scholarly journals First report of Brenneria goodwinii and Gibbsiella quercinecans bacteria, detected on weaken oak trees in Poland

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Miłosz Tkaczyk ◽  
Laura Celma ◽  
Dainis Edgars Ruņģis ◽  
Gunita Bokuma
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quercus Robur ◽  
Abiotic Factors ◽  
Oak Decline ◽  
Groundwater Levels ◽  
Oak Trees ◽  
Field Samples ◽  
The Moment ◽  
Forest District

The decline of oak stands is a phenomenon that has been observed since the beginning of the 20th century in many European countries. It can be caused both with abiotic factors such as drought and fluctuations in groundwater levels, as well as biotic factors such as infestations by insects, fungi and bacteria. Acute Oak Decline (AOD) is an dangerous disease of oak trees which was first observed in the XX century. From the moment the first symptoms were noticed, the disease is able to kill trees in up to 6 years. Two species of bacteria, Brebberia goodwinii and Gibsiella quercinecand, are considered as one of the infectious agents of AOD. The decline of Quercs robur was reported from a weakened stand from the Chojnów Forest District. Symptomatic exudates were sampled and subjected to laboratory analysis. Using the Real time PCR, bacteria Brenneria goodwinii and Gibsiella quercinecans were confirmed in two out of seven collected field samples. This report is the first information on the observation of these bacteria in weakened stands with the participation of Quercus robur in Poland. Keywords: Quercus robur, bacteria, Brenneria goodwinii, Gibsiella quercinecans, real time PCR, Acute Oak Decline

Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

2019 ◽  
Vol 20 (2) ◽  
pp. 6-11
Author(s):  
Aly El-Kenawy ◽  
Mohamed El-Tholoth ◽  
Emad A
Keyword(s):  
Real Time ◽  
Disease Virus ◽  
Real Time Pcr ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Field Samples ◽  
Feather Follicle ◽  
Positive Results

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Development and validation of a real-time PCR specific for the leukotoxin gene of Bibersteinia trehalosi

2018 ◽  
Vol 30 (4) ◽  
pp. 589-592 ◽  
Author(s):  
Halcyon J. Killion ◽  
William Edwards ◽  
Jessica Jennings-Gaines ◽  
Mary Wood ◽  
Karen Fox ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Indirect Method ◽  
Pcr Primers ◽  
Bighorn Sheep ◽  
Mixed Species ◽  
Pcr Assay ◽  
Field Samples ◽  
Bibersteinia Trehalosi ◽  
Development And Validation

A real-time PCR assay for the leukotoxin gene of Bibersteinia trehalosi was developed and validated to better identify this pathogen, which is a cause of respiratory disease in bighorn sheep. The specificity of the PCR primers was evaluated with DNA from 59 known isolates of the Pasteurellaceae family. For validation, 162 field samples were compared using both the new assay and an indirect method using 2 sets of published protocols. The real-time PCR assay was found to be specific for the leukotoxin gene of B. trehalosi and provides a rapid and direct approach for detecting leukotoxin-producing forms of this organism from samples containing mixed species of leukotoxin-positive Pasteurellaceae.

scholarly journals Factores ambientales en la distribución de la seca en la encina (Quercus ilex subsp. ballota) = Environmental Variables in the Distribution of La Seca Disease in the Holm Oak (Quercus Ilex Subsp. Ballota)

2018 ◽  
pp. 107
Author(s):  
Mario Corral Ribera ◽  
Concepción Fidalgo Hijano ◽  
Begoña Peco Vázquez
Keyword(s):  
Environmental Variables ◽  
Quercus Ilex ◽  
Phytophthora Cinnamomi ◽  
Abiotic Factors ◽  
Information Criteria ◽  
Holm Oak ◽  
Oak Decline ◽  
Topographic Wetness Index ◽  
Daphne Gnidium ◽  
Field Samples

