CN110511889B - Ass-derived salmonella AD19 strain and application thereof - Google Patents
Ass-derived salmonella AD19 strain and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to an donkey-derived salmonella AD19 strain and application thereof, wherein the preservation number of microorganisms is CGMCC N0.18046, and the preservation date is as follows: 27/6/2019, depository: china general microbiological culture Collection center. 1 gram negative bacillus is separated and screened from a disease material of aborted colt collected from a certain donkey farm in Shandong, and the separated strain is judged to be salmonella through morphological identification, biochemical identification, 16Sr DNA cloning and sequence analysis after being purified and cultured; the pathogenicity test result shows that the salmonella strain has a certain lethality to mice, and the most important of the salmonella strain can cause abortion of pregnant mice. The strain can be used for preparing an inactivated vaccine of salmonella causing donkey abortion. The inactivated vaccine prepared by the strain has good immunogenicity, high safety and good protection effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a salmonella equi AD19 strain and application thereof.
Background
Salmonellosis is one of the human and livestock comorbidities of great public health importance, the etiology of which is the Salmonella family of Enterobacteriaceae, including those that cause food poisoning, resulting in gastroenteritis, typhoid and paratyphoid. They can infect many animals including mammals, birds, etc., in addition to humans. Salmonellosis is a general term for infectious diseases of livestock and poultry groups caused by bacteria of the genus salmonella, and the disease is commonly found in pigs, chickens, cattle and other animals, wherein the salmonella abortus is a pathogen causing paratyphoid fever of horses, and is associated with abortion, neonatal sepsis and polyarthritis. The disease is easy to infect primary-producing dams and foals, most of the diseases have no obvious clinical symptoms before abortion occurs, abortion occurs suddenly, and domestic reports on salmonellosis of equine animals, particularly donkeys, are few.
Donkey is a mammal (about 360 days) with a long gestational period and mostly has a single fetus, so that in order to meet the requirement of large-scale cultivation for rapidly increasing the number of stalls, promote the rapid development of donkey breeding industry, purify the donkey disease as soon as possible, control the occurrence and prevalence of the donkey disease, prevent and block the pathogen of the donkey salmonellosis, a local donkey salmonellosis epidemic strain must be separated, research and possible application such as pathogenic biochemistry and molecular identification are enhanced, and effective prevention and treatment of the donkey abortions caused by the salmonella local are realized.
Disclosure of Invention
The donkey-derived salmonella strain AD19 has the microorganism preservation number of CGMCC N0.18046 and the preservation date: 27 days 6 months in 2019, and the classification is named as salmonella Salmonella SP.And the preservation unit: the China general microbiological culture Collection center is No. 3 Xilu No.1 Beijing, Chaoyang, North Chen, China. The bacterial colony of the bacterium cultured in a salmonella chromogenic medium is purple, medium-sized, neat in edge, smooth and moist, and round and convex. Inoculating to nutrient broth, culturing at 37 deg.C for 24 hr, and culturingThe bacterium is purified by gram staining observation, and is gram-negative brevibacterium with two blunt ends.
The 16S rDNA of the salmonella donkey strain AD19 is a nucleotide sequence shown in a sequence table 1.
16S rDNA PCR amplification of bacterial 16S rDNA Universal primers were:
F27：５′－agagttgatcctggctcag－３′（SEQID No.2），
R1492：５′－aaggaggtgatccaaccgca－３′（SEQID No.3）。
the application of the salmonella equi strain AD19 in preparation of live vaccine is provided. The salmonella equi strain AD19 is subjected to nutrient broth strain amplification by taking peptone and beef extract powder as component formulas, formaldehyde inactivation and aseptic inspection, and after the safety inspection is qualified, the salmonella equi-adjuvant is emulsified to prepare the salmonella inactivated vaccine.
Advantageous effects
The invention provides a dominant epidemic strain of salmonella of donkey origin separated from Shandong area and causing abortion. Only salmonella pathogenic bacteria are separated from the donkey abortion pathogenic material, and an accurate basis is provided for the pathogenic source causing donkey abortion. In a mouse infection experiment, the pathogenic capability of the strain of bacteria on mice is general, but the abortion causing capability of the strain of bacteria on pregnant mice is very strong, which is consistent with the abortion caused by the salmonella on donkeys during pregnancy. The inactivated vaccine prepared by the strain has good immunity, and has important value for effectively preventing donkey abortion caused by local epidemic strains.
