antibiotics by analyzing the etiological characteristics in the elderly patients with AECOPD.

Development of indigestible IgA antibody for restoring microbiome balance

Dysbiosis, especially in the gut plays a crucial role in the pathogenesis of a wide variety of diseases, including inflammatory bowel disease, colorectal cancer, cardiovascular disease, obesity, diabetes and multiple sclerosis. At mucosal surfaces, mucosal polymeric immunoglobulin A（IgA）antibodies are known to be important to regulate the gut microbiota as well as to exclude infection induced by pathogenic bacteria or virus such as influenza and SARS-CoV-2（severe acute respiratory syndrome coronavirus 2）. Since the 1970s, oral administration of IgA or IgG antibodies has been performed against infectious enteritis caused by pathogenic Escherichia coli or Clostridioides difficile. However, none of them has been successfully developed as an antibody drug up to now. Although IgA is well known to modulate the gut commensal microbiota, the therapeutic IgA drugs to treat dysbiosis has not been developed. Here, we discuss the advantages of therapeutic IgA antibodies.Alternate :抄録Dysbiosisは、健康な微生物叢と比較した微生物組成の変化であり、腸内微生物多様性の減少および微生物分類群の変化を特徴とする。腸内のdysbiosisはまた、炎症性腸疾患、結腸直腸がん、心血管疾患、肥満、糖尿病および多発性硬化症を含むさまざまな疾患の病因において重要な役割を果たすと提唱されている。腸の多量体免疫グロブリンA（IgA）抗体は、腸内微生物叢を調節するだけではなく、病原性細菌、インフルエンザやSARS-CoV-2（重症急性呼吸器症候群コロナウイルス2）などのウイルス感染を粘膜部位から排除するのに重要であることが、多くのエビデンスから示されている。1970年代以降、治療用IgAまたはIgGの経口投与試験が、主に病原性大腸菌またはディフィシル菌によって引き起こされる感染性腸炎を治療するために行われてきた。しかし、現在まで臨床応用として開発に成功したものはない。腸内病原体に対する防御機能に加えて、IgAは腸内共生微生物叢を調節して共生に導くことがよく知られているが、dysbiosisを治療するためのIgA治療薬の開発も進んでいない。本稿では、治療用IgA抗体の利点とその開発について議論する。

Efficacy evaluation of an air-assisted electrostatic disinfection device for the effective disinfection and sanitization against the spread of pathogenic infections

This research aims to check the chargeability of sodium hypochlorite and the efficacy evaluation of an air-assisted electrostatic disinfection device. Five different inanimate surfaces i.e., wood, glass, stainless steel, plastic and fabric were considered to examine the performance in terms of efficacy, survival time, off-target losses, spray coverage and the volume of disinfectant consumed. A significant charge-to-mass level of 2.43 mC/kg was achieved for sodium hypochlorite at an applied voltage of 2.0 kV, a liquid flowrate of 253 ml/min and applied air pressure of 4.0 bar. The experimental results found that 1000 mg/L of sodium hypochlorite concentration effectively eliminated Pseudomonas aeruginosa, Clostridium perfringens and Bacteriophage MS2 colonies. © 2023 Elsevier B.V.

Comorbidity of purulent meningitis with COVID‐19: A case report

To date, only a few cases of intracranial infection related to severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) were reported. Here we describe a case of coronavirus disease 2019 (COVID‐19) that was comorbid with purulent meningitis. A 62‐year‐old male patient was diagnosed with moderate COVID‐19 and had no fever or cough after treatment. However, he suffered from a head injury and experienced headache and fever immediately after the accident. Computed tomography (CT) of the brain showed bilateral frontal lobe contusion, subdural hematoma, and subarachnoid hemorrhage. In the following days, the patient suffered from recurrent fever, although chest CT did not show evidence of worsening of infection. Several lumbar punctures were made, confirming increased cerebrospinal fluid (CSF) pressure and karyocyte count. SARS‐CoV‐2 nucleic acid was not detected in CSF but revealed the presence of Escherichia coli. Thus, the patient was diagnosed with purulent meningitis, presumably caused by brain trauma or the immunologic dysfunction caused by COVID‐19, which was supported by the significant reduction of all kinds of immune cells. Since immunologic dysfunction is commonly presented in COVID‐19 patients, comorbidity with meningitis should be considered when a COVID‐19 patient presents with headache and fever. Lumbar punctures and CSF cultures may help in the diagnosis.

Quantitative Evaluation of Municipal Wastewater Disinfection by 280 nm UVC LED

UV-LED irradiation has attracted attention in water and wastewater disinfection applications. However, no studies have quantitatively investigated the impact of light intensity on the UV dosage for the same magnitude of disinfection. This study presents a powerful 280 nm UV-LED photoreactor with adjustable light intensity to disinfect municipal wastewater contaminated with E. coli, SARS-CoV-2 genetic materials and others. The disinfection performance of the 280 nm LED was also compared with 405 nm visible light LEDs, in terms of inactivating E. coli and total coliforms, as well as reducing cATP activities. The results showed that the UV dose needed per log reduction of E. coli and total coliforms, as well as cATP, could be decreased by increasing the light intensity within the investigated range (0–9640 µW/cm2). Higher energy consumption is needed for microbial disinfection using the 405 nm LED when compared to 280 nm LED. The signal of SARS-CoV-2 genetic material in wastewater and the SARS-CoV-2 spike protein in pure water decreased upon 280 nm UV irradiation. © 2023 by the authors.

