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Efficacy of natural formulations in bovine mastitis pathology: alternative solution to antibiotic treatment Full text
2020
Pașca Claudia | Mărghitaș Liviu Alexandru | Dezmirean Daniel Severus | Matei Ioana Adriana | Bonta Victorița | Pașca Ioan | Chirilă Flore | Cîmpean Adrian | Iosif Fiț Nicodim
Efficacy of natural formulations in bovine mastitis pathology: alternative solution to antibiotic treatment Full text
2020
Pașca Claudia | Mărghitaș Liviu Alexandru | Dezmirean Daniel Severus | Matei Ioana Adriana | Bonta Victorița | Pașca Ioan | Chirilă Flore | Cîmpean Adrian | Iosif Fiț Nicodim
Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis.
Show more [+] Less [-]Efficacy of natural formulations in bovine mastitis pathology: alternative solution to antibiotic treatment Full text
2020
Pașca, Claudia | Mărghitaș, Liviu Alexandru | Dezmirean, Daniel Severus | Matei, Ioana Adriana | Bonta, Victorița | Pașca, Ioan | Chirilă, Flore | Cîmpean, Adrian | Iosif Fiț, Nicodim
Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis. Two natural formulations with previously suggested in vitro antimicrobial effect were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = 20) were divided into three treatment groups: two groups (n = 8) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter being more concentrated) and one group (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated by somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit. R7 was effective in the most cases, being therapeutically bactericidal in six out of eight cows, while R4 gave good results in three out of eight cows, and conventional antibiotics cured one out of four. These results suggest the possible therapeutic potential of these natural products in bovine mastitis.
Show more [+] Less [-]Detection of white sturgeon iridovirus (WSIV) in wild sturgeons (Actinopterygii: Acipenseriformes: Acipenseridae) in Poland Full text
2020
Hofsoe-Oppermann Paulina | Kiełpińska Jolanta | Panicz Remigiusz | Bergmann Sven M.
Detection of white sturgeon iridovirus (WSIV) in wild sturgeons (Actinopterygii: Acipenseriformes: Acipenseridae) in Poland Full text
2020
Hofsoe-Oppermann Paulina | Kiełpińska Jolanta | Panicz Remigiusz | Bergmann Sven M.
White sturgeon iridovirus (WSIV) disease is caused by a virus of the eponymous family and is mostly triggered by stressful environmental conditions, i.e. high rearing density, excessive handling, or temporary loss of water. The aim of this study was to develop the most effective diagnostic method for quick and efficient confirmation or exclusion of the presence of WSIV.
Show more [+] Less [-]Detection of white sturgeon iridovirus (WSIV) in wild sturgeons (Actinopterygii: Acipenseriformes: Acipenseridae) in Poland Full text
2020
Hofsoe-Oppermann, Paulina | Kiełpińska, Jolanta | Panicz, Remigiusz | Bergmann, Sven M.
White sturgeon iridovirus (WSIV) disease is caused by a virus of the eponymous family and is mostly triggered by stressful environmental conditions, i.e. high rearing density, excessive handling, or temporary loss of water. The aim of this study was to develop the most effective diagnostic method for quick and efficient confirmation or exclusion of the presence of WSIV. A total of 42 samples (spleen, gills, intestine, skin, kidney, and brain) were collected from eight sturgeon (Acipenser gueldenstaedtii and A. oxyrinchus) aged ≤5+ farmed or caught between 2010 and 2014 in open waters (Dąbie Lake and Szczecin Lagoon). They were tested for WSIV presence using conventional PCR, qPCR, and in situ hybridisation (ISH). In gross examination, all fish appeared to be healthy. Neither species showed clinical signs typical of WSIV infection. In the majority of cases, fragments of iridoviral DNA were found using molecular methods in the kidneys, and also in the liver, gills, and skin. The detection rate using ISH was 47.37% and most commonly the brain and kidney tissues were positive. The most efficient of the methods used was real-time PCR, with 100% effectiveness in detection of WSIV DNA. The study demonstrates the capabilities for WSIV diagnosis available to sturgeon farmers and water administrators, indicating useful methods of adequate sensitivity as well as organs to sample in order to achieve the highest probability of viral detection.
