scholarly journals Rapid Detection and Typing of Actinobacillus pleuropneumoniae Serovars Directly From Clinical Samples: Combining FTA® Card Technology With Multiplex PCR

2021 ◽  
Vol 8 ◽  
Author(s):  
Oliver W. Stringer ◽  
Janine T. Bossé ◽  
Sonia Lacouture ◽  
Marcelo Gottschalk ◽  
László Fodor ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Lung Tissue ◽  
Low Cost ◽  
Actinobacillus Pleuropneumoniae ◽  
Economic Losses ◽  
Clinical Samples ◽  
High Morbidity ◽  
Swine Industry ◽  
Tissue Samples ◽  
Diagnostic Pcr

Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality, and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs. We have optimized the processing of 3-mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-multiplex PCR (mPCR) assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

scholarly journals Streptococcus pluranimalium 2N12 Exerts an Antagonistic Effect Against the Swine Pathogen Actinobacillus pleuropneumoniae by Producing Hydrogen Peroxide

2021 ◽  
Vol 8 ◽  
Author(s):  
Katy Vaillancourt ◽  
Michel Frenette ◽  
Marcelo Gottschalk ◽  
Daniel Grenier
Keyword(s):  
Hydrogen Peroxide ◽  
Culture Supernatant ◽  
Actinobacillus Pleuropneumoniae ◽  
Antagonistic Effect ◽  
Economic Losses ◽  
Protective Mechanism ◽  
Upper Respiratory Tract ◽  
Lactate Oxidase ◽  
Swine Industry

Actinobacillus pleuropneumoniae is the causal agent of porcine pleuropneumonia, a highly contagious and often deadly respiratory disease that causes major economic losses in the swine industry worldwide. The aim of the present study was to investigate the hydrogen peroxide (H2O2)-dependent antagonistic activity of Streptococcus pluranimalium 2N12 (pig nasal isolate) against A. pleuropneumoniae. A fluorimetric assay showed that S. pluranimalium produces H2O2 dose- and time-dependently. The production of H2O2 increased in the presence of exogenous lactate, suggesting the involvement of lactate oxidase. All 20 strains of A. pleuropneumoniae tested, belonging to 18 different serovars, were susceptible to H2O2, with minimal inhibitory concentrations and minimal bactericidal concentrations ranging from 0.57 to 2.3 mM. H2O2, as well as a culture supernatant of S. pluranimalium, killed planktonic cells of A. pleuropneumoniae. Treating the culture supernatant with catalase abolished its bactericidal property. H2O2 was also active against a pre-formed biofilm-like structure of A. pleuropneumoniae albeit to a lesser extent. A checkerboard assay was used to show that there were antibacterial synergistic interactions between H2O2 and conventional antibiotics, more particularly ceftiofur. Based on our results and within the limitations of this in vitro study, the production of H2O2 by S. pluranimalium could be regarded as a potential protective mechanism of the upper respiratory tract against H2O2-sensitive pathogens such as A. pleuropneumoniae.

scholarly journals Draft Genome Sequence of Actinobacillus pleuropneumoniae Serotype 7 Strain S-8

2012 ◽  
Vol 194 (23) ◽  
pp. 6606-6607 ◽  
Author(s):  
Gang Li ◽  
Fang Xie ◽  
Yanhe Zhang ◽  
Chunlai Wang
Keyword(s):  
Genome Sequence ◽  
Draft Genome ◽  
Actinobacillus Pleuropneumoniae ◽  
Draft Genome Sequence ◽  
Economic Losses ◽  
Negative Bacterium ◽  
Swine Industry ◽  
Gram Negative ◽  
Content Type ◽  
Porcine Pleuropneumonia

ABSTRACTThe Gram-negative bacteriumActinobacillus pleuropneumoniaeis the etiological agent of porcine pleuropneumonia, a respiratory disease that leads to severe economic losses in the swine industry. For years, scientists working with it have lacked a reliable genome sequence for comparison with otherActinobacillusspecies. Here, we report the draft genome sequence ofA. pleuropneumoniaeserotype 7 (strain S-8), isolated from swine lung in China in 1992.

scholarly journals Development of an accurate lateral flow immunoassay for PEDV detection in swine fecal samples with a filter pad design

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Siyi Zou ◽  
Lei Wu ◽  
Gan Li ◽  
Juan Wang ◽  
Dongni Cao ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Cross Reactivity ◽  
Rapid Diagnosis ◽  
Lateral Flow ◽  
Economic Losses ◽  
Lateral Flow Immunoassay ◽  
High Morbidity ◽  
Swine Industry ◽  
Fecal Samples ◽  
Diarrhea Virus

