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 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 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.

scholarly journals Detection and dynamics of porcine circovirus 2 shedding in semen using conventional and real-time PCR

2010 ◽  
Vol 30 (11) ◽  
pp. 918-920 ◽  
Author(s):  
Priscilla F Gerber ◽  
Flávia F Pinto ◽  
Marcos B Heinemann ◽  
Zélia I.P Lobato
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Polymerase Chain

The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

scholarly journals Construction of a Quadruple Gene-deleted Vaccine Confers Complete Protective Immunity Against Emerging PRV Variant Challenge in Piglets

2021 ◽  
Author(s):  
Leqiang Sun ◽  
Yajie Tang ◽  
Keji Yan ◽  
Huawei Zhang
Keyword(s):  
Pseudorabies Virus ◽  
Clinical Signs ◽  
Economic Losses ◽  
Swine Industry ◽  
Live Attenuated Vaccine ◽  
Specific Antibodies ◽  
Variant Strain ◽  
Attenuated Vaccine ◽  
Suckling Piglets ◽  
Full Protection

Abstract Background: Pseudorabies virus (PRV) causes Aujeszky’s disease or pseudorabies (PR) in pigs worldwide, which leads to heavy economic losses to the swine industry. Pigs are the natural host, meanwhile, animals such as dogs, cats, foxes, rabbits, cattle and sheep are susceptible to infection. In 2011, the emerging PRV variant led to the outbreak of PR in Bar-tha-K61-vaccinated pigs. The PR outbreaks demonstrated that the Bartha-K61 vaccine did not provide full protection against the emerging PRV variant. It is widely believed that PRV live-attenuated vaccines could control PRV infection. Methods: In this study, we developed a novel PRV live-attenuated vaccine by deleting its gI, gE, US9, and US2 genes through CRISPR/Cas9, which was named PRV GDFS-delgI/gE/US9/US2.Results: Safety experiments confirmed that PRV GDFS-delgI/gE/US9/US2 was safe for 5 to 7-day-old suckling piglets. Piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine did not produce PRV gE-specific antibodies but could generate PRV gB-specific antibodies and high neutralizing titers against the PRV GDFS strain (variant PRV strain) or PRV Ea strain (older PRV strain). After challenge with the emerging PRV GDFS variant, none of the piglets immunized with the PRV GDFS-delgI/gE/US9/US2 vaccine showed any clinical signs, and their rectal temperatures were normal. Moreover, the autopsy and histopathological analyses revealed that the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine group did not show apparent gross or pathological lesions. Furthermore, the piglets in the PRV GDFS-delgI/gE/US9/US2 vaccine groups did not present weight loss. According to the criteria of the OIE terrestrial manual, the results of the experiment confirmed that the PRV GDFS-delgI/gE/US9/US2 vaccine could provide full protection against the emerging PRV variant strain in piglets. Conclusions: The PRV GDFS-delgI/gE/US9/US2 strain is a potential new live-attenuated vaccine against emerging PRV variant strain infections in China.

scholarly journals Different methods of real-time PCR for detection of pseudorabies virus

2017 ◽  
Vol 47 (3) ◽  
Author(s):  
Carolina Kymie Vasques Nonaka ◽  
◽  
Antônio Augusto Fonseca Junior ◽  
Estefânia Oliveira Guedes ◽  
Régia Maria D´Ambros ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pseudorabies Virus ◽  
Animal Health ◽  
Viral Disease ◽  
Economic Importance ◽  
Swine Industry ◽  
Iso 17025 ◽  
Analysis Error ◽  
Genomic Regions

ABSTRACT: Pseudorabies (PR) is a highly contagious viral disease of great animal health and economic importance in swine industry. The aim of this study was to evaluate different genomic regions, real-time PCR chemistries and equipment for the molecular diagnosis of PR. Eight primer pairs targeting four genes (gB, gC, gE, gD), three different qPCR chemistries (SybrGreen, hydrolysis probes and plexor) and two equipment (ABI7500, Rotorgene 3000) were evaluated. Oligonucleotides targeting gB using hydrolysis probes showed the best performance after evaluating efficiency (99%), the detection limit (10-1.5 TCID50 mL-1) and diagnostic sensitivity and; therefore, those primers were selected for performance verification factors such as repeatability, reproducibility and robustness (1.39% variance between days, 24% variance between analysts and 4.07% variance in analysis error). The qPCR standardized and validated in this research proved to be reliable for the diagnosis of PR and may be used in diagnostic laboratories that follow ISO 17025 and ISO 16140.

