Molecular Detection of Bacterial Pathogens Directly from the Nasal Swabs and Lung Tissue of Sheep and Goats

2019 ◽  
Vol 15 (02) ◽  
pp. 22-25
Author(s):  
Sunaina Thakur ◽  
Subhash Verma ◽  
Prasenjit Dhar ◽  
Mandeep Sharma
Keyword(s):  
Pasteurella Multocida ◽  
Direct Detection ◽  
Bacterial Species ◽  
Economic Losses ◽  
Isolation And Identification ◽  
Sheep And Goats ◽  
Histophilus Somni ◽  
Nasal Swabs ◽  
Mycoplasma Spp

Respiratory infections of sheep and goats cause heavy morbidity and mortality, leading to huge economic losses. Conventional methods of diagnosis that include isolation and identification of incriminating microbes are time-consuming and fraught with logistic challenges. Direct detection of incriminating microbes using molecular tools is gaining popularity in clinical, microbiological settings. In this study, a total of 50 samples (44 nasal swabs and 6 lung tissues) from sheep and goats were screened for the detection of different bacterial species by in vitro amplification of genus or species-specific genes. Histophilus somni was detected in 2% goat samples, Trueperella pyogenes in 20% goat nasal swabs, whereas 22% goat nasal swab samples were found positive for Mycoplasma spp. None of the samples from sheep was detected positive for H. somni, T. pyogenes, Mycoplasma spp. Similarly, all samples, irrespective, whether from sheep or goats, showed negative results for Pasteurella multocida, Mannheimia haemolytica, and Corynebacterium pseudotuberculosis.

scholarly journals Real-time quantitative PCR for detection and identification of Actinobacillus pleuropneumoniae serotype 2

2016 ◽  
Vol 60 (3) ◽  
pp. 253-256 ◽  
Author(s):  
Arkadiusz Dors ◽  
Andrzej Kowalczyk ◽  
Małgorzata Pomorska-Mól
Keyword(s):  
Real Time ◽  
Lung Tissue ◽  
Capsular Polysaccharide ◽  
Bacterial Species ◽  
Taqman Probe ◽  
Economic Losses ◽  
Isolation And Identification ◽  
Polysaccharide Biosynthesis ◽  
Specificity And Sensitivity ◽  
Nasal Swabs

AbstractIntroduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs.Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 104 CFU/mL, for lung tissue and nasal swabs it was 1.2 × 105 CFU/mL, and for tonsils - 1.2 × 105 CFU/mL.Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.

scholarly journals Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle

2021 ◽  
Vol 8 ◽  
Author(s):  
Alicia F. Klompmaker ◽  
Maria Brydensholt ◽  
Anne Marie Michelsen ◽  
Matthew J. Denwood ◽  
Carsten T. Kirkeby ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Age Groups ◽  
Therapeutic Interventions ◽  
Respiratory Pathogens ◽  
Logistic Regression Models ◽  
Histophilus Somni ◽  
Nasal Swabs

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

scholarly journals Transmission of Novel Bacterial Pathogens through Pigs Transported from Myanmar to Mizoram

2021 ◽  
Author(s):  
C. Lalremruata ◽  
T.K. Dutta ◽  
P. Roychoudhury ◽  
Sanjeev Kumar ◽  
A. Sen ◽  
...  
Keyword(s):  
Pasteurella Multocida ◽  
Virulence Genes ◽  
Bacterial Pathogens ◽  
Bacterial Species ◽  
Microbial Pathogens ◽  
Identification System ◽  
Bacterial Strains ◽  
Illegal Migration ◽  
Isolation And Identification ◽  
Specific Pcr

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.

Isolation and Identification of Bacterial Pathogens from Respiratory Tract of Goats

2017 ◽  
Vol 12 (3) ◽  
Author(s):  
Daljeet Chhabra ◽  
Preeti Barde ◽  
U. K. Garg ◽  
R> Sharda ◽  
Supriya Shukla
Keyword(s):  
Tract Infection ◽  
Respiratory Tract ◽  
Respiratory Tract Infection ◽  
Pasteurella Multocida ◽  
Bacterial Pathogens ◽  
Isolation And Identification ◽  
Blood Samples ◽  
Pulmonary Lesions ◽  
Nasal Swabs ◽  
Different Parts

A total of 170 samples were examined for bacterial pathogens after staining with Wright’s stain. These included oral swabs, nasal swabs, blood samples, impression smears and tissues from different parts of respiratory tract showing pulmonary lesions. Out of these, only 36 samples (21.17%) collected from clinically ill animals or morbid tissues were showing respiratory tract infection suggestive of respiratory tract infection of bacterial origin which were further processed for microbiological examinations. Pasteurella multocida and E.coli were isolated from 7(19.44%) and 11 samples (30.55%) out of 36 samples respectively in pure culture. The remaining samples did not reveal any bacterial growth.

