scholarly journals Porcine Gammaherpesviruses in Italian Commercial Swine Population: Frequent but Harmless

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 47
Author(s):  
Giovanni Franzo ◽  
Michele Drigo ◽  
Matteo Legnardi ◽  
Laura Grassi ◽  
Maria Luisa Menandro ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Signs ◽  
Northern Italy ◽  
Porcine Circovirus ◽  
Swine Industry ◽  
Positive Interaction ◽  
High Prevalence ◽  
Swine Population ◽  
Clinical Conditions

Differently from alpha- and betaherpesviruses affecting swine, interest in the recently discovered Suid gammaherpesvirus 3, Suid gammaherpesvirus 4, and Suid gammaherpesvirus 5, also known as porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, and PLHV-3), has largely focused on their role as potential zoonotic agents in cases of xenotransplantation. However, their role as primary pathogens of swine or as co-factors for other lymphotropic infections has essentially been neglected. The present study aims at filling this gap, evaluating the association between PLHVs infection and different clinical conditions and/or porcine circovirus (PCV) co-infection. One hundred seventy-six samples were obtained from different animals located in a high-density pig area of Northern Italy in the period 2017–2020. The presence of PLHVs and PCVs was tested and quantified by specific real-time PCR: PLHVs were widespread among pigs (PLHV-1, PLHV-2, and PLHV-3 prevalence was 28.97%, 10.79%, and 4.54%, respectively) and detected in all considered tissues and clinical conditions. Frequent co-infections were also observed among PLHVs and with PCVs, although a significant association was not detected with the exception of a positive interaction between PLHV-1 and PLHV-3, and a negative one between PLHV-2 and PCV-2. Significantly, no association between PLHVs, alone or in co-infection, emerged with any of the considered clinical signs, their frequency being comparable between healthy and diseased animals. Based on these pieces of evidence and despite their high prevalence, PLHVs’ relevance for the swine industry appears negligible, either as primary pathogens or as predisposing factors for circovirus-induced diseases.

scholarly journals Detection of agents associated with respiratory diseases of swine by real time PCR

2015 ◽  
Vol 16 (2) ◽  
pp. 300-307 ◽  
Author(s):  
Antônio Augusto FONSECA JUNIOR ◽  
Carolina Kymie Vasquez NONAKA ◽  
Estefânia de Oliveira GUEDES ◽  
Zélia Inês Portela LOBATO ◽  
Alessandra Silva DIAS ◽  
...  
Keyword(s):  
Real Time ◽  
Pseudorabies Virus ◽  
Clinical Signs ◽  
Infectious Agent ◽  
Porcine Circovirus ◽  
Infectious Agents ◽  
Swine Industry ◽  
Specific Infectious Agent ◽  
Pooled Samples ◽  
Porcine Respiratory

Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1),Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactions—a duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.

scholarly journals Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ellen Kathrin Link ◽  
Matthias Eddicks ◽  
Liangliang Nan ◽  
Mathias Ritzmann ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Diagnostic Sensitivity ◽  
Locked Nucleic Acid ◽  
Porcine Circovirus ◽  
Amplification Efficiency ◽  
Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

scholarly journals Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the South of Vietnam

2020 ◽  
Author(s):  
Vu Thuy Duong ◽  
Le Thi Phuong Tu ◽  
Ha Thanh Tuyen ◽  
Le Thi Quynh Nhi ◽  
James I Campbell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Middle Income ◽  
Middle Income Countries ◽  
Virulence Markers ◽  
Low Middle Income Countries ◽  
High Prevalence ◽  
Pcr Assays

Abstract BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2,815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

scholarly journals Detection of Aspergillus species in bone marrow transplant patients

2010 ◽  
Vol 4 (08) ◽  
pp. 511-516 ◽  
Author(s):  
Parisa Badiee ◽  
Abdolvahab Alborzi
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Bone Marrow Transplant ◽  
Real Time Pcr ◽  
Clinical Signs ◽  
Marrow Transplant ◽  
Aspergillus Species ◽  
Pcr Assay ◽  
Blood Samples ◽  
Transplant Patients

Introduction:  Invasive aspergillosis is a severe complication of cytotoxic chemotherapies and bone marrow transplantation (BMT). The aim of this study was to assess the utility of a real-time PCR assay for the early diagnosis of Aspergillus species in blood samples from BMT patients. Methodology: Blood specimens (n = 993) from patients (n = 82) scheduled for BMT were collected prior to transplant and for 100 days post transplantation.  The specimens were later tested using an Aspergillus-specific real-time PCR assay. Cultures of clinical samples, along with sonography and computerized tomographic scans, were performed as standard of care. Results: Aspergillus DNA was positive in 94 sequential blood samples from 13 patients with clinical and radiological signs of infection. Samples from three of these patients were PCR-positive for Aspergillus in the first week of admission, prior to transplantation. Four patients with aspergillosis were cured with antifungal agents and nine died. An additional 12 patients without clinical signs of infection were PCR-positive on one occasion each, while two patients with clinical signs of infection were PCR-negative. Compared to routine methods of aspergillosis diagnosis, the respective sensitivity, specificity, negative, and positive predictive values of the PCR method by patient were 86.6%, 82%, 96.5% and 52%. Conclusions: The results show that Aspergillus infections in the blood of bone marrow transplant patients can be dectected by PCR methods. Early detection of Aspergillus infections by PCR has the potential to positively impact patient mortality rate and provide cost savings to hospitals.

