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 ◽  
...  

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.

Author(s):  
C. Lalremruata ◽  
T.K. Dutta ◽  
P. Roychoudhury ◽  
Sanjeev Kumar ◽  
A. Sen ◽  
...  

Background: Illegal migration of pigs/piglets from Myanmar to Mizoram is a common practice to meet the local demands. The migrated animals are suspected as potential carrier of various microbial pathogens. The present study was conducted on isolation, identification and molecular characterization of major bacterial pathogens (Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Mycoplasma hyopneumoniae and Pasteurella multocida) in pigs illegally migrated from Myanmar to Mizoram. Methods: A total of 209 rectal swabs and 209 nasal swabs were collected from apparently healthy migrated pigs during October 2018 to April, 2019. All the samples were processed for PCR based detection of target bacterial species followed by isolation and identification by bacteriological techniques. The bacterial species were further confirmed by BD Phoenix automated bacterial identification system and selected virulence genes of the bacterial species were determined by specific PCR assay. Result: By species specific PCR, 110 samples were found to be positive for selected bacterial species, of which 20 (9.57%), 1 (0.478%), 86 (41.15%), 2 (0.956%) and 1 (0.478%) were A. pleuropneumoniae, B. bronchiseptica, H. parasuis, M. hyopneumoniae and P. multocida, respectively. A total of 52 bacterial strains were isolated and identified, of which 11, 1, 39 and 1 were A. pleuropneumoniae, B. bronchiseptica, H. parasuis, M. hyopneumoniae and P. multocida, respectively. Virulence genes were detected in A. pleuropneumoniae and H. parasuis isolates. Based upon the published literatures, this is the first ever report of isolation and identification of pathogenic A. pleuropneumoniae and H. parasuis in pigs in India.


Author(s):  
P.N. Shastin ◽  
A.V. Kapustin ◽  
E.A. Yakimova ◽  
E.V. Ivanov ◽  
A.I. Laishevtsev

The paper presents the results of bacterial screening of goat and sheep breeding enterprises in certain regions of Russia (Tver, Moscow, Smolensk regions, as well as the Republic of Mari-El and Tatarstan), conducted in the period from 2018 to 2021. In the course of this work, 556 samples of sectional material (heart, lungs, gastrointestinal tract, spleen, kidneys, liver, lymph nodes, breast, flushes from the genitourinary system, as well as exudate from purulent lesions) were subjected to a comprehensive bacteriological study. As a result of the conducted studies, 1223 isolates belonging to 25 families (111 bacterial species) were isolated and identified (by the method of time-of-flight mass spectrometry MALDI-ToF). According to the data obtained, the incidence of Escherichia coli isolation was 10.95%, Trueperella pyogenes – 5.47%, Staphylococcus aureus – 5.31%, Proteus mirabilis – 4.08%, Mannheimia haemolytica – 4.00%, Enterococcus faecalis – 3.76%, Enterobacter cloacea and Staphylococcus haemolyticus – 3.59%, Streptococcus dysgalactiae – 3.51%, Pasteurella multocida – 3.27%, Acinetobacter lwoffii – 2.78%, Staphylococcus cohnii – 2.61%, Bibersteinia trehalosi – 2.29%, Pseudomonas aeruginosa – 2.12%, Bacillus cereus and Micrococcus luteus – 1.96%, Corynebacterium pseudotuberculosis and Staphylococcus equorum – 1.88%, Aerococcus viridans – 1.80%, Corynebacterium xerosis – 1.72%, Clostridium perfringens, Streptococcus mitis and Streptococcus pyogenes – 1.39%, Staphylococcus chromogenes and Streptococcus entericus – 1.14%, respectively. The incidence of isolation of other types of microorganisms was below 1%. The data obtained indicate the circulation of a wide range of bacteria in goat and sheep breeding enterprises of the Russian Federation, some of which should be positioned as pathogenic flora (for example, Pasteurella multocida, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, Clostridium perfringens, etc.), some as conditionally pathogenic (Trueperella pyogenes, Staphylococcus aureus, Bibersteinia trehalosi, Mannheimia haemolytica, Pseudomonas aeruginosa, Moraxella bovis, Moraxella bovoculi, etc.), as well as the normal flora of the animal body. It is worth focusing on these data when conducting a survey of livestock enterprises in order to establish an objective epizootic situation, including taking into account the possibility of circulating pathogens of factor diseases.