Show more [+] Less [-]Detection of white sturgeon iridovirus (WSIV) in wild sturgeons (Actinopterygii: Acipenseriformes: Acipenseridae) in Poland Full text
2020
Hofsoe-Oppermann, Paulina | Kiełpińska, Jolanta | Panicz, Remigiusz | Bergmann, Sven
Introduction White sturgeon iridovirus (WSIV) disease is caused by a virus of the eponymous family and is mostly triggered by stressful environmental conditions, i.e. high rearing density, excessive handling, or temporary loss of water. The aim of this study was to develop the most effective diagnostic method for quick and efficient confirmation or exclusion of the presence of WSIV. Material and Methods A total of 42 samples (spleen, gills, intestine, skin, kidney, and brain) were collected from eight sturgeon (Acipenser gueldenstaedtii and A. oxyrinchus) aged ≤5+ farmed or caught between 2010 and 2014 in open waters (Dąbie Lake and Szczecin Lagoon). They were tested for WSIV presence using conventional PCR, qPCR, and in situ hybridisation (ISH). Results In gross examination, all fish appeared to be healthy. Neither species showed clinical signs typical of WSIV infection. In the majority of cases, fragments of iridoviral DNA were found using molecular methods in the kidneys, and also in the liver, gills, and skin. The detection rate using ISH was 47.37% and most commonly the brain and kidney tissues were positive. The most efficient of the methods used was real-time PCR, with 100% effectiveness in detection of WSIV DNA. Conclusion The study demonstrates the capabilities for WSIV diagnosis available to sturgeon farmers and water administrators, indicating useful methods of adequate sensitivity as well as organs to sample in order to achieve the highest probability of viral detection.
Show more [+] Less [-]Effect of electromagnetic field exposure on mouse brain morphological and histopathological profiling Full text
2020
Zymantiene Judita | Juozaitiene Vida | Zelvyte Rasa | Oberauskas Vaidas | Spancerniene Ugne | Sederevicius Antanas | Aniuliene Albina
Effect of electromagnetic field exposure on mouse brain morphological and histopathological profiling Full text
2020
Zymantiene Judita | Juozaitiene Vida | Zelvyte Rasa | Oberauskas Vaidas | Spancerniene Ugne | Sederevicius Antanas | Aniuliene Albina
Mobile phones (MP) and other electronic and communication devices that are used daily expose users to electromagnetic fields (EMF) and contribute to an increasing incidence of neurological disorders. Brain tissue is the closest organ to the MP as it operates, thus the influence of MP radiation on brain tissue is of particular concern, although research is still inconclusive. The present study investigated the possible effect of an EMF (1,350–1,375 megahertz (MHz)) from an MP on morphological and histopathological profiles in the mouse brain.
Show more [+] Less [-]Effect of electromagnetic field exposure on mouse brain morphological and histopathological profiling Full text
2020
Zymantiene, Judita | Juozaitiene, Vida | Zelvyte, Rasa | Oberauskas, Vaidas | Spancerniene, Ugne | Sederevicius, Antanas | Aniuliene, Albina
Mobile phones (MP) and other electronic and communication devices that are used daily expose users to electromagnetic fields (EMF) and contribute to an increasing incidence of neurological disorders. Brain tissue is the closest organ to the MP as it operates, thus the influence of MP radiation on brain tissue is of particular concern, although research is still inconclusive. The present study investigated the possible effect of an EMF (1,350–1,375 megahertz (MHz)) from an MP on morphological and histopathological profiles in the mouse brain. Healthy BALB/c mice were assigned to three equal groups (a control and two experimental groups, n = 10 each). Experimental mice were exposed to EMFs continuously for 72 h, those of experimental group I to a 1,350 MHz field at a specific absorption rate (SAR) of 4.0 W/kg, and group II to a 1,375 MHz field EMF at an SAR of 4.0 W/kg. Brain segmentation and histopathological analysis were applied to detect changes in the morphometric parameters of the brain lobes and identify pathological lesions, respectively. Histopathology results revealed shrinkage of pyramidal neurons, presence of mild perivascular and perineural oedema, and some vacuolation of neurons and glial cells derived from mouse great hemispheres. The lesions also included reduction of Purkinje cells, vacuolisation of neurons and glial cells, and interstitial oedema in the cerebellum. MP distance of 3 cm from the cage may induce appreciable morphological changes in mouse brain structures; therefore, more comprehensive research is essential for assessment of safe distance. These pronounced effects may interfere with the results of laboratory tests on murine experimental models in veterinary or biomedical research.
