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To demonstrate the relationship between tumour development and virus replication, eight specific-pathogen-free pullets of line P2 (Group P; 14 weeks old) and five adult chickens (Group A; 96 weeks old) were inoculated with virulent Marek’s disease virus (vMDV). Five chickens of Group P died or were euthanised due to moribund condition following the development of neoplastic lesions between days 53 and 91. On histopathological examination, these lesions were characterised by the proliferation of lymphoid cells of variable size. On analysis by polymerase chain reaction (PCR), the MDV meq gene was detected in Group P from day 21, and it was continuously identified in five chickens until they died or were euthanised. Abnormal signs and histopathological changes were not observed in chickens of Group A. The MDV meq gene was temporarily detected in some chickens of Group A, but it remained almost undetectable throughout the experimental period. In older chickens inoculated with vMDV, the onset of MD lymphoma development tended to be delayed as compared with the young chicks. The relationship between MD lymphoma development and virus replication in older chickens has been suggested. Our data might indicate the underlying existence of an age-related resistance to vMDV challenge.

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Acta Veterinaria Hungarica
Authors:
Károly Erdélyi
,
János Gál
,
László Sugár
,
Krisztina Ursu
,
Petra Forgách
,
Levente Szeredi
, and
Theodora Steineck

Oval, firm, cutaneous tumours with a rough, hairless, pigmented surface, exhibiting a moderately pronounced papillary structure were detected on the abdominal skin of two young red deer ( Cervus elaphus ). One animal was shot in Lower Austria in 2004, the other at a deer farm in Hungary in 2007. Histological examination of both samples classified the tumours as fibropapillomas, showing marked proliferation of fibroblasts and connective tissue, accompanied by hyperkeratosis, parakeratosis and acanthosis of the overlaying epidermis, and occasional foci of inflammation. The distribution of cytokeratin and vimentin was characterised in the lesion. The presence of papillomavirus (PV) antigen was demonstrated by immunohistochemistry in both cases. Papillomavirus-specific DNA was successfully amplified by PCR from one sample. The obtained partial nucleotide sequence of the L2 ORF exhibited the highest critical identity values with the homologous regions of Delta-papillomaviruses, especially the Roe deer papillomavirus (93%). Phylogenetic analysis of the partial L2 ORF sequence alignment of 10 papillomaviruses by both neighbour-joining and maximum parsimony method confirmed that the Red deer PV is very closely related to the Western roe deer papillomavirus (CcPV1).

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Acta Veterinaria Hungarica
Authors:
Zsuzsanna Tapaszti
,
Petra Forgách
,
Csaba Kővágó
,
László Békési
,
Tamás Bakonyi
, and
Miklós Rusvai

Microsporidiosis (nosema disease) of the European honeybee ( Apis mellifera L.) is present in bee colonies worldwide. Until recently, Nosema apis had been regarded as the causative agent of the disease, which may have many negative effects on the colony and cause heavy economic losses in apicultures. Another microsporidium species, Nosema ceranae , was reported to infest the Asian honeybee ( Apis ceranae ), but both honeybee species are susceptible to both microsporidia. In the European honeybee N. ceranae was first detected in Spain in the year 2006. As it is difficult to distinguish N. ceranae and N. apis morphologically, a rapid and accurate assay has been developed to differentiate N. apis and N. ceranae based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the partial large subunit ribosomal RNA. The assay was tested on 38 Nosema -infested bee samples, which were collected from geographically distant Hungarian bee colonies representing all regions of the country. Only one sample contained N. apis , and in the other 37 samples N. ceranae was detected, which indicates the dominance of N. ceranae in Hungarian apiaries. This is the first report on the presence of N. ceranae in Hungary.

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The genus Geranium (Geraniaceae); with about 320 species throughout the temperate regions, is chemically characterised by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. It is necessary to optimise the extraction protocols to reduce the effects of the presence of these compounds to the lowest level.

The present study compares the plant genomic DNA extraction Kit (DNP™ Kit), CTAB DNA extraction method by Murray and Thompson and Sahu et al., from the extracting DNA point of view Geranium species. The results showed significant differences in DNA contents between the three methods. Quantity and quality of extracted genomic DNAs were compared by employing the spectrophotometer, Nano-Drop, agarose gel electrophoresis, and polymerase chain reaction (PCR) methods and molecular marker such as (ITS and trnL-F) and ISSR. The method of Sahu et al., provided the best results (200 ng/µL) in terms of quantity and quality of DNA, therefore, this method was taken and optimised for DNA extraction. Our results proposed that this method could be effective for plants with same polysaccharides, proteins and polyphenols components. The advantage of this method is that it omits the use of liquid nitrogen and toxic phenols which are expensive. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Geranium species group and the reliability of this method has been discussed.

