CN111961627A - Separation and culture method of lawsonia intracellularis - Google Patents
Separation and culture method of lawsonia intracellularis Download PDFInfo
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- CN111961627A CN111961627A CN202010880403.5A CN202010880403A CN111961627A CN 111961627 A CN111961627 A CN 111961627A CN 202010880403 A CN202010880403 A CN 202010880403A CN 111961627 A CN111961627 A CN 111961627A
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Abstract
The invention discloses a separation and culture method of lawsonia intracellularis, which comprises the following steps: preparing lawsonia intracellularis isolate; subculturing McCoy cells; inoculating and culturing bacteria; passage and freezing storage of lawsonia intracellularis; and (4) identifying lawsonia intracellularis. The McCoy cell is used as a host cell, and is used for separating and culturing the lawsonia intracellularis, so that the blank that the lawsonia intracellularis is not successfully separated and cultured from a pig body in China is filled, and a foundation is laid for the research on the lawsonia intracellularis attenuated vaccine and the research on the pathogenic mechanism.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a separation and culture method of lawsonia intracellularis.
Background
Lawsonia Intracellularis (LI) is a common enteric pathogenic bacterium in the pig industry, is a pathogen of Porcine Proliferative Enteritis (PPE), and LI mainly causes slow growth and development of growing and fattening pigs, feed conversion ratio reduction and the like, is ubiquitous in pig farms all over the world, and becomes an important enteric disease in the modern pig industry. Losses due to PPE are reported to be as high as $ 2000 ten thousand and $ 400 thousand per year in the united states and the united kingdom, respectively. In Europe, the disease causes economic losses of more than 1 Euro per fattening pig per year. In addition, the disease is prevalent on a world scale, the infection rate is very high, LI infection is caused in more than 20 countries such as canada, finland, brazil, france, south africa, greece, thailand, india, japan, korea and china, and the incidence rate is high particularly in european and american countries. In nature, LI can be transmitted by a variety of routes, such as animal transmission of birds, mice, insects, etc., and fluid transmission of people, equipment, vehicles, etc. In view of the serious threat of PPE to the healthy development of the pig industry, the successful isolation of LI from clinical samples, the development of effective vaccines and the deep research on the pathogenic mechanism of LI are necessary to provide an effective new control strategy for PPE.
Lawsonia intracellularis is in a curved shape, a short rod shape or a comma shape and has polymorphism, the tail end is tapered or blunt, the length is 1.25-1.75 μm, the width is 0.25-0.43 μm, the lawsonia intracellularis is a typical gram-negative bacterium with three outer membranes, and the lawsonia intracellularis is strictly intracellular parasitism, microaerophilic and can not grow in an in vitro common culture medium, so that the lawsonia intracellularis extremely difficult to separate and culture. LI can only grow on intestinal epithelial cells such as IEC-18, McCoy, Hep-2 and IPEC-J2; in addition, the growth of LI requires a special gas atmosphere, i.e., 8% O2、8.8％CO2And 83.2% N2. Because of the special culture conditions, only a few laboratories in the world have the ability to separate the bacteria, but no relevant report about successful LI separation and culture exists at home and now, in view of the above, a method for separating and culturing LI from intestinal tract samples of diseased pigs is provided, and the method has important significance for prevention and control of PPE.
Disclosure of Invention
The invention aims to provide a technical scheme of a separation and culture method of lawsonia intracellularis, and lays a foundation for the research of lawsonia intracellularis vaccines and the research of pathogenic mechanisms.
The purpose of the invention can be realized by the following technical scheme:
a method for separating and culturing Lawsonia intracellularis comprises the following steps:
step 1: preparing lawsonia intracellularis isolate;
step 2: subculturing McCoy cells;
and step 3: diluting the lawsonia intracellularis isolate obtained in the step 1 and the lawsonia intracellularis inoculation culture solution according to a ratio of 1:10, adding the diluted lawsonia intracellularis isolate and the diluted lawsonia intracellularis inoculation culture solution into McCoy cells for culture, replacing the diluted lawsonia intracellularis inoculation culture solution after 3 hours, continuously culturing for 7 days, replacing the culture solution every 2-3 days, harvesting bacteria after 7 days, inoculating the bacteria to fresh McCoy cells, and continuing to perform next-generation culture;
and 4, step 4: carrying out passage and cryopreservation on lawsonia intracellularis;
and 5: identifying lawsonia intracellularis;
the lawsonia intracellularis inoculation culture solution comprises the following components in percentage by total volume:
the culture solution for culturing the lawsonia intracellularis comprises the following components in terms of the total volume of the culture solution:
% represents volume percent.