Show more [+] Less [-]Characterisation of porcine enteropathogenic Escherichia coli isolated in northeastern India Full text
2020
Kylla Hosterson | Dutta Tapan Kumar | Roychoudhury Parimal | Subudhi Prasant Kumar | Lalhruaipuii | Lalsiamthara Jonathan | Mandakini Rajkumari
Characterisation of porcine enteropathogenic Escherichia coli isolated in northeastern India Full text
2020
Kylla Hosterson | Dutta Tapan Kumar | Roychoudhury Parimal | Subudhi Prasant Kumar | Lalhruaipuii | Lalsiamthara Jonathan | Mandakini Rajkumari
Enteropathogenic Escherichia coli (EPEC) is one of the main pathotypes causing gastroenteritis, particularly in young immunocompromised hosts. The study reports the prevalence, characterisation, and molecular epidemiology of EPEC from piglets in northeastern India.
Show more [+] Less [-]Characterisation of porcine enteropathogenic Escherichia coli isolated in northeastern India Full text
2020
Kylla, Hosterson | Dutta, Tapan Kumar | Roychoudhury, Parimal | Subudhi, Prasant Kumar | Lalhruaipuii, | Lalsiamthara, Jonathan | Mandakini, Rajkumari
Enteropathogenic Escherichia coli (EPEC) is one of the main pathotypes causing gastroenteritis, particularly in young immunocompromised hosts. The study reports the prevalence, characterisation, and molecular epidemiology of EPEC from piglets in northeastern India. A total of 457 faecal samples were collected, from which 1,286 E. coli strains were isolated and screened by PCR. The resultant EPEC strains were serotyped and phenotypically characterised for resistance against 15 antimicrobials. Also, the phylogenetic sequence was analysed for 11 selected strains. A total of 42 strains (3.26%) belonged to atypical EPEC, of which, 15 (35.71%, and 2.29% of the 654 strains from this farm type) were isolated from organised and 27 (64.29%, and 4.27% of the 632 strains from this farm type) from unorganised farms; further, 5 (11.90% of the EPEC strains and 1.51% of the 330 strains from this breed) were isolated from the indigenous breeds and 37 (88.10%, and 3.87% of the 956 strains from this breed) from crossbred piglets. Serogroups O111 (11.9%) and O118 (7.14%) were the most prevalent of the 10 present. Sequence analysis of a length of the eaeA gene of 11 isolates of the region showed them to have 100% homology with each other and their identity ranged from 99.4% to 99.7% with GenBank reference sequences. All the EPEC isolates were multi-drug resistant, showing the highest resistance to amoxicillin (80.9%) and cephalexin (76.19%). The study highlighted the association of EPEC with piglet’s diarrhoea in northeastern India. EPEC isolates belonged to many serotypes and phenotypically all were multi-drug resistant with close genetic homology.
Show more [+] Less [-]In-house validation method for quantification of amoxicillin in medicated feedingstuffs with the use of HPLC-DAD technique Full text
2020
Patyra Ewelina | Kwiatek Krzysztof
In-house validation method for quantification of amoxicillin in medicated feedingstuffs with the use of HPLC-DAD technique Full text
2020
Patyra Ewelina | Kwiatek Krzysztof
A high-performance liquid chromatographic–diode array detector (HPLC-DAD) method for the determination of amoxicillin in medicated feedingstuffs was developed and validated. The method was used to investigate the quality requirements of animal feedingstuffs (declared content of active substance and feed homogeneity).