AbstractPorcine epidemic diarrhea virus (PEDV), as the main causative pathogen of viral diarrhea in pigs, has been reported to result in high morbidity and mortality in neonatal piglets and cause significant economic losses to the swine industry. Rapid diagnosis methods are essential for preventing outbreaks and transmission of this disease. In this study, a paper-based lateral flow immunoassay for the rapid diagnosis of PEDV in swine fecal samples was developed using stable color-rich latex beads as the label. Under optimal conditions, the newly developed latex bead-based lateral flow immunoassay (LBs-LFIA) attained a limit of detection (LOD) as low as 103.60 TCID50/mL and no cross-reactivity with other related swine viruses. To solve swine feces impurity interference, by adding a filtration unit design of LFIA without an additional pretreatment procedure, the LBs-LFIA gave good agreement (92.59%) with RT-PCR results in the analysis of clinical swine fecal samples (n = 108), which was more accurate than previously reported colloidal gold LFIA (74.07%) and fluorescent LFIA (86.67%). Moreover, LBs-LFIA showed sufficient accuracy (coefficient of variance [CV] < 15%) and stable (room temperature storage life > 56 days) performance for PEDV detection, which is promising for on-site analysis and user-driven testing in pig production system.

A Report on Massive Outbreaks of FMD with Concurrent Mannhaemia hemolytica Infection among Cattle in Tamil Nadu

2020 ◽  
Author(s):  
K. Manimaran ◽  
S. Hemalatha ◽  
A. Ramesh ◽  
S. Jaisree ◽  
K. Kumanan ◽  
...  
Keyword(s):  
Mortality Rate ◽  
Multiplex Pcr ◽  
Tamil Nadu ◽  
Large Scale ◽  
Sandwich Elisa ◽  
Economic Losses ◽  
High Morbidity ◽  
Serum Samples ◽  
Adult Cattle ◽  
Serotype O

Back ground: India has a huge resource of livestock and stands first globally in milk production, but reemergence of diseases like foot and mouth disease (FMD) causes a great concern and economic losses to the farmers. Three serotypes of FMD virus (O, A and Asia 1) are prevalent and serotype O was responsible for 80 per cent of confirmed outbreaks /cases in India. The highest incidence of the disease was seen in Southern region (31.5%) and during the month of September. Highest number of outbreaks (201) occurred in Tamil Nadu in the year 2007-08 caused by serotype O virus. FMD causes very high morbidity and mortality rate among young animals and the mortality rate is generally low in adult animals and seen with manifestation of infection limited to epithelial sites. However, In the present study an unusually high mortality among adult cattle due to FMD, complicated with Mannheimia hemolytica infection with specific myocardial lesions were discussed in several outbreaks of FMD in the state of Tamil Nadu, India. Methods: Outbreaks of FMD occurred in various districts of Tamil Nadu during the period of October to December in the year 2013 causing large scale mortality among adult cross bred (Jersey cross and Holstein Friesian cross) cows, heifers and calves and detailed disease investigations were carried out .Samples were collected from ailing animals and dead animals for laboratory confirmation of the disease. The collected samples were subjected to isolation of bacteria, serology and molecular confirmation of the virus. Result:Affected animals showed characteristic lesions of FMD with multiple, focal to coalescing ulcers and erosions covered with shreds of epithelium and fibrin on the dorsum and lateral margins of the tongue, dental pad, hard palate, mucosa of lips and gums. Samples of vesicular fluid, oral and tongue epithelium and heart confirmed the presence of FMD type ‘O’ virus based on sandwich ELISA and multiplex PCR. Cultural examination of bacteriological samples revealed presence of Mannheimia hemolytica which was further confirmed by multiplex PCR. Out of 83 serum sample collected, 45 sera were positive for FMD by LPB ELISA, while 32 serum samples were positive for FMD by DIVA test. The sudden deaths among adult cattle and heifers during the FMD outbreak might be attributed to the severe myocarditis and the disease was controlled by following appropriate treatment and preventive measures.

scholarly journals Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

2010 ◽  
Vol 192 (21) ◽  
pp. 5625-5636 ◽  
Author(s):  
Zhuofei Xu ◽  
Xiabing Chen ◽  
Lu Li ◽  
Tingting Li ◽  
Shengyue Wang ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Severe Disease ◽  
Gene Clusters ◽  
Actinobacillus Pleuropneumoniae ◽  
Etiologic Agent ◽  
Economic Losses ◽  
Comparative Genomic ◽  
Swine Industry ◽  
Genomic Characterization ◽  
Reference Strains

ABSTRACT The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.

scholarly journals Molecular characterization of Pasteurella multocida isolates from swine lungs by Randomly Amplified Polymorphic DNA

2015 ◽  
Vol 46 (1) ◽  
pp. 119-125
Author(s):  
Cristiane Silva Chitarra ◽  
Mayara Inácio Vincenzi da Silva ◽  
Laila Natasha Santos Brandão ◽  
Francielle Cristina Kagueyama ◽  
Stefhano Luis Candido ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Pasteurella Multocida ◽  
Economic Losses ◽  
Clinical Samples ◽  
Atrophic Rhinitis ◽  
Randomly Amplified Polymorphic Dna ◽  
Swine Industry ◽  
Mato Grosso ◽  
Swine Herds