Clinical presentation, epidemiological findings, diagnosis, immunity, prevention and control of postweaning multisystemic wasting syndrome (PMWS)

2003 ◽  
Vol 2003 ◽  
pp. 223-223
Author(s):  
J. Segalés ◽  
M. Domingo
Keyword(s):  
Clinical Signs ◽  
Porcine Circovirus Type ◽  
Postweaning Multisystemic Wasting Syndrome ◽  
Porcine Circovirus ◽  
Full Spectrum ◽  
Wasting Syndrome ◽  
And Control ◽  
Porcine Respiratory

Postweaning multisystemic wasting syndrome (PMWS) was initially described in 1991 in Saskatchewan (Canada) and has now been described in all continents rearing pigs but Oceania. Porcine circovirus type 2 (PCV2) is the aetiology of this disease, which has also been called porcine circovirosis in some countries. Although the full spectrum of clinical signs and lesions observed in natural cases of PMWS is very difficult to reproduce under experimental infections using PCV2 alone, little doubt exists on the causal relationship between the virus and the wasting syndrome. Furthermore, the clinical and pathological scope of PCV2 infection has been expanded since 1991, and it has been implicated in other conditions: reproductive disorders, porcine dermatitis and nephropathy syndrome (PDNS), the so-called porcine respiratory disease complex (PRDC), proliferative and necrotising pneumonia (PNP), and congenital tremors. The role of PCV2 in these conditions has not been fully clarified and, in some of these cases, it remains as a controversial issue. The objective of this presentation is to review some practical aspects of PMWS.

scholarly journals Diagnostic investigation of porcine periweaning failure-to-thrive syndrome

2011 ◽  
Vol 24 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Yanyun Huang ◽  
Henry Gauvreau ◽  
John Harding
Keyword(s):  
Influenza A ◽  
Clinical Signs ◽  
Case Fatality ◽  
Failure To Thrive ◽  
Torque Teno Virus ◽  
Porcine Circovirus ◽  
Respiratory Syndrome Virus ◽  
Transmissible Gastroenteritis Virus ◽  
Swine Industry ◽  
Diagnostic Investigation

Porcine periweaning failure-to-thrive syndrome (PFTS), an increasingly recognized syndrome in the swine industry of North America, is characterized by the anorexia of nursery pigs noticeable within 1 week of weaning, and progressive loss of body condition and lethargy during the next 1–2 weeks. Morbidity caused by PFTS is moderate, but case fatality is high. The etiology of PFTS is presently unknown and may include infectious agent(s), noninfectious factors, or both. PFTS was identified in a high health status farm with good management in early 2007. A diagnostic investigation was undertaken to identify the pathological lesions of, and infectious agents associated with, pigs demonstrating typical clinical signs. Affected (PFTS-SICK) and unaffected (PFTS-HLTHY) pigs from an affected farm, and unaffected pigs from 2 unaffected farms, were examined. The most prevalent lesions in PFTS-SICK pigs were superficial lymphocytic fundic gastritis, atrophic enteritis, superficial colitis, lymphocytic and neutrophilic rhinitis, mild nonsuppurative meningoencephalitis, and thymic atrophy. Rotavirus A and Betacoronavirus 1 (Porcine hemagglutinating encephalomyelitis virus) were identified only in PFTS-SICK pigs, but the significance of the viruses is uncertain because PFTS is not consistent with the typical presentation following infection by these pathogens. Porcine reproductive and respiratory syndrome virus, Porcine circovirus-2, Influenza A virus, Alphacoronavirus 1 (Transmissible gastroenteritis virus), Torque teno virus 1, Brachyspira hyodysenteriae, and Brachyspira pilosicoli were not identified in PFTS-SICK pigs. Suid herpesvirus 2 (Porcine cytomegalovirus), Porcine enteric calicivirus, Torque teno virus 2, pathogenic Escherichia coli, and coccidia were detected in both PFTS-SICK and PFTS-HLTHY pigs. It was concluded that there is a lack of compelling evidence that PFTS is caused by any of these pathogens.

scholarly journals Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Yang Yang ◽  
Xiaodong Qin ◽  
Yingjun Sun ◽  
Guozheng Cong ◽  
Yanmin Li ◽  
...  
Keyword(s):  
Real Time ◽  
Virus Type ◽  
Pseudorabies Virus ◽  
Classical Swine Fever ◽  
Porcine Circovirus Type ◽  
Recombinase Polymerase Amplification ◽  
Fluorescent Detection ◽  
Porcine Circovirus ◽  
Respiratory Syndrome Virus ◽  
Mouth Disease Virus

Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

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