Combined in-vitro and on-farm evaluation of commercial disinfectants used against Brachyspira hyodysenteriae

Abstract Background Swine dysentery (SD) is a severe infectious disease with a relevant impact on pig production usually caused by Brachyspira hyodysenteriae, although B. hampsonii causes an identical clinical picture. SD control relies on antimicrobials, good management practices and strict biosecurity with cleaning and disinfection as crucial tools to avoid the pathogen transmission. This study evaluates the in-vitro efficacy of an array of commercial disinfectants against a collection of B. hyodysenteriae isolates using broth tests. The efficacy of cleaning and disinfection protocols was also evaluated on two farms with endemic SD using surface swabs collected in emptied pens before and after cleaning and disinfection procedures, using both real-time PCR and bacterial microbiological culture. Results Most of the commercial disinfectants evaluated were effective against all B. hyodysenteriae isolates tested, with a reduction of more than 5.00 log10 CFU/mL (bactericidal efficacy of 99.999%). However, some isolates exhibited reduced susceptibility to Virkon-S and Limoseptic disinfectants. The evaluation of cleaning and disinfection protocols on farms with SD outbreaks showed that approximately half the pens tested (n = 25) were positive by real-time PCR after pigs removal (mean B. hyodysenteriae counts 5.72 ± 1.04 log10 CFU/mL) while almost 20% of the pens remained positive after cleaning (n = 7) and disinfection (n = 5) procedures although with significantly lower, mean estimates (4.31 ± 0.43 log10 CFU/mL and 4.01 ± 0.55 log10 CFU/mL, respectively). Conclusions These results show the efficacy of disinfectants against B. hyodysenteriae but also stress the need to implement adequately the cleaning and disinfection protocols on pig farms and review and revise their efficiency periodically.

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Evaluation of the Time Period for which Real-Time Polymerase Chain Reaction Detects Dead Bacteria

Polish Journal of Microbiology ◽

10.33073/pjm-2014-053 ◽

2014 ◽

Vol 63 (4) ◽

pp. 393-398 ◽

Cited By ~ 2

Author(s):

HYONMIN CHOE ◽

YUTAKA INABA ◽

NAOMI KOBAYASHI ◽

YUSHI MIYAMAE ◽

HIROYUKI IKE ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Chain Reaction ◽

Bacterial Dna ◽

Methicillin Resistant ◽

Time Period ◽

Before And After ◽

Polymerase Chain

Real-time polymerase chain reaction (PCR) is currently widely used for the diagnosis of infections. We evaluated the time after treatment during which real-time PCR can detect dead bacteria. The presence of bacterial DNA was identified by real-time PCR through methicillin-resistant Staphylococcus (MRS)-PCR and universal PCR. Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, and Escherichia coli were each killed with alcohol, antibiotics, or heat treatment in vitro. The detection periods of MRS-PCR for MRSA treated by alcohol, vancomycin, linezolid, and heat were found to be less than 16, 8, 12, and 8 weeks, respectively. The detection period of universal PCR for S. epidermidis treated by alcohol, cefazolin, and heat was less than 20, 20, and 4 weeks, whereas that for E. coli was 8, 20, and 4 weeks, respectively. The presence of detectable bacterial DNA in infected arthroplasty patients before and after successful treatment was also assessed by MRS- and universal PCR. MRS-PCR was positive in 6 patients before treatment and all became negative after a mean interval of 20.8 weeks (95% confidential interval, 13.2 to 33.7) after treatment. Universal PCR detected remnant bacterial DNA in 4 patients at a mean of 15.2 weeks (95% CI, 12.4 to 18.0) after treatment and was negative in 7 patients at a mean of 17.3 weeks (95% CI, 10.6 to 24.0) after treatment. Our studies revealed that real-time PCR detects dead bacteria for several weeks, but this capability decreases with time and is likely lost by 20 weeks after treatment.