Drawings
FIG. 1 is a gram stain pattern of Salmonella equi strain AD 19;
FIG. 2 shows PCR amplification product M: marker; 1: strain AD19 was isolated.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1
1 materials and methods
1.1 pathological material
In 2019, large-scale donkey abortion occurred in Dong donkey farm in Shandong in Dong in Shandong, 200 donkey in total in the small donkey farm, 45 donkey in abortion, and abortion at 6-11 months of gestational age are all the first childbearing. The aborted donkey has symptoms such as body temperature rise and the like, and then dies. Necropsy of dead donkeys on both ends showed diffuse hemorrhagic inflammation of the cecum and large colon, liver fibrosis in the lung, and necrotic lesions in the kidneys and liver. Tissue samples of mediastinal lymph nodes, heart, lung, kidney, liver, spleen, brain and colon of 1 dead colt are collected for bacteriological detection, virus screening and other tests.
1.2 Primary reagents
The salmonella chromogenic medium, the common agar and the common nutrient broth medium are purchased from Qingdao Haibobo biology, Inc.; the biochemical identification tube is purchased from Qingdao Haibobo biology Co., Ltd; the drug sensitive tablets were purchased from Oxoid.
1.3 isolation and culture of the Strain
And (3) placing the pathogenic tissue collected in the step (1.1) in a superclean workbench, taking the inner part of the pathogenic tissue, the inner wall of the intestinal tract and contents by using an inoculating loop, inoculating the pathogenic tissue, the inner wall of the intestinal tract and the contents into a salmonella chromogenic culture medium, placing the salmonella chromogenic culture medium in an incubator at 37 ℃ for 24 hours, and observing the colony morphology. Selecting purple colonies in a chromogenic culture medium, inoculating the purple colonies to a nutrient broth culture medium, placing the nutrient broth culture medium in a 37 ℃ incubator for culturing for 24 hours, performing gram staining microscopic examination on the culture, and observing morphological characteristics and purification of the strain.
1.4 Biochemical identification
Inoculating the separated and purified strain to trisaccharide iron agar, lysine decarboxylase culture medium, indigo substrate (tryptophan broth), urease, potassium cyanide contrast and amino acid decarboxylase contrast, culturing in a 37 ℃ incubator for 24h, observing the color change and gas production conditions of the bottom and the inclined plane, and judging the result according to the GB4789.4-2010 standard.
1.5 PCR sequencing of isolate 16S rDNA
1.5.1 preparation of PCR template the isolated strain pathogen was picked up and single colony was pure cultured, after enrichment in the general nutrient broth medium, the DNA of the bacterial liquid was extracted with the EasyPure bacterionic Genomic DNA Kit.
1.5.216 PCR amplification of bacterial 16S rDNA
F27: 5'-AGAGTTGATCCTGGCTCAG-3', R1492: 5'-AAGGAGGTGATCCAACCGCA-3', respectively; the size of the fragment to be amplified is about 1500bp, and the primer is synthesized by Beijing Optimalaceae Biotechnology limited; reaction (25 μ L): 2 xTaq PCR Master Mix 12.5. mu. L, ddH2O9. mu.L, 1. mu.L of each of the upstream and downstream primers (10 mmol/L), and 1.5. mu.L of the template; the reaction program comprises 95 ℃ for 5min, 95 ℃ for denaturation 40s, 56 ℃ for annealing 40s, 72 ℃ for extension 90s, 35 cycles, and 72 ℃ for extension 8 min. The PCR product was observed by electrophoresis on a 1% agarose gel.
1.5.3 sequencing, comparing and analyzing the single strip PCR product, sending the product to Huada Gen-Kun Co., Ltd for sequencing, the sequence is shown as SEQID No. 1. The sequencing results were aligned in Gen-Bank by Blast (https:// Blast. ncbi. nlm. nih. gov /).
1.7 mouse pathogenicity test
1.7.1 Experimental animals SPF Kunming mouse, 42 mice, the weight of which is about 20 g, half of male and female, 12 SPF Kunming pregnant mice, purchased from the experimental animals center of Shandong university.
1.7.2 determination of LD50 mice were randomly divided into 7 groups of 6 trials, 1 control group of 6 mice each, hermaphrodite and bacterial suspension at 1.8X 109-1.8×104CFU/mL. Intraperitoneal injection is adopted, 0.2mL of bacterial solution cultured for 8 hours is inoculated to mice with different concentrations in equal volumes, and the control group is injected with the same amount of normal saline. After challenge, the observation was continued for 10 days.
1.7.3 pregnant mice were divided into 3 groups of 5 out of 2 trials, according to the results calculated in 1.7.2; 1 control group, 2; selection 1.8X 109And 1.8X 108And (5) detecting the abortion caused by the salmonella.
1.7.4 mice are necropsied, bacteria are recovered, and dead mice are identified by PCR, and the mice are observed for pathological changes of organs of tissues and abortion embryos. Inoculating the liver and spleen of a mouse and an embryo of an aborted mouse into a salmonella chromogenic medium, recovering bacteria, and extracting bacterial DNA by using the kit. PCR was performed to detect the presence of Salmonella in the organs and aborted mice.