Acaricidal Activity of Mongolian Plants Against Dermacentor nuttalli

2019 ◽  
Vol 28 (03) ◽  
pp. 26-33
Author(s):  
Banzragchgarav O ◽  
Battur B ◽  
Battsetseg B ◽  
Myagmarsuren P ◽  
Murata T ◽  
...  
Keyword(s):  
Tick Species ◽  
Amaranthus Retroflexus ◽  
Acaricidal Activity ◽  
Economic Losses ◽  
Isolation And Identification ◽  
Ephedra Sinica ◽  
Start Line ◽  
Potential Activity ◽  
First Time

Dermacentor nuttalli is the most commonly distributed tick species in Mongolia, and responsible for huge economic losses related to tick-borne diseases in livestock industry. Many rural herders have used plants to control tick which is noted in few documents, mostly transferred orally through generations. The aim of the present study was to determine acaricidal activity of Mongolian plants against D. nuttalli. We checked acaricidal activity of 113 crude extracts of 76 plant species included in 26 families. Among them Amaranthus retroflexus root (40%), Ephedra sinica leaf (40%), Erigeron acer root (40%), Ranunculus japonicus root (42%), and Spiraea salicifolia leaf (43.5%) showed potential activity against D. nuttalli. Acaricidal activity of Mongolian plants were experimented in vitro condition in first time. Furthermore, the study will continue isolation and identification active ingredients of potential candidates.  We hope this study will be start line of acaricidal activity research in our country. Монгол орны ургамлын Акарицид (хачиг үхүүлэх) идэвхийн судалгаа Монгол орны бүх бүс нутагт Dermacentor nuttalli зүйл хачиг өргөн тархсан төдийгүй мал аж ахуйд хамгийн их хор хөнөөл учруулдаг. Малчид хачиг, шавжтай тэмцэхэд зарим ургамлыг ханд, цацлага, утлага хэлбэрээр хэрэглэж ирсэн уламжлалтай ч түүний шинжлэх ухааны үндэслэлтэй эсэхийг тогтоох судалгаа төдийлөн хийгдээгүй байна. Бид энэ удаа D. nuttalli хачиг дээр турших цуврал судалгааны ажлыг эхлүүлэх зорилго тавин ажиллав. Нийт 26 овогт хамаарагдах 76 зүйл ургамлын 113 дээж дээр акарицид идэвхийг шалгахад, тэдгээрээс Урвуу гагадайн (Amaranthus retroflexus) үндэс, Нангиад зээргэний (Ephedra sinica) навч, Хахуун цийлэгийн (Erigeron acer) үндэс тус бүр 40%, Япон холтсон цэцгийн (Ranunculus japonicus) үндэс 42%, Бургас навчит тавилганы (Spiraea salicifolia) навч 43.5% буюу бусад ургамлаас илүү идэвх үзүүлж байв. Энэхүү судалгаагаар анх удаа Монгол орны хачиг дээр in vitro орчинд туршин сорих арга зүйг нэвтрүүллээ. Энэ нь цаашид ургамлаас хачгийн эсрэг үйлдэлтэй бодис, нэгдэлийг хайж илрүүлэх, цуврал суурь судалгааны ажлын эхлэл болж өгч байгаагаараа чухал ач холбогдолтой юм. Түлхүүр үг: акарицид (хачиг үхүүлэх) идэвх, хачиг, Dermacentor nuttalli, ургамал                

scholarly journals Isolation and molecular characterization of Mycoplasma spp. in sheep and goats in Egypt

2019 ◽  
Vol 12 (5) ◽  
pp. 664-670 ◽  
Author(s):  
Mounier M. Abdel Halium ◽  
Fayez A. Salib ◽  
S. A. Marouf ◽  
Emil S. Abdel Massieh
Keyword(s):  
Phylogenetic Analysis ◽  
Small Ruminants ◽  
Mycoplasma Species ◽  
African Countries ◽  
Biochemical Tests ◽  
Isolation And Identification ◽  
Sheep And Goats ◽  
Pcr Product ◽  
Nasal Swabs ◽  
Bronchial Wash