2018 ◽  
Vol 63 (4) ◽  
pp. 759-765 ◽  
Author(s):  
V.R. Kundave ◽  
Hira Ram ◽  
Partha S. Banerjee ◽  
Rajat Garg ◽  
K. Mahendran ◽  
...  

Abstract This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10−6, 10−6 and 10−4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.


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 ◽  
...  

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.


2017 ◽  
Vol 25 (4) ◽  
pp. 1569-1575 ◽  
Author(s):  
Nadia Rajabzadeh ◽  
Mohsen Naeemipour ◽  
Mohsen Seyedabadi

2011 ◽  
Vol 236-238 ◽  
pp. 2803-2809
Author(s):  
Ai Li ◽  
Guo Xing Yang ◽  
Wei Zhang

To establish a rapid, sensitive and specific multiplex PCR method for the simultaneous detection ofStaphylococcus aureus,Salmonellaspp, andlisteria monocytogenes. Three pairs of primers have been designed according to theStaphylococcus aureusnucgene,Salmonellaspp IpaBgene,listeria monocytogenes inlAgene. Orthogonal experimental design was used to determine Multiplex PCR amplification system for Food-borne Bacterial Pathogens of four factors (Taq DNA polymerase, Mg2+, dNTP and primers) from four levels; three DNA fragments of 210bp,280bp and 476bp were amplified. The specificity and the sensitivity of this method was valued. Template was prepared using FTA filter; the three food-borne Bacterial Pathogens were simultaneously detected by the multiplex PCR technology which have been designed; The sensitivity of this method was 3.0×102cfu/mL forStaphylococcus aureus, 2.0×102cfu/mL forSalmonellaspp, and 3.5×102cfu/mL forlisteria monocytogenes. This method lies on its accuracy, rapidity and efficiency in the diagnosis, so it could be a useful method for the simultaneous detection of the three species of bacteria in food.


2021 ◽  
Vol 14 (4) ◽  
pp. 986-995
Author(s):  
Heba Roshdy ◽  
Azhar G. Shalaby ◽  
Ahmed Abd Elhalem Mohamed ◽  
Heba Badr

Background and Aim: Rabbits are a highly sensitive species and susceptible to various bacterial pathogens that may be causative agents for early embryonic death. This study aimed to explore the administration of different bacterial agents in does suffering from early embryonic death. Furthermore, identification of genes associated with virulence was performed to identify the phenotypic and genotypic antimicrobial resistance patterns that may increase the virulence of pathogens and lead to early embryonic death. Materials and Methods: We isolated and identified bacterial agents in 106 samples from live and dead female rabbits that had undergone early embryonic death, including liver and intestine tissue, aborted fetuses, discharges, and vaginal swabs. Conventional polymerase chain reaction (PCR) was conducted to confirm the identity of the isolated bacterial strains and their virulence. Moreover, antibiotic resistance was studied phenotypically and genotypically. Results: We isolated Escherichia coli, Salmonella, Staphylococcus aureus, Pasteurella multocida, and Listeria monocytogenes. PCR confirmed typical identification except in P. multocida, which was confirmed as Gallibacterium spp. in some cases. The final percentage of detection was 34%, 30.2%, 16.9%, 13.2%, and 11.3%, respectively. Virulence properties were investigated using different designated genes. All Salmonella strains harbored invA, stn, avrA, and ompf genes, while the sopE gene was identified in 31.25%. E. coli strains harboring the iss gene lacked the shiga toxin (stx1) gene. L. monocytogenes and S. aureus strains harbored the hemolysin gene (66.7% and 33.4%, respectively). Multidrug resistance was detected phenotypically and genotypically in most strains. Each bacterial pathogen had a different antibiotic resistance profile. Conclusion: Multiple bacterial species may contribute to early embryonic death in does. Furthermore, the combined infection could be the main cause of early embryonic death. Thus, monitoring programs should bear this in mind and focus on the early detection of these bacterial agents in female rabbits to avoid embryonic death.


2004 ◽  
Vol 67 (1) ◽  
pp. 27-33 ◽  
Author(s):  
YONG LI ◽  
AZLIN MUSTAPHA

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157: H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 × 10−1 CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 × 101 CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.


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