1.8 mouse Immunity test
1.8.1 adding 0.4% formaldehyde into the separated and amplified bacteria for inactivation for 36-48 h, centrifuging and discarding the clear solution. The precipitate was diluted to 1X 10 with sterile PBS (10 mmol pH 7)6CFU/mL. And emulsifying the qualified inactivated bacterial liquid and equivalent Freund's complete adjuvant and Freund's incomplete adjuvant after sterile inspection and safety inspection to prepare immunogen.
1.8.2 mice of 15g weight were divided into 2 groups of 6 mice, 6 mice were injected subcutaneously with 0.3mL of the prepared inactivated antigen, and 6 mice were injected subcutaneously with 0.3mL of 0.9% physiological saline, each group was given 2 immunizations 20 days after the first immunization, and the immune protection test was given 1.7 days after the 2 immunizations.
Example 2
2 results and analysis
2.1 results of Strain culture
The strain is named as AD19, and the bacterial colony cultured in a salmonella chromogenic culture medium with the preservation number (CGMCC N0.18046) is purple, medium-sized, neat in edge, smooth and moist and round and convex. After culturing the strain inoculated into the nutrient broth at 37 ℃ for 24 hours, the strain was purified to gram-negative Brevibacterium with blunt ends by gram stain observation (FIG. 1).
2.2 Biochemical test results
2.3 PCR amplification analysis of 16S rDNA of Strain
The PCR amplification product of the AD19 strain 16S rDNA shows a remarkable target band at about 1500bp (figure 2). The isolated strain is subjected to 16S rDNA PCR sequencing, and the sequence is subjected to Blast comparison, so that the result shows that the 16S rDNA sequence of the isolated strain is salmonella.
2.4 results of pathogenicity test of Strain
2.4.1 determination of Strain LD50 the Salmonella strain was able to infect mice and the inoculation concentration was 1.8X 10 h after inoculation9The mice of the CUF/mL test group have the first disease symptoms and have small diseaseThe mice had rough hair, listlessness, and poor appetite, and the infection to mice is shown in Table 1. According to the Reed-Muench method, the LD50 value of AD19 for mice was calculated to be 2.29X 107CFU/mL。
TABLE 1 lethality of mice infected with various doses of Salmonella
2.4.2 abortion test results of pregnant mice, the salmonella strain can cause abortion of pregnant mice, and the abortion of pregnant mice is shown in table 2. When the inoculation amount is 1.8X 109And the pregnant mouse is inoculated with the salmonella strain, the disease symptoms appear after 4 hours, the pregnant mouse is completely aborted within 5-11 hours, and the abortion rate is 100%, which indicates that the salmonella strain can cause the abortion of the pregnant mouse.
TABLE 2
2.3 examination of mice by dissection and identification of recovered bacteria
Performing a cesarean examination on the infected mouse, and finding that the spleen and the intestinal tract of the infected mouse are swollen; inoculating salmonella chromogenic culture medium again from the liver, spleen and embryo of aborted mouse, culturing at 37 deg.C for 24h, growing purple colony in the culture medium, and performing gram staining microscopy to obtain the strain with the same shape as that of the injected mouse. No other pathogenic microorganism is detected in the disease material, and the salmonella AD19 is considered to be the pathogenic bacterium causing the abortion of the donkey.
2.4 Immunity Effect of inactivated vaccine
AD19 infection of digestive tract causes depression, dyspnea, death and abortion in mice, and infection causes enlargement and necrosis of mouse spleen. The AD19 infection causes abortion in pregnant mice, and research shows that Salmonella infection can cause abortion in pregnant donkeys, but no report on abortion caused by Salmonella in mice is found, and the reason and mechanism of the abortion are needed to be further researched. As a result, all the negative control groups had abortion, and the strain was usedAfter nutrient broth culture enrichment (culture at 36 +/-1 ℃ for not less than 6 h), the serum antibody titer of the prepared inactivated vaccine (0.4% formaldehyde) after two times of immunization is 1:5.2 × 104The abortion symptom does not appear in the immune group, and the immune protective capacity of the immune group can reach 100%.