Show more [+] Less [-]In-house validation method for quantification of amoxicillin in medicated feedingstuffs with the use of HPLC-DAD technique Full text
2020
Patyra, Ewelina | Kwiatek, Krzysztof
A high-performance liquid chromatographic–diode array detector (HPLC-DAD) method for the determination of amoxicillin in medicated feedingstuffs was developed and validated. The method was used to investigate the quality requirements of animal feedingstuffs (declared content of active substance and feed homogeneity). Two-gram samples were extracted by potassium phosphate buffer solution. Extracts were filtered and directly analysed by HPLC-DAD without further clean-up. Amoxicillin was separated by acetonitrile and 0.01M phosphate buffer (pH 5.0) on a Phenomenex Luna C18 column. This method provided average recoveries of 76.1 to 81.6% with coefficients of variation (CV, %) for repeatability and reproducibility in the ranges of 3.7–7.2% and 5.3–7.6%, respectively. The limit of detection was 51.2 mg/kg and limit of quantification was 103.0 mg/kg. The method was successfully validated and proved to be efficient, precise, and useful for quantification of amoxicillin in medicated feedingstuffs.
Show more [+] Less [-]Staphylococcus spp. from wild mammals in Aragón (Spain): antibiotic resistance status Full text
2020
García Leticia Alcalá | Torres Carmen | López Antonio Rezusta | Rodríguez Carmelo Ortega | Espinosa Jesús Orós | Valencia Carmen Simón
Staphylococcus spp. from wild mammals in Aragón (Spain): antibiotic resistance status Full text
2020
García Leticia Alcalá | Torres Carmen | López Antonio Rezusta | Rodríguez Carmelo Ortega | Espinosa Jesús Orós | Valencia Carmen Simón
Antimicrobial resistance is a global health threat. It has been studied in humans and domestic animals, but there is a lack of data on wild animals. The objective of this study is the elucidation of its patterns in Staphylococcus spp. isolated from wild mammals of the Autonomous Community of Aragón (Spain).
Show more [+] Less [-]Staphylococcus spp. from wild mammals in Aragón (Spain): antibiotic resistance status Full text
2020
García, Leticia Alcalá | Torres, Carmen | López, Antonio Rezusta | Rodríguez, Carmelo Ortega | Espinosa, Jesús Orós | Valencia, Carmen Simón
Antimicrobial resistance is a global health threat. It has been studied in humans and domestic animals, but there is a lack of data on wild animals. The objective of this study is the elucidation of its patterns in Staphylococcus spp. isolated from wild mammals of the Autonomous Community of Aragón (Spain). A total of 103 mammals (Artiodactyla, Carnivora, Chiroptera, Erinaceomorpha, and Lagomorpha) were studied. A recovery centre provided 32 and hunting 71. Nasal and faecal samples yielded 111 staphylococci, which were identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. A susceptibility test to 11 antibiotics was carried out, and statistical analysis was performed. Some differences were detected in bacterial prevalence depending on how the mammal fed. Artiodactyla, mainly hunted, were predisposed to carry coagulase-positive staphylococci. The staphylococci species recovered were resistant to at least two classes of antibiotics, and were disseminated in all of the geographical areas studied. Resistant staphylococci are widely distributed in the wild mammals in the areas of the study, but the resistance quantified in them is lower than that to be expected if the use of antibiotics in farms had a direct influence on the wildlife and its environment. On the other hand, resistance to antibiotics restricted to human use was widely disseminated in various wild animal species.