ABSTRACT: Swine respiratory diseases such as atrophic rhinitis and bronchopneumonia caused by Pasteurella (P.) multocida cause important economic losses to the modern swine industry. The purpose of this study was to characterize P. multocida strains isolated from swine lungs by RAPD (Randomly Amplified Polymorphic DNA) to demonstrate their genetic diversity. Ninety-four samples of fragments from lungs with pneumonia and sixty one samples without pneumonia were collected in slaughterhouses in Mato Grosso during the period from December 2009 to March 2010. Clinical cases in 2012 and 2013 were also included in this study. Among the lung fragments with macroscopic lesions, without macroscopic lesions and clinical samples, 40.42%, 4.49% and 100% were positive for P. multocida, respectively. Bacterial identification culturing was confirmed by PCR (polymerase chain reaction) by means of the amplification of the gene kmt1. RAPD technique was performed for 46 isolates, and in every isolate, a total of 7 to 11 amplification bands were detected, composed of 8 clusters based on genetic similarity. Thus, treatment, control and preventive measures should consider the genetic diversity of P. multocida populations in swine herds in order to improve the development of new protocols to produce antimicrobials and vaccines.

scholarly journals Binding of Actinobacillus pleuropneumoniae to Phosphatidylethanolamine

2003 ◽  
Vol 71 (8) ◽  
pp. 4657-4663 ◽  
Author(s):  
Marie-Eve Jeannotte ◽  
Maan Abul-Milh ◽  
J. Daniel Dubreuil ◽  
Mario Jacques
Keyword(s):  
Bacterial Colonization ◽  
Actinobacillus Pleuropneumoniae ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
Economic Losses ◽  
Respiratory Pathogens ◽  
Swine Industry ◽  
Molybdenum Blue ◽  
O Antigen ◽  
Serotype 1

ABSTRACT The gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of A. pleuropneumoniae serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve as receptors for several bacteria, including respiratory pathogens. To study this effect, we used thin-layer chromatography overlay binding assays to test commercial phospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine (PE). Our results indicate that A. pleuropneumoniae serotype 1 binds to PE but not to the other phospholipids tested. Serotypes 5b and 7, which, along with serotype 1, are the most prevalent serotypes of A. pleuropneumoniae in North America, share the ability to bind PE. Inhibition of binding with a monoclonal antibody against A. pleuropneumoniae serotype 1 O antigen and the use of isogenic lipopolysaccharide (LPS) mutants of A. pleuropneumoniae serotype 1 showed that the O antigen seems to be implicated in the binding to PE, at least for A. pleuropneumoniae serotype 1. A. pleuropneumoniae was also shown to bind to a phospholipid extracted from swine lungs by using the method of Folch. Chemical staining with molybdenum blue and ninhydrin, migration with neutral, acidic, and basic solvent systems, and mass spectrometry analysis all indicated that this lipid is PE. This study is, to the best of our knowledge, the first description of A. pleuropneumoniae binding to phospholipids. Our data also suggest that LPS O antigens could be involved in binding to PE.

scholarly journals Antigens of Actinobacillus pleuropneumoniae and their use in the design of vaccines, especially glycoconjugates

2018 ◽  
Vol 72 ◽  
pp. 471-480
Author(s):  
Sylwia Przybyl ◽  
Wojciech Jachymek
Keyword(s):  
Actinobacillus Pleuropneumoniae ◽  
Economic Losses ◽  
Swine Industry ◽  
Carbohydrate Antigens ◽  
Microbial Structures ◽  
Protein Conjugates ◽  
Contagious Diseases ◽  
Young Pigs ◽  
Potential Vaccine ◽  
Prevention Methods

Actinobacillus pleuropneumoniae (further: A. pleuropneumoniae) is microaerophilic, Gram-negative bacteria belonging to the Pasteurellaceae family. This pathogenic microorganism is a major cause of porcine pleuropneumonia and fibrinous pleurisy, highly contagious diseases of the respiratory tract, affecting predominantly young pigs. Pleuropneumonia and fibrinous pleurisy can be diagnosed due to cough combined with a high mortality and the common infection route is direct transfer of bacteria by aerosol. A. pleuropneumoniae is a significant factor for economic losses in the swine industry all over the world. Progress made in research concerning a new potential vaccine enables the development of technology for designing safe, new candidates which could provide full protection against most of A. pleuropneumoniae serotypes. Several immunogenic factors of A. pleuropneumoniae have been found, including carbohydrate antigens, protein molecules and lipostructures. Carbohydrate antigens, as capsular polysaccharides (CPS) and lipopolysaccharides (LPS), have a special place due to their properties and wide potential. Some of these microbial structures were used to create subunit vaccines containing polysaccharide-protein conjugates, which seem to be a promising solution. The prevention methods, such as vaccines, should minimize medical expenses and financial losses. This article shows a wide repertoire of A. pleuropneumoniae antigens and focuses on one of the most promising strategies in vaccine design – glycoconjugate vaccines.