First report of wild boar susceptibility to Porcine circovirus type 3: High prevalence in the Colli Euganei Regional Park (Italy) in the absence of clinical signs

2018 ◽  
Vol 65 (4) ◽  
pp. 957-962 ◽  
Author(s):  
Giovanni Franzo ◽  
Claudia Maria Tucciarone ◽  
Michele Drigo ◽  
Mattia Cecchinato ◽  
Marco Martini ◽  
...  
Keyword(s):  
Wild Boar ◽  
Clinical Signs ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
First Report ◽  
High Prevalence ◽  
Type 3

scholarly journals Objective pathogen monitoring in nursery and finisher pigs by monthly laboratory diagnostic testing

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Nicole B. Goecke ◽  
Maja Kobberø ◽  
Thomas K. Kusk ◽  
Charlotte K. Hjulsager ◽  
Ken Steen Pedersen ◽  
...  
Keyword(s):  
Real Time ◽  
San Francisco ◽  
Influenza A Virus ◽  
High Throughput ◽  
Real Time Pcr ◽  
Influenza A ◽  
Clinical Signs ◽  
Swine Influenza ◽  
Porcine Cytomegalovirus ◽  
Swine Influenza A Virus

Abstract Background Infectious diseases are of great economic importance in commercial pig production, causing both clinical and subclinical disease, with influence on welfare, productivity, and antibiotic use. The causes of these diseases are often multifactorial and laboratory diagnostics are seldom routinely performed. The aim of the present study was to explore the benefits of monthly pathogen monitoring in nursery and finisher herds and to examine association between laboratory results and observed clinical signs, including coughing and diarrhoea. Three monthly samplings were conducted in three different age groups in six nursery and four finisher production units. For each unit, two pens were randomly selected in each age group and evaluated for coughing and diarrhoea events. Furthermore, faecal sock and oral fluid samples were collected in the selected pens and analysed for 18 respiratory and enteric viral and bacterial pathogens using the high-throughput real-time PCR BioMark HD platform (Fluidigm, South San Francisco, USA). Results In total, 174 pens were sampled in which eight coughing events and 77 diarrhoeic events were observed. The overall findings showed that swine influenza A virus, porcine circovirus 2, porcine cytomegalovirus, Brachyspira pilosicoli, Lawsonia intracellularis, Escherichia coli fimbria types F4 and F18 were found to be prevalent in several of the herds. Association between coughing events and the presence of swine influenza A virus, porcine cytomegalovirus (Cq ≤ 20) or a combination of these were found. Furthermore, an association between diarrhoeic events and the presence of L. intracellularis (Cq ≤ 24) or B. pilosicoli (Cq ≤ 26) was found. Conclusions The use of high-throughput real-time PCR analysis for continuous monitoring of pathogens and thereby dynamics of infections in a pig herd, provided the veterinarian and farmer with an objective knowledge on the distribution of pathogens in the herd. In addition, the use of a high-throughput method in combination with information about clinical signs, productivity, health status and antibiotic consumption, presents a new and innovative way of diagnosing and monitoring pig herds and even to a lower cost than the traditional method.

scholarly journals Simultaneous detection of porcine pseudorabies virus, porcine parvovirus and porcine circovirus type 2 by multiplex real-time PCR and amplicon melting curve analysis using SYBR Green I

2018 ◽  
Vol 63 (No. 8) ◽  
pp. 358-366
Author(s):  
LL Zheng ◽  
XH Jin ◽  
FS Wei ◽  
CQ Wang ◽  
HY Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pseudorabies Virus ◽  
Simultaneous Detection ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Porcine Parvovirus ◽  
Porcine Pseudorabies Virus

Porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 can cause reproductive failure in pigs, and swine are often simultaneously infected by combinations of the three viruses. We here report the development of a SYBR Green I-based multiplex real time PCR assay for simultaneous detection of porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2. Three pairs of specific primers were designed for the porcine parvovirus-VP2, porcine pseudorabies virus-gH and porcine circovirus type 2-ORF2 genes. Viral genomes were identified based on their distinctive melting temperatures in singleplex PCR reactions. The melting temperature was 74.5 °C for the 313 bp amplicon of porcine parvovirus-VP2 gene, 87.5 °C for the 355 bp amplicon of porcine pseudorabies virus-gH gene and 80.5 °C for the 171 bp amplicon of the porcine circovirus type 2-ORF2 gene, respectively. The detection limit of the method ranged from 0.01–0.03 TCID<sub>50</sub>/ml for the three viruses. In addition, porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 viral loads were measured in 100 field samples, and the result showed that the concordance between real-time PCR and conventional PCR was 60.42%. The sensitivity and specificity of real-time PCR were 100% and 100%, while those of conventional PCR were 40.83% and 72.22%, respectively.

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