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Prevalence of Yersinia enterocolitica in Market Weight Hogs in the United States†

Journal of Food Protection ◽

10.4315/0362-028x-71.6.1162 ◽

2008 ◽

Vol 71 (6) ◽

pp. 1162-1168 ◽

Cited By ~ 22

Author(s):

IRENE V. WESLEY ◽

SAUMYA BHADURI ◽

ERIC BUSH

Keyword(s):

Real Time ◽

Yersinia Enterocolitica ◽

Real Time Pcr ◽

Management Practices ◽

Animal Health ◽

The United States ◽

Individual Animal ◽

Enrichment Broth ◽

Fecal Samples ◽

On Farm

Pigs are the major animal reservoir for Yersinia enterocolitica strains, which are potentially pathogenic for humans. The goals of this study were (i) to estimate the individual animal and on-farm prevalences of Y. enterocolitica in hogs based on tonsil samples collected during National Animal Health Monitoring System Swine 2002 study and (ii) to use these data with data previously published for fecal samples to determine on-farm risk factors for Y. enterocolitica. Tonsil swabs (1,218) and fecal samples (2,847) were collected on 124 farms located in the top 17 pork-producing states. Ten percent of tonsils (122 of 1,218 samples) were positive in irgasan-tiracillin-chlorate (ITC) enrichment broth by real-time PCR, but only 5.6% of samples (68 of 1,218) were positive after subculture on the more selective cefsulodin-irgasan-novobiocin (CIN) agar. For tonsils, the on-farm prevalence based on real-time PCR detection of the ail gene in ITC enrichment broth cultures was 32% (32 of 100 premises sampled); the prevalence based on subculture in CIN agar was 19.6% (20 of 102 premises). Results of bacteriological isolation and real-time PCR analysis of tonsils and feces were combined to estimate prevalence (individual animal and farm), which was subsequently correlated with 40 farm management practices. Four factors and their accompanying odds ratios (ORs) were identified in the final regression model: location in a central state (OR =0.3), vaccination for Escherichia coli (OR = 3.0), percentage of deaths due to scours (OR = 3.5), and presence of meat or bone meal in grower-finisher diet (OR = 4.1).

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The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽

10.3390/cancers12092341 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2341

Author(s):

Normann Steiner ◽

Karin Jöhrer ◽

Selina Plewan ◽

Andrea Brunner-Véber ◽

Georg Göbel ◽

...

Keyword(s):

Multiple Myeloma ◽

Tyrosine Kinase ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Tyrosine Kinase Receptor ◽

Bone Marrow Biopsies ◽

Flt3 Inhibitors ◽

Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

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A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs

F1000Research ◽

10.12688/f1000research.20639.1 ◽

2019 ◽

Vol 8 ◽

pp. 1702

Author(s):

Arkadiusz Dors ◽

Ewelina Czyżewska-Dors ◽

Grzegorz Woźniakowski

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Finishing Pigs ◽

Lawsonia Intracellularis ◽

Fecal Samples ◽

Brachyspira Pilosicoli ◽

Brachyspira Hyodysenteriae ◽

Veterinary Research ◽

Two Samples ◽

Intestinal Pathogens

Background: The major pathogenic intestinal spirochetes affecting pigs during the growing- finishing stage of production include Brachyspira hyodysenteriae and Brachyspira pilosicoli. Infections by these pathogens, which affect the economics of pig production, can result in mortality, growth rate losses and substantial antibiotic costs. The aim of this study was to assess the current occurrence of B. hyodysenteriae and B. pilosicoli in Polish pig herds. Moreover, associations between the presence of diarrhea or other intestinal pathogens and occurrence of B. hyodysenteriae and B. pilosicoli in pigs were investigated. Methods: Between January 2017 and August 2019, a total of 401 samples of pig feces from 95 different herds were submitted to the National Veterinary Research Institute of Poland. These samples were obtained from pigs older than 7 weeks. All the received fecal samples were examined for the presence of B. hyodysenteriae, B. pilosicoli and Lawsonia intracellularis by real-time PCR. Results: For B. pilosicoli, 4.5% (95% CI, 2.5–7.0%) of samples and 13.7% (95% CI, 7.5–22.3%) of herds were positive. Out of 12 samples, B. pilosicoli was detected simultaneously with L. intracellularis, B. hyodysenteriae and B. pilosicoli were detected alone in two samples each. In terms of B. hyodysenteriae, 7.0% of samples (95% CI, 4.7–9.9%) from 18.9% of herds (95% CI, 11.6–28.3%) were positive in real time PCR. The presence of B. hyodysenteriae in fecal samples was associated with the presence of diarrhea in pigs. Conclusions: This study confirmed that B. pilosicoli infections occur in Polish pig herds, but the prevalence is at a low level and the presence of B. pilosicoli is not associated with the development of diarrhea in pigs. B. hyodysenteriae is still a common cause of diarrhea among pigs from Polish herds.