Show more [+] Less [-]Molecular characterisation of the Mycobacterium bovis causing bovine tuberculosis outbreaks in Poland Full text
2020
Krajewska-Wędzina Monika | Kozińska Monika | Radulski Łukasz | Lipiec Marek | Augustynowicz-Kopeć Ewa | Weiner Marcin | Szulowski Krzysztof
Molecular characterisation of the Mycobacterium bovis causing bovine tuberculosis outbreaks in Poland Full text
2020
Krajewska-Wędzina Monika | Kozińska Monika | Radulski Łukasz | Lipiec Marek | Augustynowicz-Kopeć Ewa | Weiner Marcin | Szulowski Krzysztof
Since 2009, Poland has been recognised as a country officially free of bovine tuberculosis (bTB), although in each year of the last five there were from 8 to 18 outbreaks of the disease. In 2008–2016, the largest number of cattle infected with bovine mycobacteria were eliminated in the Masovian Province (the central region of Poland) and the largest number of outbreaks of this zoonosis were recorded in this area. The close proximity of farms where bTB was found led to the suspicion that tuberculosis could have been transmitted between the affected herds. The aim of the study was the molecular characterisation of the pertinent M. bovis/caprae strains and determination of the epidemiological relationship of various bTB outbreaks.
Show more [+] Less [-]Molecular characterisation of the Mycobacterium bovis causing bovine tuberculosis outbreaks in Poland Full text
2020
Krajewska-Wędzina, Monika | Kozińska, Monika | Radulski, Łukasz | Lipiec, Marek | Augustynowicz-Kopeć, Ewa | Weiner, Marcin | Szulowski, Krzysztof
Since 2009, Poland has been recognised as a country officially free of bovine tuberculosis (bTB), although in each year of the last five there were from 8 to 18 outbreaks of the disease. In 2008–2016, the largest number of cattle infected with bovine mycobacteria were eliminated in the Masovian Province (the central region of Poland) and the largest number of outbreaks of this zoonosis were recorded in this area. The close proximity of farms where bTB was found led to the suspicion that tuberculosis could have been transmitted between the affected herds. The aim of the study was the molecular characterisation of the pertinent M. bovis/caprae strains and determination of the epidemiological relationship of various bTB outbreaks. The material for microbiological tests came from 119 cattle (Bos taurus) from nine herds located in five provinces, neighbouring the Masovian Province. Laboratory tests of tissue material gave results confirming tuberculosis in 54 (45%) animals. All strains belonged to the Mycobacterium bovis species. A two-step analysis of genetic affinity allowed 50 strains to be identified as phylogenetically closely related and separated between three genetic clusters consisting of 2 to 27 strains. Based on the results of genotyping, bTB outbreaks were found in three herds, and three transmission chains were identified among these herds.
Show more [+] Less [-]Cloning and identification of PK15 cells for enhanced replication of classical swine fever virus Full text
2020
Yin Mei | Hu Dongfang | Li Peng | Kong Lingyun | Ning Hongmei | Yue Feng | Jiang Jinqing | Wang Xuannian
Cloning and identification of PK15 cells for enhanced replication of classical swine fever virus Full text
2020
Yin Mei | Hu Dongfang | Li Peng | Kong Lingyun | Ning Hongmei | Yue Feng | Jiang Jinqing | Wang Xuannian
Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs, leading to economic losses around the world. Attenuated live vaccines with CSFV antigens have played an important role in the prevention and control of the disease. Porcine kidney 15 (PK15) cells have been widely used for the propagation of CSFV, but this cell line is not efficient or homogeneously susceptible to viral infection.
Show more [+] Less [-]Cloning and identification of PK15 cells for enhanced replication of classical swine fever virus Full text
2020
Yin, Mei | Hu, Dongfang | Li, Peng | Kong, Lingyun | Ning, Hongmei | Yue, Feng | Jiang, Jinqing | Wang, Xuannian
Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs, leading to economic losses around the world. Attenuated live vaccines with CSFV antigens have played an important role in the prevention and control of the disease. Porcine kidney 15 (PK15) cells have been widely used for the propagation of CSFV, but this cell line is not efficient or homogeneously susceptible to viral infection. To achieve a homogeneous PK15 cell line which enabled high titre replication of CSFV, we used the limiting dilution cell cloning method. We developed two cell clones, PK15-1A6 and PK15-3B1, which respectively have high- and low-permissive phenotypes to CSFV infection. The PK15-1A6, PK15-3B1, and PK15 parent cells showed different characteristics in cell proliferation rate, susceptibility to CSFV infection, and CSFV production. The mean virus titres per millilitre reflected by TCID₅₀ values in PK15-1A6, PK15-3B1, and PK15 parent cells were 106.85, 103.63, and 104.74, respectively. The PK15-1A6 cell clone is more permissive to CSFV infection than the PK15 parent cells. The screened high-permissive cells will be useful for CSFV propagation and vaccine development in vitro, and facilitate research on the pathogenicity of CSFV.