scholarly journals Identification and Distribution of Causative Agents of Pleurisy in the Brazilian Pig Farming

2018 ◽  
Vol 46 (1) ◽  
pp. 8
Author(s):  
Enio Rivelino Maria Nascimento ◽  
Ricardo Zanella ◽  
Lucas Fernando Dos Santos ◽  
Rosalí Ebertz ◽  
Diego Finamor Nascimento ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Pasteurella Multocida ◽  
The State ◽  
Economic Losses ◽  
Swine Industry ◽  
Tissue Samples ◽  
Commercial Laboratory ◽  
Stage Of Development ◽  
Causative Agents ◽  
Clinical Cases

Background: Pleurisy is defined as an inflammation of the pleural membranes, usually caused by bacterial infections. It is considered the second most common reason for condemnations of swine carcass during slaughter. Prior to slaughter, pleurisy can causes discomfort to the animals, and during the slaughter, the carcass from affected animals, has to be trimmed, reducing the value to both the farmer and the plant, increasing the costs of disposal, and line speeds are reduced, increasing processing costs. The objective of this study was to verify the prevalence and to identify the bacterial agents associated with pleurisy lesions in pigs.Materials, Methods & Results: In this study, were used tissue samples from 4,536 piglets collected from the main swine producing regions of Brazil between the years 2013/2014. Samples were collected from animals that presented respiratory, enteric or nervous symptoms. Samples were sent to pathology and bacteriology examinations in a commercial laboratory. The pathogen isolates were classified according to the agent Pasteurella multocida (P. multocida), Haemophilus parasuis (HPS), Streptococcus suis (S. Suis), Actinobacillus pleuropneumoniae (APP) and Bordetella bronchiseptica (BB) stage of development and regional location of the case. Data were analyzed based on the frequency according to the age group using Fisher-test. Of the total number of animals with clinical cases investigated in this study, 10.63% of them presented pleurisy lesions. When investigating the pleurisy frequency among the states or regions, we have observed that the state of Santa Catarina had the higher prevalence of pleurisy with 35.06% of cases, followed by the state of Minas Gerais with 35.31%, Rio Grande do Sul with 17.22% Paraná with 9.75% and the Midwest region had the lower prevalence with 6.02% of cases. Out of the total isolates, HPS were isolated in 55.0% of the total; P. multocida 31.0%; S. suis 7.0%; APP 6.0% and BB 1.0%. The higher prevalence of HPS was identified in southern states, while in MG, Southeast and Midwest there was equality between HPS and P. multocida isolations. The prevalence of HPS (P < 0.05) was in the nursery phase. P. multocida and APP prevailed in the growth and fattening phases.Discussion: In the swine industry, respiratory problems are responsible for huge economic losses caused to the animals, including treatment, condemnation of carcass up to the death of the animal, without including the animal pain and discomfort condition. Our data support the previous findings that P. multocida, was observed in higher frequency in the phase of growth (54.08%) and finishing (29.85%), being responsible to 83.93% of the cases in those phases. Therefore it could be considered the most economically important agent, since the HPS lesions are repaired over time, having low identification at later ages and slaughter of the pig. Similar findings were observed with APP. The S. suis and BB have been identified in sporadic forms. Knowing the possible agente causing the lesion based on the animal age and country region it is easier to predict with higher chances better management techniques to prevent the pathogen occurrence or even indicate a better treatment to reduce the lesion levels. Our data supported hypotheses of the identification of clinical cases and the animal growth stage for the most accurate classification for the success of animal treatment.

scholarly journals Detection of porcine circoviruses in clinical specimens using multiplex PCR in Hubei, central China

2018 ◽  
Author(s):  
Keli Yang ◽  
Zuwu Jiao ◽  
Danna Zhou ◽  
Rui Guo ◽  
Zhengying Duan ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Infection Rate ◽  
Target Genes ◽  
Limit Of Detection ◽  
Central China ◽  
Clinical Samples ◽  
Monitoring And Control ◽  
Pcr Assay ◽  
Tissue Samples ◽  
Multiplex Pcr Assay

In order to detect and simultaneously discriminate PCV1, PCV2 and PCV3, a multiplex PCR assay was developed and used to detect clinical samples in this study. Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 and PCV3. The tissue samples from eight pig farms were detected using the multiplex PCR assay. The results showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and Real-time PCR methods. It proved that this multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei Province, Central China.

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