Balantidium coli in pig farms suspected of porcine circovirus type 2 (PCV2) associated enteritis

Abstract Introduction Diarrhoea in growing-finishing pigs is a common problem of commercial pig farms. Among many causative factors, porcine circovirus type 2 (PCV2) is one considered an important pathogen in modern pig production. The aim of the study was to verify if PCV2 was responsible for antibiotic non-responsive diarrhoea and wasting in pigs. Material and Methods A total of 13 dead pigs aged between 12 and 15 weeks from three Polish farms with persistent herd symptoms suggestive of PCV2 infection were provided for evaluation. Sections of lymph nodes and intestines were analysed by in situ hybridization (ISH) for PCV2 and histopathological examination. Faeces and intestinal scrapings were tested for Lawsonia intracellularis and Brachyspira hyodysenteriae by real-time PCR and for parasitic infection by flotation and decantation. Results ISH and histopathological examination showed that all pigs were PCV2 systemic disease negative. Swine dysentery was confirmed by real-time PCR on two farms, and proliferative enteropathy on one farm. In histological examinations, erosions of the caecal and colonic mucosa were found, together with cysts and trophozoites of Balantidium coli. The protozoa were present in the intestinal lumen and mucosa. B. coli cysts were identified in faeces from all examined pigs. Conclusion These results suggest that monitoring of B. coli infections should be an additional measure of control and prevention of gastrointestinal tract disorders in modern swine husbandry.

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

Virology Journal ◽

10.1186/s12985-021-01541-z ◽

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.

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Comparison of Multiplex Real-Time PCR and PCR-Reverse Blot Hybridization Assays for the Direct and Rapid Detection of Porcine Circovirus Type 2 Genotypes

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.00200 ◽

2020 ◽

Vol 7 ◽

Author(s):

Hye-young Wang ◽

Joong Ki Song ◽

Seongho Shin ◽

Hyunil Kim

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Blot Hybridization ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Hybridization Assays

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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

Veterinární Medicína ◽

10.17221/3/2018-vetmed ◽

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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Real-Time PCR Detection Patterns of Porcine Circovirus Type 2 (PCV2) in Polish Farms with Different Statuses of Vaccination against PCV2

Viruses ◽

10.3390/v11121135 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1135 ◽

Cited By ~ 6

Author(s):

Aleksandra Woźniak ◽

Dagmara Miłek ◽

Piotr Matyba ◽

Tomasz Stadejek

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Quantitative Real Time Pcr ◽

Viral Loads ◽

Oral Fluids ◽

Significant Difference

Porcine circovirus type 2 (PCV2) is a globally spread pathogen controlled with generally highly efficacious vaccination protocols. In order to compare PCV2 detection profiles in farms with different vaccination statuses, serum (359) and fecal pools (351) and oral fluids (209) from four farms that do not vaccinate against PCV2 (NON-VAC) and from 22 farms that do vaccinate (VAC) were tested with quantitative real-time PCR. Additionally, nucleotide sequences of ORF2 of the virus were obtained from selected samples. Three genotypes, PCV2a, PCV2b, and PCV2d, were detected. Significant differences (p < 0.05) in PCV2 prevalence and quantities between the VAC and NON-VAC farms were evident. In five VAC farms, no viremia or shedding in feces was detected. On the other hand, in four VAC farms, the results were very similar to those from NON-VAC farms. No significant difference in PCV2 prevalence in oral fluids was observed between VAC and NON-VAC farms. An examination of viremia can be recommended for the detection of vaccination efficacy issues. The median of the PCV2 viral loads >6.0 log10 copies/mL in pooled sera from the vaccinated population should be considered a very strong indication that the vaccination protocol needs revision.

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