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Nidzworski D.,Medical University of Gdańsk | Wasilewska E.,Medical University of Gdańsk | Smietanka K.,The National Veterinary Research Institute | Szewczyk B.,Medical University of Gdańsk | Minta Z.,The National Veterinary Research Institute
Acta Biochimica Polonica | Year: 2013

Newcastle disease virus (NDV), member of the Paramyxoviridae family and avian influenza virus (AIV), member of the Orthomyxoviridae family, are two main avian pathogens causing serious economic problems in poultry health. Both are enveloped, single-stranded, negativesense RNA viruses and cause similar symptoms, ranging from sub-clinical infections to severe diseases, including decrease in egg production, acute respiratory syndrome, and high mortality. Similar symptoms hinder the differentiation of infection with the two viruses by standard veterinary procedures like clinical examination or necropsy. To overcome this problem, we have developed a new duplex real-time PCR assay for the detection and differentiation of these two viruses. Eighteen NDV strains, fourteen AIV strains, and twelve other (negative control) strains viruses were isolated from allantoic fluids of specific pathogen-free (SPF), embryonated eggs. Four-weeks-old SPF chickens were co-infected with both viruses (NDV - LaSota and AIV - H7N1). Swabs from cloaca and trachea were collected and examined. The results obtained in this study show that by using duplex real-time PCR, it was possible to detect and distinguish both viruses within less than three hours and with high sensitivity, even in case a bird was co-infected. Additionally, the results show the applicability of the real-time PCR assay in laboratory practice for the identification and differentiation of Newcastle disease and influenza A viruses in birds.


Durkalec M.,Wrocław University of Environmental and Life Sciences | Durkalec M.,The National Veterinary Research Institute | Szkoda J.,The National Veterinary Research Institute | Kolacz R.,Wrocław University of Environmental and Life Sciences | And 3 more authors.
International Journal of Environmental Research | Year: 2015

We used wild boars and roe deer as biomonitors of lead (Pb), cadmium (Cd), and mercury (Hg) contamination in two major industrial sites in Poland with different levels of toxic metal pollution. Masurian Lakes District, located far away from industry, was used as the reference site. Levels of Pb, Cd, and Hg in liver, kidney and muscle samples and in the stomach content of the animals were determined using atomic absorption spectroscopy (AAS) methods. We calculated also the mean concentration factors in the animal tissues versus their concentration in the gastric or rumen content. Our results indicate that area affected by metal smelting was more contaminated than brown coal mining area and the reference site, as indicated by higher levels of Pb and Cd in tissues and stomach contents of the animals. High levels of those metals in the offal of game animals may pose a threat to consumers of venison. © 2015, University of Tehran. All rights reserved.


PubMed | The National Veterinary Research Institute
Type: Journal Article | Journal: The Journal of parasitology | Year: 2014

The FLOTAC technique is a quantitative coproscopic method for the diagnosis of parasitic infection that is based on the centrifugation of a fecal sample to levitate helminth eggs with a flotation solution in a proprietary apparatus. Determination of the efficacy of the FLOTAC method and multiplication factors for calculation of the number of Toxocara, Trichuris, and Ascaris eggs in 1 g of feces on the basis of the number of detected eggs is presented. An investigation was conducted using feces samples enriched with a known number of parasite eggs: 3, 15, 50, or 100 parasite eggs of 3 nematode genera (Toxocara, Trichuris, and Ascaris) per 1 g (EPG) of feces. In addition, 80 samples of dog feces were prepared consisting of 20 repetitions for each level of contamination. The samples were analyzed using the FLOTAC basic technique. The limit of detection was calculated as the lowest level of egg content at which at least 50% of repetitions were positive. Multiplication factors for estimating the true number of parasite eggs in the samples were derived from regression coefficients that illustrated the linear relationship between the number of detected eggs and the number of eggs added to the sample. The percentages of recovered eggs for 1 chamber and for the whole apparatus ranged from 11.67 to 21.90% and from 21.33 to 40.10%, respectively, depending on dose enrichment and genus of parasite. The limit of detection calculated for the whole FLOTAC device was 3 EPG and was 15 EPG for 1 chamber for each of the 3 parasite genera. The limit of quantification calculated for whole FLOTAC was 15 EPG for each of 3 kinds of eggs. For 1 chamber, the limit of quantification was 15 EPG for Ascaris and Toxocara eggs and 50 EPG for Trichuris eggs. Multiplication factors for calculation of the number of eggs in 1 g of feces calculated for whole FLOTAC were 3 (for Toxocara and Ascaris eggs) and 4 (for Trichuris eggs). Experimentally calculated parameters of the method differ significantly from the theoretical assumptions of the authors of the FLOTAC technique and can significantly affect the reliability of the results. This does not alter the fact that the FLOTAC technique is the most effective parasitological quantitative method, which can be used to detect parasitic forms in feces. However, the results of our study emphasized the need for validation of the method before using it in the laboratory.


PubMed | The National Veterinary Research Institute
Type: Evaluation Studies | Journal: Journal of virological methods | Year: 2013

Swine influenza virus (SIV) causes a contagious and requiring official notification disease of pigs and humans. In this study, a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on primer-probe energy transfer (PriProET) for the detection of SIV RNA was developed. The assay uses matrix gene-specific primers and an Oregon Green-labeled fluorescent probe and was employed for the detection of SIV in clinical samples to identify outbreaks and to monitor the prevalence of disease. The PriProET technology was used to obtain a probe melting profile for confirmation of the specific product amplification. The assay is specific for influenza virus with a sensitivity of detection limit of approximately 10 copies of RNA by PCR. Based on serial dilutions of SIV, the detection limit of the assay was approximately 0.003 TCID(50)/ml for H1N1 A/Swine/Poland/KPR9/2004 virus. The PriProET RT-PCR was suitable for the detection of SIV RNA isolated directly from clinical samples. The assay detected SIV RNA in pre-clinical swab samples as early as 2 days post-infection (dpi). The PriProET RT-PCR assay is an alternative to the existing diagnostic assays and could have enhanced applicability for clinical diagnosis.

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