La combinación de factores bióticos y abióticos (clima, orografía, litología, competencia interespecífica, etc.) y la patogenicidad de agentes como el hongo Phytophthora cinnamomi Rands han generado la enfermedad fitosanitaria denominada en España como la seca de encinas y alcornoques de la Península Ibérica. El objetivo del presente estudio es el análisis que desempeñan las variables ambientales en el desarrollo de dicha enfermedad y determinar cuáles tienen una mayor influencia en su propagación. Se toma como área de estudio el Monte de Valdelatas, Alcobendas, Madrid (hoja 534 del Mapa Topográfico Nacional a escala 1:50.000. Coordenadas UTM X: X: 442592.51, Y: 4487266.63 zona 30T). Se obtivieron muestras en campo de 100 árboles de Quercus ilex subsp. ballota (50 con apariencia saludable y 50 con síntomas aparentes de enfermedad) azarosamente ubicados en 7 transectos. En relación al modelo estadístico se empleó un modelo binomial cuya variable respuesta se definió como sano/enfermo, según la apariencia del árbol. Así mismo, se manejó el Criterio de Información de Akaike (AIC) con el fin de establecer el mejor modelo. En su caso, presentando un 44% de la varianza (D2 = 0.437) y un valor de 89.68 en AIC. Las principales variables ambientales que interfieren en la enfermedad fueron: proximidad a cursos fluviales, índice de humedad topográfico, cobertura de Daphne gnidium y Quercus ilex subsp. ballota y la edad de los individuos muestreados. Dada la pérdida en la masa forestal de Quercus debido a la enfermedad de la seca, es necesario comprender el comportamiento de dicha enfermedad, así como conocer los factores ambientales que contribuyen a su expansión, pudiendo llegar de tal modo a identificar algún factor inhibidor. The combination of biotic and abiotic factors (climate, orography, lithology, interspecific competition, etc.) and the pathogenicity of agents such as the fungus Phytophthora cinnamomi Rands has generated the phytosanitary disease known as the oak decline (la seca) in holm oak and cork oak stand of the Iberian Peninsula. The aim of this study is to analyse the role played by environmental variables in the development of this disease, and to determine which have a stronger influence in the spread of the disease. Data has been obtained from Monte de Valdelatas, Alcobendas, Madrid (sheet 534 national topographic map, scale 1:50.000, UTM coordinates X: 442592.51, Y: 4487266.63 zone 30T). Field samples were taken from 100 randomly selected Quercus ilex subsp. ballota individuals (50 with a healthy appearance and 50 that showed disease symptoms), located within 7 transects. In relation to the statistical model, in this work a binomial model was used. On it, the response variable was defined as healthy/unhealthy, depending on the appearance of each tree. Akaike Information Criteria (AIC) was used to define the quality of the model. We selected model obtained a score of 89.68 and accounts for approximately 44% of data variability (D2 = 0,437). The main environmental variables that impact disease were: proximity to river course, topographic wetness index, cover of Daphne gnidium and Quercus ilex subsp. ballota, and the age of the individuals. Given the loss of Quercus forest mass caused by the spread of the oak decline disease, detected in several countries, including Spain, it becomes necessary to adequately understand the different factors that contribute and, in some cases, is the cause of the expansion of the disease, as well as to identify any inhibiting factors.

Inter-laboratory and inter-assay comparison on two real-time PCR techniques for quantification of PCV2 nucleic acid extracted from field samples

2009 ◽  
Vol 133 (1-2) ◽  
pp. 172-178 ◽  
Author(s):  
C.K. Hjulsager ◽  
L. Grau-Roma ◽  
M. Sibila ◽  
C. Enøe ◽  
L. Larsen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Field Samples ◽  
Pcr Techniques

scholarly journals Pseudomonas putida Isolation and Quantification by Real-Time PCR in Agricultural Soil Biodegradable Mulching

Agriculture ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 782
Author(s):  
Stefania Fontanazza ◽  
Alessia Restuccia ◽  
Giovanni Mauromicale ◽  
Aurelio Scavo ◽  
Cristina Abbate
Keyword(s):  
Pseudomonas Putida ◽  
Real Time ◽  
Real Time Pcr ◽  
Abiotic Factors ◽  
Polymer Surface ◽  
Subterranean Clover ◽  
Pcr Analysis ◽  
Plastic Pollution ◽  
Soil Particles ◽  
Mulch Films

To reduce the plastic waste problem in agriculture, biodegradable plastic (BP) mulch films have become of key importance thanks to their biodegradability and beneficial effects on crops. However, at present, BPs cannot always replace conventional plastics, because biodegradation is governed by many biotic and abiotic factors under field conditions. This research aimed at isolating and identifying, from soil particles directly attached to the surface of BP samples, the microorganisms responsible of degradation through a combined approach based on biodegradation and molecular tests. For this purpose, a field trial within a Mediterranean apricot orchard was carried out to study the biodegradation of a commercial BP mulch with respect to a no-BP, a conventional apricot management, following the standard agricultural practices, and a subterranean clover cover cropping, either incorporating or leaving its dead mulches on the soil surface. After BP film appeared visibly degraded in field, we isolated from soil particles attached to the polymer surface, a mesophilic bacterium with certain degradative potential assessed by plate and liquid assays, identified by sequencing as Pseudomonas putida. Quantitative real time PCR analysis showed the P. putida was significantly more abundant in PB plots than the other plot treatments. These preliminary results are potentially applicable to accelerate the degradation of BP mulch films and decrease the plastic pollution in agriculture.

scholarly journals Development of a Real-Time PCR based protocol for quantifying Radopholus similis in field samples

1970 ◽  
pp. 52-60
Author(s):  
P B Krishna, S J Eapen
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Its Region ◽  
Pcr Primers ◽  
Soil Samples ◽  
Radopholus Similis ◽  
Efficient Tool ◽  
Field Samples ◽  
Burrowing Nematode

The burrowing nematode, Radopholus similis, is an obligate migratory endoparasite. Currently detection of this nematode is carried out mostly by physically extracting them from soil and then observing under a light microscope. To identify this nematode, a thorough knowledge about their morphological features is quite indispensable. Developing a DNA based detection technique makes it more convenient and accurate in detection. Though PCR based methods have been reported by earlier workers, developing a Real-Time PCR based method will enable estimating their population in field samples. In this study, Real-Time PCR primers were designed using the DNA sequences from the ITS region of R. similis. It can detect R. similis up to the limit of 100 fg μL-1 DNA. The real time PCR based detection serves as an efficient tool for the detection and estimation of this nematode from soil samples.

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