scholarly journals Investigation of the Food-Transmitted Parasites Trichinella spp. and Alaria spp. in Wild Boars in Greece by Classical and Molecular Methods and Development of a Novel Real-Time PCR for Alaria spp. Detection

Animals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2803
Author(s):  
Dimitris Dimzas ◽  
Taxiarchis Chassalevris ◽  
Zanda Ozolina ◽  
Chrysostomos I. Dovas ◽  
Anastasia Diakou
Keyword(s):  
Wild Boar ◽  
Real Time ◽  
Real Time Pcr ◽  
Large Subunit ◽  
Taqman Probe ◽  
Amplification Efficiency ◽  
Wild Boars ◽  
The Usa ◽  
Wild Boar Meat ◽  
Low Prevalence

Foodborne parasitic diseases represent a major threat to public health. Trichinellosis, caused by the nematode parasite Trichinella spp., is one of the most important foodborne diseases, while alariosis, caused by the trematode parasite Alaria spp., is less common in humans, and rare cases have been reported only in the USA and Canada. Both parasites can infect humans via the consumption of raw or undercooked wild boar meat. In order to investigate the prevalence of these parasites in wild boar meat in Greece, samples from the diaphragm pillars and the region of the mandibular angle from 128 wild boars, hunted in Greece, were collected. The samples were examined by classical parasitological (compression, artificial digestion, and Alaria spp. migration) and by molecular (real-time PCR) methods. For Trichinella spp. an existent real-time PCR detecting all species likely to be present in Greece was applied, while for Alaria spp. a real-time PCR was developed, employing an LNA TaqMan probe targeting the large subunit ribosomal RNA gene. All examined wild boar samples from Greece resulted negative for Trichinella and Alaria species, indicating a low prevalence of infection in the examined population. The novel real-time PCR for Alaria spp. has 81.5% amplification efficiency and is able to detect 0.12 larvae per 50 g of tissue and could be utilized as a complementary to AMT diagnostic tool in surveillance.

scholarly journals Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

2004 ◽  
Vol 70 (7) ◽  
pp. 3988-3995 ◽  
Author(s):  
Hebe M. Dionisi ◽  
Christopher S. Chewning ◽  
Katherine H. Morgan ◽  
Fu-Min Menn ◽  
James P. Easter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Subunit ◽  
Coal Tar ◽  
Dry Weight ◽  
Amplification Efficiency ◽  
Gene Sequences ◽  
Pcr Assay ◽  
Sediment Samples ◽  
Dioxygenase Genes

ABSTRACT We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 103 to (2.9 ± 0.3) × 105 copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 105 to (5.4 ± 0.4) × 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.

scholarly journals Leptospira fainei Detected in Testicles and Epididymis of Wild Boar (Sus scrofa)

Biology ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 193
Author(s):  
Giovanni Cilia ◽  
Fabrizio Bertelloni ◽  
Domenico Cerri ◽  
Filippo Fratini
Keyword(s):  
Wild Boar ◽  
Real Time ◽  
Reproductive System ◽  
Real Time Pcr ◽  
Sus Scrofa ◽  
Rrna Gene ◽  
Pathogenic Leptospira ◽  
Wild Boars ◽  
First Time ◽  
Opening Up

Leptospirosis is a re-emerging and worldwide diffused zoonosis. Recently, the high importance of their epidemiology was explained by the intermediate Leptospira strains. Among these strains, Leptospira fainei was the first intermediate strain detected in domestic and wild swine. Wild boars (Sus scrofa) are well known as a reservoir, as well as all swine, for pathogenic Leptospira, but very little information is available concerning intermediate Leptospira infection. The investigation aim was to evaluate if intermediate Leptospira can infect the reproductive systems of wild boars hunted in the Tuscany region (Italy), as previously demonstrated for pathogenic ones. The reproductive system tissue (testicles, epididymides, uteri), and placentas and fetuses, were collected from 200 regularly hunted animals. Bacteriological examination and real-time PCR were performed to detect intermediate Leptospira DNA. Unfortunately, no isolates were obtained. Using real-time PCR, in six (3%) male organs (both testicles and epididymis), intermediate Leptospira DNA was found. The amplification of the 16S rRNA gene identified that all DNA obtained belong to Leptospira fainei. The results of this investigation highlighted for the first time the localization of Leptospira fainei in the male wild boar reproductive system, opening up a new avenue to further investigate.

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 Trichinosis in Lublin Province in 2003-2010 on a Background of Wild Boar`S Population Dynamics

2012 ◽  
Vol 56 (1) ◽  
pp. 43-46
Author(s):  
Marian Flis
Keyword(s):  
Wild Boar ◽  
Fold Increase ◽  
Wild Boar Population ◽  
Wild Boars ◽  
Hospitalised Patients ◽  
Species Population ◽  
Wild Boar Meat ◽  
Population Interaction ◽  
Domestic Swine ◽  
Epidemiological Situation

Abstract The research aimed at evaluating the epizootic and epidemiological situation of trichinosis during the last 8 years in Lublin province on a background of progressing increase in wild boar population within the region and in the whole country. Data for the study were taken from the report on the results of the official examination of slaughter animals and meat, poultry, game, lagomorphs and aquaculture animals and six reports on the number of trichinosis cases found at wild boars and domestic swine. In order to evaluate the trichinosis epidemiological situation within the region, reports of the National Institute of Public Health-National Institute of Hygiene on the number of identified trichinosis cases in people, as well as the number of hospitalised patients were presented. In addition, information on the population and hunting achievement of wild boars in hunting circuits of Lublin province during the last 8 years was enclosed. The number of identified trichinosis cases in meat of wild boars from Lublin region increased 9 times, while the percentage of trichinosis occurrence in reference to the number of examined carcasses almost 3-fold. At the same period, the number of porcine carcasses, in which trichinosis was found, decreased by over 4 times. Over double increase in wild boar population on the studied area was observed during the evaluation. Dynamic increase in the population size - in an aspect of the species population interaction with the living habitat, and in the form of the increase in the number of damages of crops and cultivation fields - contributed to intensified hunting pressure towards the species expressed as almost 3-fold increase of wild boar hunting. Analysis of epizootic and epidemiological situation of Poland indicates that wild boar meat was the principal source of trichinosis during the studied period. Considering Lublin province, the number of identified trichinosis cases is still high as compared to eastern and central provinces. Meanwhile, when compared to western and northern Poland, the level of trichinosis invasion can be considered as low. Furthermore, the trichinosis morbidity among people, that does not exceed 0.18/100 thousand inhabitants, can be regarded as low. Nevertheless, the fact of underestimating the necessity of both wild boar’s and swine’s meat examination seems to be alarming

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Evaluation of droplet digital PCR for the detection of black canker disease in tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Li Wang ◽  
Tian Qian ◽  
Pei Zhou ◽  
Wenjun Zhao ◽  
Xianchao Sun
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Detection Threshold ◽  
Digital Pcr ◽  
Taqman Probe ◽  
Tomato Seed ◽  
Canker Disease ◽  
Probe Set

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

scholarly journals Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3031-3040 ◽  
Author(s):  
Shabnam Rahimi-Khameneh ◽  
Sanni Hsieh ◽  
Renlin Xu ◽  
Tyler J. Avis ◽  
Sean Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phylogenetic Analyses ◽  
Similarity Index ◽  
Taqman Probe ◽  
Economic Losses ◽  
In Planta ◽  
Pcr Assay ◽  
Blind Test ◽  
Reliable Detection

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

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