Inactivation of Pathogenic Microorganisms on Polymeric Materials through Ozonation

Ozone-based technologies have been evaluated to inactivate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on surfaces. However, the vast diversity of information makes it difficult to establish common ground for determining the best practices for using this technology. The objective of this work is to evaluate the success of N95 mask decontamination by ozonation, determining the specific parameters for process control. To quantify the effectiveness of the process, a disinfection protocol was initially developed based on two bacterial species (Escherichia coli and Staphylococcus pseudintermedius), followed by another disinfection assay using the murine hepatitis coronavirus (MHV-3), in a laboratory-scale prototype. Ozone is an effective candidate for use against SARS-CoV-2 or other viruses to disinfect personal protection equipment (PPE). © 2023 Wiley-VCH GmbH.

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system.

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

Complex of Ceftriaxone with Mg(II): Synthesis, Structure, Spectral and Antibacterial Properties

Аннотация. Получен и охарактеризован магниевый комплекс цефтриаксона методами атомно-эмиссионного и элементного анализов, ТГА, ИК- и КР-спектроскопии, РФА и расчетов теории функционала плотности. Цефтриаксон координируется к иону магния через кислород триазинового цикла в шестом положении, азот аминогруппы тиазольного цикла и атомы кислорода карбоксильной и лактамной групп. Динатриевая соль цефтриаксона и комплекс магния были исследованы на антибактериальную активность в отношении Staphylococcus aureus, Escherichia coli и Pseudomonas aeruginosa.Alternate abstract:Magnesium complex of ceftriaxone was obtained and characterized by atomic-emission and elemental analysis, TGA, FTIR and Raman spectroscopy, X-ray diffraction and density functional theory calculations. Ceftriaxone was coordinated to the magnesium ion by the oxygen of the triazine cycle in the 6th position, the nitrogen of the amine group of the thiazole ring, and oxygen atoms of the lactam carbonyl and carboxylate groups. The disodium salt of ceftriaxone and magnesium complex were screened for antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

Expression of SARS-CoV-2 Nucleocapsid (N) Recombinant Protein Using Escherichia coli System

One of the main antigen that can be used for serological testing is the nucleocapsid (N) which is the most abundant viral-derived protein in SARS-CoV-2 where this virus can cause COVID19 disease. The aim of this study was to develop the SARS-CoV-2 N recombinant protein using Escherichia coli expression system. A total of 1,089 nucleotides encoding 362 amino acids of SARS-CoV-2 N was cloned to pET-14b vector. The plasmid then expressed in E. coli BL21 (DE3) and induced with 1.0 mM IPTG (Isopropyl-β-d-1-thiogalactopyranoside). The cell was harvested using denaturation lysis buffer due to inclusion body formation of SARS-CoV-2 N protein. Dialysis processed and concentrated using PEG-6000 resulted 0.992 mg/ml protein yield. Analysis of SARS-CoV-2 N recombinant protein using SDS-PAGE technique showed approximately 37.0 kDa specific band target protein. Application of this SARS-CoV-2 N recombinant protein to vaccinated and non-vaccinated antibody serum samples using ELISA technique indicated the significant result of optical density mean at 0.603 and 0.135, respectively. This study revealed that the production of SARS-COV-2 N recombinant protein could be carried out in E. coli expression system under denatured conditions, therefore the methods are more effective in producing the protein as a basic material in immuno-diagnostic assay. © 2023, Bogor Agricultural University. All rights reserved.

Truncated expression of S1 protein of Feline infectious peritonitis virus and preparation of its monoclonal antibody

In order to develop monoclonal antibody against Feline infectious peritonitis virus (FIPV) S1 protein, the truncated S1 protein (rS1) was expressed through Escherichia coli and subsequently purified. Then BALB/c mice were immunized with purified rSl. Three hybridoma cell strains, named 2D7,3D8 and 5G1, stably secreting antibodies against rSl were obtained by cell fusion and indirect ELISA screening. The identification of antibody subtype showed that antibody subtypes of 2D7,5G1 and 3d8 strains were IgG2a,IgG2a and IgGl,respectively. And the light chain of those three hybridoma cell strains was Kappa. Result of karyotype identification of hybridoma cells showed that the chromosome numbers of those three hybridoma cells were about 102,101 and 103, which was belonged to the karyotype of hybridoma. The titer of ascites antibody for indirect ELISA was 1 : 204 800, and monoclonal antibodies were purified. Moreover, all of 2D7,3D8 and 5G1 could react with rS1 by Western-blot and FIPV in cells by IFA. These data suggest that three monoclonal antibodies against rSl with good activities were ideal materials in the study of early diagnosis of FIPV and the biological function of FIPV in the future.

The microbial-killing Ag nanoparticles in food supply chain: How it was applied and what a consumer should know?

Moving to 133 years of the synthesis of citrate-stabilized silver nanoparticles (AgNPs) by M. C. Lea (published in Am. J. Sci, 1889), a myriad of scholarly works and patents were published globally demonstrating the applicability of this microbial-killing nanoparticles in various industries. One of the favorite applications is on the food supply chain whereby AgNPs serve to improve food safety and quality. In this paper, the adaptation of AgNPs in each phase of a typical food supply chain is disclosed, doubts associated with the potential risks brought by this technology, and what a consumer shall be aware of are highlighted.

Differentiating infectious from non-infectious respiratory disease in the cat and dog

This paper describes the clinical signs and use of differential laboratory diagnostic techniques (computed tomography, cytology, histopathology, antigen/antibody detection and polymerase chain reaction) for infectious (viral, bacterial, fungal and parasitic) and non-infectious (inflammatory/immune mediated, neoplastic, cardiac, malformation, foreign body, smoke inhalation, aspiration of caustic material, non-cardiogenic, pulmonary oedema, traumativ, pneumothorax, pulmonary contusions and idiopathic) causes of respiratory diseases in cats and dogs in Ontario, Canada.

One-step multiple TaqMan real-time RT-PCR for simultaneous detection of swine diarrhea viruses

Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.