Background and Aim: Different species of Mycoplasma are associated with many pathological problems in small ruminants including respiratory manifestation, this problem results in significant losses, especially in African countries. This study aimed to (I) study some epidemiological aspects of Mycoplasma species infections in Egyptian sheep and goats at Giza Governorate, (II) diagnosis of Mycoplasma species affections using bacterial isolation and identification, (III) apply the polymerase chain reaction (PCR) for typing of different Mycoplasma species, and (IV) illustrate the phylogenetic tree for the isolated Mycoplasma species and other species from GenBank using the purified PCR product. Materials and Methods: A total of 335 samples were collected from sheep and goats from Giza Governorate in Egypt as 142 nasal swabs from clinically affected animals, 167 pneumonic lungs, 18 samples from tracheal bifurcation, and 8 samples by bronchial wash were cultured on pleuropneumonia-like organisms (PPLOs) media for cultivation of Mycoplasma species. PCR and sequencing and phylogenetic analysis were adopted to identify and classify the isolated Mycoplasma species. Results: A total of 24 Mycoplasma isolates were isolated on PPLO media, identified by biochemical tests, and confirmed and typed by PCR using specific primers. 10 isolates were confirmed as Mycoplasma arginini, four isolates as Mycoplasma ovipneumoniae by PCR, and 10 isolates as undifferentiated Mycoplasma species. A purified isolate of M. arginini and M. ovipneumoniae was sequenced and phylogenetic analysis was illustrated. Conclusion: M. arginini and M. ovipneumoniae are prevalent in Egyptian sheep and goats. Further studies on M. arginini are required due to its high frequency of isolation from pneumonic sheep and goats and also from animals suffer from different respiratory manifestations.

Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

2020 ◽  
Author(s):  
Jyoti Kumar ◽  
G. G. Sonawane ◽  
Fateh Singh ◽  
S. Jegaveera Pandian ◽  
Rajiv Kumar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Bacterial Species ◽  
Sybr Green ◽  
Economic Losses ◽  
Conventional Pcr ◽  
Specific Detection ◽  
Pcr Assay ◽  
Tissue Samples

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

scholarly journals A ten-year (2000–2009) study of antimicrobial susceptibility of bacteria that cause bovine respiratory disease complex—Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni—in the United States and Canada

2012 ◽  
Vol 24 (5) ◽  
pp. 932-944 ◽  
Author(s):  
Ellen Portis ◽  
Cynthia Lindeman ◽  
Lacie Johansen ◽  
Gillian Stoltman
Keyword(s):  
Pasteurella Multocida ◽  
Inhibitory Concentration ◽  
The United States ◽  
Mannheimia Haemolytica ◽  
In Vitro Susceptibility ◽  
Histophilus Somni ◽  
Bovine Respiratory Disease Complex ◽  
Year 2000 ◽  
Over Time

Bovine isolates of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, collected from 2000 to 2009, were tested for in vitro susceptibility to ceftiofur, penicillin, danofloxacin, enrofloxacin, florfenicol, tetracycline, tilmicosin, and tulathromycin. Ceftiofur remained very active against all isolates. Penicillin retained good activity against P. multocida and H. somni isolates with no appreciable changes in susceptibility or minimal inhibitory concentration (MIC) distributions with time. While there was no obvious trend, the percent of M. haemolytica that were susceptible to penicillin ranged from 40.9% to 66.7%. Danofloxacin MIC50 and MIC90 values for M. haemolytica and P. multocida did not change beyond a single dilution over the 6 years it was included in the testing panel. The MIC90 for H. somni increased beyond 1 dilution. Enrofloxacin MIC50 values for the 3 pathogens also did not change over time, unlike the MIC90 values, which increased by at least 4-doubling dilutions. Ninety percent or more of M. haemolytica and H. somni isolates were susceptible to florfenicol, while susceptibility among P. multocida was 79% or greater. Less than 50% of the isolates tested as susceptible to tetracycline in many of the years. All 3 organisms showed declines in tilmicosin and tulathromycin MIC50 and MIC90 values over the years in which they were tested.

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