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Nanopages
Authors:
Imrich Barák
,
Massimo Barbaro
,
Dušan Blaškovič
,
Annalisa Bonfiglio
,
Andrea Alessandrini
,
Denisa Mullerová
,
Paolo Facci
, and
Luigi Raffo

Number of pathogenic tick-borne bacteria cause diseases in humans and animals. Wide range of conventional methods as PCR, immuno-based detection, cultivation, reverse line blot or microscopy is available for identification of bacterial pathogens. Although, these methods are used often in laboratories, they do not allow simultaneous detection of wide spectrum of bacteria. DNA chips represent technology for fast, sensitive, relatively cheap and simultaneous detection of microorganisms in biological samples. We have developed the oligo-chip for detection of different tick-borne pathogens. This method is based on detection of fluorescent signal after hybridization reaction by using laser scanner. We are also working on preparation of electronic device for detection of specific contact of two single complementary strands of DNA. This method will allow simple detection without using the fluorescent probes and expensive laser scanner. For this purpose a novel solid-state sensor for detection of bio-molecular processes was developed. The device is compatible with a standard CMOS process, providing fully electronic readout and large-scale of integration of biosensors on a single chip. A model of the device was developed and simulated.

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Acta Microbiologica et Immunologica Hungarica
Authors:
Reza Ranjbar
,
Ali Naghoni
,
Shohreh Farshad
,
Hadi Lashini
,
Ali Najafi
,
Nourkhoda Sadeghifard
, and
Caterina Mammina

We evaluated the performances of a newly designed real-time polymerase chain reaction (PCR) assay using TaqMan® probes to detect Salmonella Typhi. TaqMan® real-time PCR assays were performed by designed primers and probe based on the staG gene for detecting S. Typhi. The specificity of the assay was evaluated on 15 Salmonella serovars. The analytical specificity was evaluated on 20 non-Salmonella microorganisms. The analytical sensitivity was assessed using decreasing DNA quantities of S. Typhi ATCC 19430. Finally the detection capability of the TaqMan® real-time PCR assay on isolates recovered from patients with Salmonella infections was compared to the conventional PCR assay. Only S. Typhi strain had positive results when subjected to the assay using Typhi-specific real-time PCR. No amplification products were observed in real-time PCR with any of the non-Salmonella microorganisms tested. The TaqMan® real-time PCR was more sensitive than the conventional PCR. In conclusion, we found that the easy-to-use real-time PCR assays were faster than conventional PCR systems. The staG-based TaqMan® real-time PCR assay showed to be specific and sensitive method for the safe and rapid detection of the S. Typhi.

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The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

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Kidney samples from chickens diagnosed with acute nephritis and gout were subjected to histological and electron microscopic examination. The investigations revealed cytoplasmic inclusion bodies in the tubular epithelial cells containing round virions of about 30 nm in diameter. Since avian nephritis virus (ANV) is known as a potential causative agent of the so-called baby chick nephropathy, an RT-PCR assay was developed for the molecular detection of ANV-specific nucleic acid in the specimen. The specificity of the assay was confirmed by direct sequencing of the amplicon obtained in the reaction. The nucleotide sequence of the PCR product showed 92% identity with the reference ANV sequence deposited in the GenBank database. After having been validated on some other suspicious cases of avian nephritis, the PCR method described in this study can be a potential tool for routine diagnostic examination of samples submitted from cases of gout and nephropathy in chickens.

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Acta Veterinaria Hungarica
Authors:
Sergio Villanueva-Saz
,
Jacobo Giner
,
Antonio Fernández
,
María Magdalena Alcover
,
Cristina Riera
,
Roser Fisa
,
Andrés Yzuel
,
Ana González
,
Diana Marteles
, and
Maite Verde

detect parasitic nucleic acids by polymerase chain reaction (PCR) including conventional PCR, nested PCR and quantitative PCR; and finally serological methods based on the detection of a specific IgG response against L. infantum , including enzyme

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The aim of the present study was to assess the frequency of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) infection during pregnancy. 100–100 blood samples were collected from pregnant and non-pregnant women, then nucleic acid was isolated from both plasma and leukocytes fraction. Nested and real-time PCR were used to detect and differentiate HHV-6A and HHV-6B DNA and to determine viral loads. Reverse transcription PCR (RT-PCR) for HHV-6 U79/80 mRNA was performed in order to reveal active HHV-6 replication.HHV-6A and HHV-6B active infections were not detected in blood samples neither from pregnant nor from non-pregnant women. Frequency of HHV-6B and HHV-6A latency did not show difference between the studied groups (15% vs. 16%). HHV-6B latency was dominant in both studied groups (14/15 and 15/16). Beside these results, in leukocyte samples of one pregnant and three non-pregnant women high HHV-6A viral loads (1.28 × 105 − 5.07 × 105 GEq / 1.5 × 106 leukocytes) were detected, and viral DNA was also found in plasma samples. Although RT-PCR did not confirm virus replication, but chromosomal integration was also not proved unequivocally, the number of 0.08–0.33 HHV-6 copy / 1 leukocyte refers more to postnatal infection.

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