Show more [+] Less [-]Investigation and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from companion birds in eastern Turkey Full text
2020
Adiguzel Mehmet Cemal | Timurkan Mehmet Ozkan | Cengiz Seyda
Investigation and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from companion birds in eastern Turkey Full text
2020
Adiguzel Mehmet Cemal | Timurkan Mehmet Ozkan | Cengiz Seyda
Avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV) induce contagious and persistent diseases that affect the beaks, feathers, and immune systems of companion birds. APV causes hepatitis, ascites, hydropericardium, depression, feather disorders, abdominal distension, and potentially death. PBFDV can induce progressive beak deformity, feather dystrophy, and plumage loss. We conducted the first prevalence survey of both APV and PBFDV infections in companion birds in eastern Turkey.
Show more [+] Less [-]Investigation and sequence analysis of avian polyomavirus and psittacine beak and feather disease virus from companion birds in eastern Turkey Full text
2020
Adiguzel, Mehmet Cemal | Timurkan, Mehmet Ozkan | Cengiz, Seyda
Avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV) induce contagious and persistent diseases that affect the beaks, feathers, and immune systems of companion birds. APV causes hepatitis, ascites, hydropericardium, depression, feather disorders, abdominal distension, and potentially death. PBFDV can induce progressive beak deformity, feather dystrophy, and plumage loss. We conducted the first prevalence survey of both APV and PBFDV infections in companion birds in eastern Turkey. A total of 113 fresh dropping samples from apparently healthy companion birds were collected in a random selection. The dropping samples were analysed for PBFDV and APV by PCR. Positive samples were sequenced with the Sanger method. The sequence was confirmed through alignment and the phylogenetic tree generated through the maximum likelihood method computationally. PBFDV and APV were detected in a respective 48.7% and 23.0% of samples. Coinfection was found in 12.4% of the samples, these all being from budgerigars (Melopsittacus undulatus). APV and PBFDV were detected in budgerigar and cockatiel (Nymphicus hollandicus) samples. This report provides a foundation for future studies on the influence of these viruses on the health of companion birds. These high positive rates for both pathogens emphasise that healthy M. undulatus and N. hollandicus in eastern Turkey may be prone to the emergence and spread of APV and PBFDV with subclinical potential.
Show more [+] Less [-]A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms Full text
2020
Zhang Shi-Jun | Wang Lu-Lu | Lu Shi-Ying | Hu Pan | Li Yan-Song | Zhang Ying | Chang Heng-Zhen | Zhai Fei-Fei | Liu Zeng-Shan | Li Zhao-Hui | Ren Hong-Lin
A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms Full text
2020
Zhang Shi-Jun | Wang Lu-Lu | Lu Shi-Ying | Hu Pan | Li Yan-Song | Zhang Ying | Chang Heng-Zhen | Zhai Fei-Fei | Liu Zeng-Shan | Li Zhao-Hui | Ren Hong-Lin
The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms.
Show more [+] Less [-]A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms Full text
2020
Zhang, Shi-Jun | Wang, Lu-Lu | Lu, Shi-Ying | Hu, Pan | Li, Yan-Song | Zhang, Ying | Chang, Heng-Zhen | Zhai, Fei-Fei | Liu, Zeng-Shan | Li, Zhao-Hui | Ren, Hong-Lin
The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R²) of the standard curve was 0.999. The sensitivity of the method was 10³ CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.
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