Rapid virulence typing of Pasteurella multocida by multiplex PCR

2009 ◽  
Vol 87 (3) ◽  
pp. 355-357 ◽  
Author(s):  
Sina Atashpaz ◽  
Jalal Shayegh ◽  
Mohammad Saied Hejazi
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Virulence Typing

scholarly journals Molecular identification by multiplex polymerase chain reaction of Pasteurella multocida in cattle and buffaloes in Baghdad

2014 ◽  
Vol 38 (1) ◽  
pp. 99-106
Author(s):  
Ihab G. M. AL-Shemmari
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Pasteurella Multocida ◽  
Type B ◽  
Chain Reaction ◽  
Blood Samples ◽  
Nasal Swabs ◽  
Polymerase Chain ◽  
B 31

The aim of this study was to identify pasteurella multocida and their types by PCR in cattle’s and buffaloesi bagdad from March to August 2012 on 204 animals , including 102 cattle and 102 buffaloes at slaughter houses from Baghdad .Blood samples and nasal swaps were collected , before slaughtering and lung tissues of slaughtered animal , and from 54 clinically suspected cases of pasteurellosis , including 27 bovines ,and 27 buffaloes the samples taken included blood and nasal swabs . Pasteurellamultocida were isolated from 94 animals include 49 cattle 45 buffaloes. The typing of the isolates by multiplex PCR for genotyping Pasteuerllamultocida revealed 93 isolates of type B , 31 from cattle and 62 from buffaloes ,and 81 isolates of type A , 55 from cattle and 26 from buffaloes .

scholarly journals Development of a multiplex PCR for simultaneous detection of Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes

2017 ◽  
Vol 65 (3) ◽  
pp. 327-339 ◽  
Author(s):  
Wenlong Zhang ◽  
Xiaodan Liu ◽  
Mengcheng Liu ◽  
Bo Ma ◽  
Li Xu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genomic Dna ◽  
Pasteurella Multocida ◽  
Bacterial Pathogens ◽  
Simultaneous Detection ◽  
Laboratory Diagnosis ◽  
Mannheimia Haemolytica ◽  
Bacterial Strains ◽  
Trueperella Pyogenes ◽  
Enrichment Technology

Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes are three bacterial pathogens closely associated with the bovine respiratory disease complex (BRDC). In the current study, a multiplex PCR for the simultaneous detection of these three bacteria in cultures was established. After serial optimisation, the detection limit of the method for the genomic DNA of the three bacteria was 40 pg/μl. The method could detect the genomic DNA of these three bacteria but not the genomic DNA of seven other bacterial strains. Together with the bacterial enrichment technology, the multiplex PCR could be used for detecting the three bacteria in animal tissues. This method might be valuable for speeding up laboratory diagnosis and directing the treatment of BRDC to these three bacterial pathogens.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

scholarly journals Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR

2013 ◽  
Vol 33 (2) ◽  
pp. 177-182 ◽  
Author(s):  
Thales Q. Furian ◽  
Karen A. Borges ◽  
Silvio L.S. Rocha ◽  
Everton E. Rodrigues ◽  
Vladimir P. do Nascimento ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
High Frequency ◽  
Respiratory Diseases ◽  
Pasteurella Multocida ◽  
Virulence Genes ◽  
Simultaneous Detection ◽  
Fowl Cholera ◽  
Avian Diseases ◽  
Genetic Profiles ◽  
Current Systems

The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC) is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25), the sodA and nanH genes were present in 96% (24/25), ptfA was present in 92% (23/25), and pfhA was present in 60% (15/25). Gene toxA was not identified in any of the samples studied (0/25). Five different genetic profiles were obtained, of which P1 (negative to toxA) was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

scholarly journals Pasteurella multocida capsular: lipopolysaccharide types D:L6 and A:L3 remain to be the main epidemic genotypes of pigs in China

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Songtao Liu ◽  
Lin Lin ◽  
Hao Yang ◽  
Wenqing Wu ◽  
Long Guo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Multiplex Pcr ◽  
Virulence Factor ◽  
Pasteurella Multocida ◽  
Pcr Detection ◽  
Respiratory Disorders ◽  
Resistance Profile ◽  
Antimicrobial Resistance Profile ◽  
Pcr Assays ◽  
Encoding Genes