Sequence listing
<110> institute of zootechnics of academy of agricultural sciences of Shandong province
<120> salmonella donovani AD19 strain and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1356
<212> DNA
<213> Salmonella ass strain AD19
<400> 1
tacttctttt gcaacccact cccatggtgt gacgggcggt gtgtacaagg cccgggaacg 60
tattcaccgt ggcattctga tccacgatta ctagcgattc cgacttcatg gagtcgagtt 120
gcagactcca atccggacta cgacgcactt tatgaggtcc gcttgctctc gcgaggtcgc 180
ttctctttgt atgcgccatt gtagcacgtg tgtagccctg gtcgtaaggg ccatgatgac 240
ttgacgtcat ccccaccttc ctccagttta tcactggcag tctcctttga gttcccggcc 300
taaccgctgg caacaaagga taagggttgc gctcgttgcg ggacttaacc caacatttca 360
caacacgagc tgacgacagc catgcagcac ctgtctcaca gttcccgaag gcaccaatcc 420
atctctggaa agttctgtgg atgtcaagac caggtaaggt tcttcgcgtt gcatcgaatt 480
aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt catttgagtt ttaaccttgc 540
ggccgtactc cccaggcggt ctacttaacg cgttagctcc ggaagccacg cctcaagggc 600
acaacctcca agtagacatc gtttacggcg tggactacca gggtatctaa tcctgtttgc 660
tccccacgct ttcgcacctg agcgtcagtc tttgtccagg gggccgcctt cgccaccggt 720
attcctccag atctctacgc atttcaccgc tacacctgga attctacccc cctctacaag 780
actcaagcct gccagtttcg aatgcagttc ccaggttgag cccggggatt tcacatccga 840
cttgacagac cgcctgcgtg cgctttacgc ccagtaattc cgattaacgc ttgcaccctc 900
cgtattaccg cggctgctgg cacggagtta gccggtgctt cttctgcggg taacgtcaat 960
tgctgcggtt attaaccaca acaccttcct ccccgctgaa agtactttac aacccgaagg 1020
ccttcttcat acacgcggca tggctgcatc aggcttgcgc ccattgtgca atattcccca 1080
ctgctgcctc ccgtaggagt ctggaccgtg tctcagttcc agtgtggctg gtcatcctct 1140
cagaccagct agggatcgtc gccttggtga gccgttacct caccaactag ctaatcccat 1200
ctgggcacat ctgatggcaa gaggcccgaa ggtccccctc tttggtcttg cgacgttatg 1260
cggtattagc caccgtttcc agtagttatc cccctccatc aggcagtttc ccagacatta 1320
ctcacccgtc cgccactcgt cagcgaagca gcaagc 1356
<210> 2
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 2
agagttgatc ctggctcag 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 3
aaggaggtga tccaaccgca 20
Claims (2)
1. The salmonella donkey strain AD19 is characterized in that the preservation number of the microorganism is CGMCC N0.18046, and the preservation date is as follows: 27/6/2019, depository: china general microbiological culture Collection center;
the 16S rDNA of the salmonella donkey strain AD19 is a nucleotide sequence shown in SEQ ID No. 1.
2. The method for preparing the donkey-derived salmonella strain AD19 fire-extinguishing vaccine for salmonella abortus according to claim 1 comprises the steps of carrying out nutrient broth strain amplification on the donkey-derived salmonella strain AD19 with a formula comprising peptone and beef extract powder as components, inactivating formaldehyde, carrying out aseptic inspection, and emulsifying with an adjuvant in an equal amount after passing the safety inspection to prepare the salmonella inactivated vaccine.
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| CN111374094B (en) * | 2020-03-02 | 2022-04-19 | 扬州大学 | Method for establishing non-pregnant mouse uterine infection model caused by subcutaneous administration of donkey-derived salmonella abortus |
| CN113881589B (en) * | 2021-09-09 | 2023-06-02 | 山东省农业科学院畜牧兽医研究所 | Klebsiella pneumoniae KP21 strain and application thereof |
| CN114712528A (en) * | 2022-04-11 | 2022-07-08 | 江苏靶标生物医药研究所有限公司 | Method for evaluating toxicity of oncolytic bacteria on mice and application of method |
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| CN108220183A (en) * | 2016-12-22 | 2018-06-29 | 新疆农业大学 | One plant of Salmonella abortus equi bacterial strain SMXJ-97 and its application in Salmonella abortus equi vaccine |
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| RO75474A2 (en) * | 1979-05-14 | 1981-03-30 | Institutul De Cercetari Veterinare Si Biopreparate "Pasteur",Ro | VACCINE AGAINST SALMONELIC TREATMENT TO THEIR EACH AND PROCESS OF PREPARING THEM |
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| RU2178713C2 (en) * | 2000-02-21 | 2002-01-27 | Якутский научно-исследовательский институт сельского хозяйства СО РАСХН | Method to prevent salmonella-caused abortion in mares |
| CN108220183A (en) * | 2016-12-22 | 2018-06-29 | 新疆农业大学 | One plant of Salmonella abortus equi bacterial strain SMXJ-97 and its application in Salmonella abortus equi vaccine |
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