As a preferred method of the present invention, the method for preparing the Lawsonia intracellularis isolate in step 1 comprises: cleaning a suspected porcine ileum sample with typical symptoms of PPE, longitudinally splitting an intestinal tube, scraping intestinal mucus by using a sterile glass slide into a centrifuge tube, adding pancreatin into the centrifuge tube for digestion, adding a SPG solution containing 10% FBS into the digested intestinal mucus, homogenizing the diluted intestinal mucus in a tissue homogenate tube, filtering the homogenate liquid by using a 200-mesh cell screen, collecting filtrate in the centrifuge tube, sequentially passing through 1.2, 0.8 and 0.65 mu m filter membranes, finally adding DMSO with the final concentration of 10% into the filtrate, and subpackaging the filtrate in an EP tube at-80 ℃ for preservation in a refrigerator.
Preferably, in the step 2, the subculturing of the McCoy cells specifically comprises:
aseptically transferring the recovered McCoy cells into a cell culture flask, adding HDMEM containing 10% FBS, and standing at 37 deg.C and 5% CO2Culturing in an incubator, digesting with 0.25% pancreatin after the cells grow into a monolayer, stopping digestion when a small amount of cells fall off, discarding digestive juice, adding HDMEM cell culture solution containing 10% FBS, repeatedly beating for several times until the cells are dispersed into single cells, sucking a proper amount of cell sap into another cell bottle for subculture, and using after the cells adapt to a culture environment.
In a preferred embodiment of the present invention, the density of the McCoy cells in step 3 is 30 to 35%, and the concentration of the McCoy cells is 5% CO at 37 ℃2The culture time in the incubator should not exceed 24 h.
Preferably, the culture in step 3 is carried out in a three-gas culture box at 37 ℃ and 8% O2、8.8％CO2And 83.2% N2Under microaerophilic conditions.
Preferably, the lawsonia intracellularis inoculation culture solution in the step 3 comprises the following components:
preferably, the culture solution for culturing lawsonia intracellularis in step 3 comprises the following components:
as a preferred mode of the present invention, the identification of Lawsonia intracellularis in step 5 refers to the identification of the isolated strain using transmission electron microscopy, general PCR, indirect immunofluorescence and peroxidase monolayer assay, respectively.
Lawsonia intracellularis obtained according to the method described above.
Preferably, the lawsonia intracellularis is preserved in the China center for type culture Collection with the preservation date of 2020, 7 and 21, and the preservation number of CCTCC NO: v202046.
The lawsonia intracellularis disclosed by the invention can be applied to preparation of vaccines for resisting lawsonia intracellularis infection.
The invention has the following beneficial effects:
1. according to the method for separating and culturing lawsonia intracellularis, provided by the invention, by optimizing an LI culture solution, the pollution of other mixed bacteria in intestinal tracts is avoided, so that the success rate of separating the lawsonia intracellularis is increased;
2. the separation and culture method of lawsonia intracellularis provided by the invention has the advantages that the tissue homogenate is subjected to pancreatin digestion and then is homogenized, so that the number of separated bacteria is greatly increased.
Drawings
FIG. 1 is a transmission electron microscope observation result diagram of McCoy cells after the isolated strain LJS19051 acts on the McCoy cells for 3 h; arrows show Lawsonia intracellularis
FIG. 2 shows the result of PCR detection of the LI aspA gene in isolate strain LJS 19051; m: DL2000 Marker; 1-3: isolating genomic DNA of strain LJS 19051; 4: an LI positive control; 5: negative control
FIG. 3 shows the result of detection of isolate LJS19051 IFA; panels A-B refer to LJS19051 cultured on McCoy cells for 12h and 24h, respectively; and (C) figure: a positive control; FIG. D: negative control
FIG. 4 shows the result of detection of isolated strain LJS19051 IPMA; FIGS. A-B: McCoy cells seeded with LJS 19051; and (C) figure: a positive control; FIG. D: negative control
FIG. 5 shows the clinical manifestations and intestinal tract autopsy changes of mice after LJS19051 challenge; FIG. A: feces of mice in each group after 21d of challenge; arrow indicates that the feces are soft and moist, and the expansion chart B is as follows: ileum of each group of mice after 21d of challenge; arrow indicates intestinal hemorrhage
FIG. 6 shows the PCR detection results of LI-specific genes in feces at different time points after infection of mice with LJS 19051;
m: DNA Marker; 1-6 refer to the genomic DNA of mice in test groups 7d, 14d, 21d, 28d and 35d after challenge respectively; 7: control group mouse fecal genomic DNA
FIG. 7 shows the ileum H & E staining results of various groups of mice; FIG. A: ileum 21d after challenge in the test group; and B: the control group was drenched with DMEM 21d ileum; note: arrow indicates local villous intestinal epithelium disappearance of tissue
FIG. 8 shows the ileum IHC test results of various groups of mice; FIG. A: the ileum of 21d mice after challenge in the test group; and B: the ileum of the control group after being infused with DMEM 21 d; note: the arrow indicates Lawsonia intracellularis reacted with LI Omp2 monoclonal antibody
Table 1 shows the results of detection of LI antibody in serum at different time points after mice were infected with LJS19051 strain
Biological material preservation information
Lawsonia intracellularis LJS19051, preserved in China center for type culture Collection, the preservation place of Wuhan university, Wuhan, China, the preservation date is 2020, 7 and 21, and the preservation number is CCTCC NO: v202046.