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The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

Frontiers in Oncology ◽

10.3389/fonc.2021.752573 ◽

2021 ◽

Vol 11 ◽

Author(s):

Dong Chen ◽

Yaqin Wang ◽

Feiya Yang ◽

Adili Keranmu ◽

Qingxin Zhao ◽

...

Keyword(s):

Prostate Cancer ◽

Real Time ◽

Expression Pattern ◽

Real Time Pcr ◽

Bioinformatic Analysis ◽

Biological Functions ◽

Quantitative Real Time Pcr ◽

Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

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Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2014-0004 ◽

2014 ◽

Vol 58 (1) ◽

pp. 23-28 ◽

Cited By ~ 3

Author(s):

Krzysztof Wąsowicz ◽

Piotr Podlasz ◽

Małgorzata Chmielewska ◽

Katarzyna Łosiewicz ◽

Jerzy Kaleczyc ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Experimental Colitis ◽

Expression Level ◽

Colonic Wall ◽

Brachyspira Hyodysenteriae ◽

Expression Levels ◽

Gapdh Expression ◽

Galanin Receptors ◽

Different Levels

Abstract The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

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Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis

Journal of Medical Microbiology ◽

10.1099/jmm.0.008169-0 ◽

2009 ◽

Vol 58 (5) ◽

pp. 648-655 ◽

Cited By ~ 39

Author(s):

Kristel Lourdault ◽

Florence Aviat ◽

Mathieu Picardeau

Keyword(s):

Guinea Pig ◽

Real Time ◽

Real Time Pcr ◽

Guinea Pigs ◽

Infection Model ◽

Avirulent Strain ◽

Leptospira Interrogans ◽

Quantitative Real Time Pcr ◽

Guinea Pig Model

The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD50, rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5×104 leptospires ml−1. The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

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Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

Protocols used in Molecular Biology ◽

10.2174/9789811439315120010006 ◽

2020 ◽

pp. 15-34

Keyword(s):

Gene Expression ◽

Real Time ◽

Tissue Regeneration ◽

Real Time Pcr ◽

Molecular Level ◽

Northern Blotting ◽

Tissue Constructs ◽

Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

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(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/β-catenin

The American Journal of Chinese Medicine ◽

10.1142/s0192415x20500172 ◽

2020 ◽

Vol 48 (02) ◽

pp. 341-356

Author(s):

Chiu-Mei Lin ◽

Wei-Jen Fang ◽

Bao-Wei Wang ◽

Chun-Ming Pan ◽

Su-Kiat Chua ◽

...

Keyword(s):

Myocardial Infarction ◽

Real Time ◽

Real Time Pcr ◽

Myocardial Infarct ◽

Epigallocatechin Gallate ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Rat Cardiomyocytes

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/[Formula: see text]-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and [Formula: see text]-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50[Formula: see text]mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and [Formula: see text]-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/[Formula: see text]-catenin pathways.

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Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

BioMed Research International ◽

10.1155/2019/5715180 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Cyril C. Y. Yip ◽

Siddharth Sridhar ◽

Kit-Hang Leung ◽

Andrew K. W. Cheng ◽

Kwok-Hung Chan ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Test Performance ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Clinical Specimens ◽

Varicella Zoster ◽

Simplex Virus ◽

Hsv 1

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

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