Identification of Pasteurella multocida isolates of ruminant origin using polymerase chain reaction and their antibiogram study
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Pasteurella multocida (p.m.) is a gram negative coccobacilli bacterial pathogen, classified into three subspecies, five capsular serogroups, which is the causative agent of a range of diseases in domestic animals of economic significant worldwide. The aims of this study are isolation of P. multocida from sheep and identification by biochemical tests and polymerase chain reaction technique (PCR) technique. Seventy samples were isolated from sheep lung lesions and were cultured for P. multocida using conventional method and isolated colonies were subjected to biochemical tests and for identification by PCR. The results in the present study showed that the biochemical tests provided an approximate 99.9% (an excellent identification for P. multocida). The PCR analysis applied on direct bacterial colonies using specific primer gave an amplified product (219 bp) specific for P. multocida, this result confirmed the results of biochemical tests and provided obvious results for the identification of this bacteria using the specific primer PMout for omp gene of P. multocida. The PCR assay was found to provide rapid and accurate diagnosis of P. multocida isolates obtained from sheep respiratory infections during the investigation of suspected hemorrhagic septicemia outbreaks. PCR technique is recommended to be used for identification procedure to be applicable in all veterinarian laboratories for bacterial identification; likewise the production of veterinary vaccines productions companies. In addition RAPD technique is reliable means of discriminating between P. multocida isolates obtained from sheep.
Key words: Biochemical tests, Pasteurella multocida, PCR analysis, Genotyping.
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A set of specific primers, XZ21, XZ22 were designed according to the sequences of Genebank No. U51470 of avian Pasteurella Mulutocida. Thirteen reference strains and nine isolates of Pasteurella Multocida and 5 Mammalian pasteurella Multocida were detected by PCR using this set of primers. A avian Pasteurella Multocida-specific 488 base pair DNA product was amplified by these primers from the 13 reference strains and 9 isolates, but not from 5 Mammalian Pasteurella Multocida and 6 other avian pathogenic viruses and bacteria.As little as 10pg of Avian Pasteurella Multocida DNA was detected Using gel electrophoresis.
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Пастереллез (Pasteurellosis) - контагиозное инфекционное заболевание, характеризующееся воспалительно-геморрагическими процессами во внутренних органах и явлениями септицемии на серозных и слизистых оболочках [5, 6, 9, 13].
Чаще возбудителем инфекции является Pasteurella multocida - мелкая, грамотрицательная, неподвижная и не образующая спор бактерия, располагающаяся изолированно, парами и реже - в виде цепочек. Размер и вид бактерий варьируют в зависимости от происхождения штамма. Пастереллы относятся к факультативным анаэробам и являются факультативными аэробами, хорошо растущими на обычных питательных средах (мясопептонном бульоне и агаре) при температуре 37°С [1, 3, 4, 10, 14].
Pasteurella multocida - патогенна для многих животных, включая человека [7, 12].
Успехи, достигнутые в изучении этого заболевания, позволили резко сократить заболеваемость животных пастереллезом, однако её уровень еще остается сравнительно высоким. Пастереллез сельскохозяйственных и диких животных по-прежнему занимает одно из ведущих мест в инфекционной патологии и наносит значительный экономический ущерб. Поэтому необходимо дальнейшее совершенствование методов диагностики пастереллеза животных.
Авторы данной работы провели нижеперечисленные исследования и достигли следующих результатов. Изоляты выделяли из патологического материала (печени, легких, селезенки и почек), отобранного от сайгака и крупного рогатого скота. Идентификация бактерий проводилась на основании культурально-морфологических характеристик. В ходе исследований для идентификации Pasteurella multocida было применено секвенирование по Сенгеру.
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A total of one hundred and seventy one indigenous birds from smallholder farms and those traded in market centers in Nairobi were examined for the presence of Pasteurella multocida . Of these, 135 were farmed and 36 were market birds. They comprised of 117 indigenous chickens and 54 ducks. Three hundred and forty two oropharyngeal and cloacal swabs were collected from them and cultured onto blood agar and other media. The recovered isolates were characterized using colonial morphology, biochemical and other tests. Twenty three P. multocida isolates were recovered: 11/135 (8%) from farm and 12/36 (33%) from the market birds. Majority of the P. multocida isolates were Pasteurella multocida gallicida 11/23 (48%), followed by Pasteurella multocida multocida 7/23 (30%) and Pasteurella multocida septica 5/23 (22%). Pasteurella multocida gallicida isolates were encountered more in the market birds, while Pasteurella multocida multocida isolates were more in farm birds. Ducks had more isolates than chickens. The concentration of the birds at market areas appeared to favor the maintenance of P. multocida in the cages, crates and pens. Market birds may, therefore, play a major role in the spreading of P. multocida. The Kenya Veterinarian Vol. 29 2005: pp. 45-47
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Pasteurella multocida infections. II. Pasteurella multocida infection in man unrelated to animal bite. W T Hubbert, and M N RosenCopyRight https://doi.org/10.2105/AJPH.60.6.1109 Published Online: August 29, 2011
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動物血液の ast. multocida に対する発育阻止作用と動物感受性の関係を調べる為, 12種動物と12菌株を用いて実験を行い次の如き成績を得た. 1. 山羊, 緬羊, 黄牛及び馬血液の Past. multocida 発育阻止作用は最も強い. 2. 水牛, 豚, 鶏, 鵝鳥及び生蕃鴨(Muscovy duck)血液の阻止作用は弱いか或はない. 3. 犬, 在来鴨, 兎, 天竺鼠血液の阻止作用は上記両群動物の中間にある. 4. 近縁動物を比較すれば在来鴨は生蕃鴨及び鵝鳥より, 天竺鼠は兎より, 黄牛は水牛よりも阻止作用が強い. 5. 各種動物の阻止作用は菌株差は見られたが, 菌系差はなかった. 6. 中間阻止帯現象が兎及び氷牛では1%血液に, 天竺鼠では0.5%血液に観察された. その発生の強さは動物の種類差の外個体差も見られた. 7. 動物血液の阻止作用と台湾に於ける各種動物の Past. multocida に対する感受性が検討された.
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The objective of this study was to develop a sensitive and specific PCR method for the detection of Pasteurella multocida from porcine.A pair of primers were designed according to the plpE genes of Pasteurella multocida published in GenBank.Then the specific PCR method on the basis of the plpE gene was developed and optimized for rapid detection of Pasteurella multocida.The sensitivity of the method were evaluated with the constructed recombinant plasmid of Pasteurella multocida and other strains.Finally the PCR method established was employed on the detection of 64 suspected samples collected from Sichuan province.Results showed the method based on the plpE gene can amplify the genome of Pasteurella multocida only,not for others and the detection limitation reached 5×101 copies.Thirty-six samples were infected with Pasteurella multocida and the positive rate reached 56.2%.These results indicate that the PCR method on the basis of the plpE gene is specific,repeatable and sensitive and can be used for the detection of Pasteurella multocida.
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Five DNA probes directed against different regions of the gene that encodes the dermonecrotic toxin of Pasteurella multocida subsp. multocida were examined for their ability to identify toxigenic P. multocida subsp. multocida strains. The specificities of the probes were studied with 96 strains of P. multocida subsp. multocida and 22 strains of 11 other bacterial species. Results of colony hybridization assays using these probes indicated that two of the five probes have potential diagnostic value.
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