AbstractPasteurella multocida is a leading cause of respiratory disorders in pigs. This study was designed to understand the genotypical and antimicrobial resistant characteristics of P. multocida from pigs in China. To achieve this, we briefly investigated 158 P. multocida isolates from pigs with respiratory disorders in China between 2019 and 2020. Genotyping through multiplex PCR assays assigned these 158 isolates into capsular genotypes A (60.13%, 95/158), D (35.44%, 56/158), F (4.43%, 7/158), and/or lipopolysaccharide (LPS) genotypes L3 (28.48%, 45/158) and L6 (66.46%, 105/158). In addition, eight isolates (5.06%, 8/158) were found to be nontypable using the LPS genotyping method. When combining the capsular genotypes and the LPS genotypes, D: L6 (34.81%, 55/158) and A: L6 (31.65%, 50/158) were the predominant genotypes, followed by A: L3 (24.05%, 38/158). PCR detection of virulence factor-encoding genes showed that over 80% of the isolates were positive for exbB, tonB, exbD, ompH, ptfA, fimA, sodA, sodC, fur, ompA, oma87, plpB, hsf-2, nanH and hgbB, suggesting the presence of these genes were broad characteristics of P. multocida. We also found approximately 63.92% (101/158), 51.27% (81/158), 8.86% (14/158), 7.59% (12/158), 3.16% (5/158), 0.63% (1/158), and 0.63% (1/158) of the isolates grew well in media with the presence of colistin (4 μg/mL), tetracycline (16 μg/mL), tigecycline (1 μg/mL), ampicillin (32 μg/mL), chloramphenicol (32 μg/mL), cefepime (16 μg/mL), and ciprofloxacin (1 μg/mL), respectively. This study contributes to the understanding of genotypes and antimicrobial resistance profile of P. multocida currently circulation in pigs of China.

scholarly journals Application of PCR technique in diagnosis of four respiratory pathogenic bacteria in pigs at the slaughterhouse

2019 ◽  
Vol 18 (03) ◽  
pp. 35-40
Author(s):  
Han M. Ly
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Pathogenic Bacteria ◽  
Pasteurella Multocida ◽  
Bordetella Bronchiseptica ◽  
Ho Chi Minh City ◽  
Haemophilus Parasuis ◽  
Chain Reaction ◽  
Injured Lung ◽  
Polymerase Chain

The polymerase chain reaction (PCR) for Actinobacillus pleuropneumonia (App), Haemophilus parasuis (Hps), Pasteurella multocida (Pm) and Bordetella bronchiseptica (Bb) were performed in pure colonies isolated from 114 lung specimens with lesions collected from the Vissan slaughterhouse in Ho Chi Minh City from July 2018 to May 2019. The aim of the experiment was to identify the four respiratory pathogenic bacteria in pigs at slaughterhouse by using PCR technique. The criteria for evaluating the results included the proportion of positive samples with multiplex PCR and percentage of samples co-infected with 2, 3, and 4 bacteria. Among a total of 114 injured lung samples, 21% of the samples was positive to at least one of the four bacteria, 3 samples (2.63%) were positive for App, 2 samples (1.75%) were positive for Hps, 7 samples (6.14%) were for Pm, and 12 lungs (10.53%) were positive for Bb. One sample (0.88%) was found co-infected with Pm and Hps.

scholarly journals Comparison of Phenotypic and Genotypic Identification Methods of Pasteurella multocida Serotypes Isolated from Pigs

2018 ◽  
Vol 44 (1) ◽  
pp. 5
Author(s):  
Amanda Figueiredo Amaral ◽  
Raquel Rebelatto ◽  
Catia Silene Klein ◽  
Karine Ludwig Takeuti ◽  
David Emilio Santos Neves de Barcellos
Keyword(s):  
Staphylococcus Aureus ◽  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Weighted Kappa ◽  
Type A ◽  
Kappa Coefficient ◽  
Identification Methods ◽  
Type D ◽  
Weighted Kappa Coefficient ◽  
Capsular Typing

Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs.  Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques.Materials, Methods & Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus was heavily streaked at right angles to the P. multocida lines and the plates were incubated at 37°C for 24 h. Type A isolates were then identified as those with smaller colonies in the region adjacent to the Staphylococcus aureus streak (negative satellitism). For acriflavine test, the isolates were inoculated into tubes containing 2 mL of BHI, incubated at 37°C for 18-24 h, centrifuged (500 g for 15 min) and 1.5 mL of the supernatant was discarded. A 1:1000 solution of acriflavine neutral (0.5 mL) was then added to the residual broth containing bacteria and kept at room temperature. Solutions of acriflavine were freshly prepared each week and stored protected from light. Type D strains were identified by the appearance of a heavy flocculent precipitate within 5 min. DNA extraction by heat shock was performed prior to multiplex PCR for the detection of capsular genes hyaD-hyaC (capsular typing A) and dcbF (capsular typing D). Test of symmetry and a weighted kappa coefficient were used to evaluate correlations and to assess agreement of the  results between the identification methods, respectively. Phenotypic tests showed that two isolates were type D (4.55%), 40 were type A (90.9%) and two (4.55%) were untypable isolates (4.55%) while PCR showed that 38 isolates were type A (86.36%) and six were type D (13.64%). The correlation analysis between the phenotypic and genotypic tests showed that 90.9% of the strains were identified as belonging to the same serotype by both tests and the weighted kappa coefficient (K = 0.633) indicates a substantial agreement between the two tests.Discussion: There was a disagreement between the phenotypic and genotypic results in four of the isolates (9.09%). The phenotypically untypable isolates were classified as type D by multiplex PCR. Nonetheless, we conclude that PCR testing is a more reliable method to differentiate between P. multocida serotypes A and D.

Phenotypic and genotypic characterisation of Pasteurella multocida strains isolated from pigs in Hungary

2007 ◽  
Vol 55 (4) ◽  
pp. 425-434 ◽  
Author(s):  
Zsuzsanna Varga ◽  
Boglárka Sellyei ◽  
T. Magyar
Keyword(s):  
Multiplex Pcr ◽  
Ornithine Decarboxylase ◽  
Pasteurella Multocida ◽  
Regional Distribution ◽  
Type A ◽  
Capsule Type ◽  
Type D ◽  
Biochemical Characterisation ◽  
River Danube ◽  
Swine Herds

A total of 146 Pasteurella multocida strains isolated from swine in Hungary in the last 20 years were examined. Biochemical characterisation and PCR-based techniques were used to determine species, subspecies, biovar, capsule type and presence of the toxA gene. Eighty-seven percent of the isolates belonged to P. multocida ssp. multocida , and 98% of these had biovar 3 or were trehalose-or lactose-fermenting or ornithine decarboxylase negative variants of that. Ten percent of the strains were P. multocida ssp. septica , and within this group 80% of the strains showed sorbitol-negative biovars (5, 6 and 7). The rest of the strains (20%) were lactose positive. Only 3% of the porcine isolates were P. multocida ssp. gallicida and 3 out of the 4 strains belonged to the dulcitol-fermenting biovar 8. Using a capsule-specific multiplex PCR, 60% of the strains belonged to capsule type D, 38% to capsule type A, and only 1 isolate had capsule type F. In contrast with data published in the literature, only 3% of capsule type D isolates carried the toxA gene, while this ratio was 41% for the type A strains. A remarkable regional distribution of toxA gene positive strains was observed. All but two isolates were found in swine herds located in the Transdanubian region, separated from other parts of Hungary by the river Danube.

scholarly journals Development of three multiplex-PCR assays for virulence profiling of different iron acquisition systems in Escherichia coli

2020 ◽  
Author(s):  
Hamideh Kalateh Rahmani ◽  
Gholamreza Hashemi Tabar ◽  
Mahdi Askari Badouei ◽  
Babak Khoramian
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Current Knowledge ◽  
Iron Acquisition ◽  
E Coli ◽  
Essential Nutrient ◽  
Pcr Assays ◽  
Virulence Typing ◽  
Different Origin

Background and Objectives: Escherichia coli is responsible for various enteric and extraintestinal infections in animals and humans. Iron as an essential nutrient, has a proven role in pathogenicity of E. coli. Pathogenic E. coli benefits of having complicated systems for iron acquisition but our current knowledge is limited because of complexity of these systems. In the present study, three multiplex-PCR assays were developed to screen nine different virulence genes related to diverse iron acquisition systems in E. coli. Materials and Methods: The multiplex-PCR systems were designed and optimized in three panels. Each panel includes a triplex-PCR cocktail. The panels are as follow: panel 1: iroN, iutA and fecA; panel 2: fyuA, sitA and irp2; and panel 3: iucD, chuA and tonB. A total of 39 pathogenic E. coli was screened according to the designed multiplex-PCR. Results: In total, the top three frequent genes were tonB (100%), fecA (66.6%) and sitA (58.9%). With the exception of fecA and tonB, comparing the prevalence of genes among different origin of isolates (human, cattle, poultry and pigeon) showed significant associations (P < 0.05). Moreover, the iroN, sitA and iucD genes were significantly prevalent (P < 0.05) among members of extraintestinal pathogenic E. coli in comparison with the group of diarrheagenic E. coli. Conclusion: The current multiplex-PCR assays could be a valuable, rapid and economic tool to investigate diverse iron acquisition systems in E. coli for more precise virulence typing of pathogenic or commensal strains.

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