Detailed Description
In order that the invention may be more readily understood, the invention will now be further described with reference to specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, and that specific experimental procedures not mentioned in the following examples are generally conducted according to conventional experimental procedures.
Sources of materials for use in the present invention
HDMEM medium and fetal calf serum were purchased from GIBCO.
1.2/0.8 and 0.65 μm filters from Whatman;
mouse fibroblast-like cells (McCoy) were purchased from ATCC;
lawsonia intracellularis positive strain from bouling invager.
The LI Omp2 protein monoclonal antibody and its polyclonal antibody were prepared in this laboratory;
FITC-labeled goat anti-mouse antibody and HRP-labeled goat anti-rabbit antibody are all products of Sigma company;
the AEC color development kit is purchased from Nanjing Biotechnology Ltd.
The bacterial genome DNA extraction kit is a product of Germany Qigen company;
amphotericin B, neomycin, and vancomycin were purchased from Shanghai assist, Biotech Co., Ltd;
DAPI and hematoxylin staining solutions were purchased from kayki biotechnology limited;
female SPF-grade ICR mice at 4 weeks of age were purchased from Nanjing Qinglongshan animal farms;
the fecal genomic DNA extraction kit is purchased from the International Biogene technology Co., Ltd of Beijing Zhuang;
the rabbit IgG-immunohistochemical kit SABC ready-to-use immunohistochemical kit is a product of biological engineering Limited company of doctor Wuhan. Other reagents not listed are commercially available from regular sources;
EXAMPLE 1 preparation of Lawsonia intracellularis isolates
A suspected porcine ileum sample with typical symptoms of PPE was taken out of a-80 ℃ refrigerator, washed 3 times with PBS for rapid thawing, the intestinal tube was longitudinally split, intestinal mucus was scraped off on a sterile slide glass into a 50mL centrifuge tube, pancreatin was added thereto, digestion was carried out at 37 ℃ for 30min, 30mL of SPG solution containing 10% FBS was added to the digested intestinal mucus, the diluted intestinal mucus was placed in a tissue homogenate tube and homogenized, the homogenate was filtered using a 200 mesh cell screen, the filtrate was collected in 50mL centrifuge tube, followed by 1.2, 0.8 and 0.65 μm filter membrane in sequence, finally DMSO was added to the filtrate to a final concentration of 10% to obtain Lawsonia intracellularis isolate, 1mL of which was dispensed into 1.5mL EP tubes and stored in a-80 ℃ refrigerator.
Example 2: isolation and culture of Lawsonia intracellularis
1) Recovery and subculture of McCoy cells
The McCoy cells were taken out of the liquid nitrogen, immediately placed in a 37 ℃ water bath to be thawed, and the thawed cells (cell density about 5X 10)6one/mL) was added to 8mL of 10% FBS-containing HDMEM culture medium at 37 ℃ in 5% CO2Culturing under the condition, after the cells grow into a monolayer, washing with sterile PBS for 3 times, digesting with 0.25% pancreatin, stopping digestion when a small amount of cells fall off, removing digestive juice, adding DMEM cell culture solution containing 10% FBS, repeatedly blowing and beating for several times until the cells are dispersed into single cells, sucking a proper amount of cell liquid to another cell bottle for subculture, and using the cells after adapting to a culture environment.
2) Bacterial inoculation and culture
Will be 1 × 105After McCoy cells were cultured at a concentration of about 30% per mL, the culture medium was discarded, the L.intracellularis isolate prepared in example 1 and diluted 10 times in the LI-inoculated culture medium was added, and the mixture was incubated at 37 ℃ under 8% O2、8.8％CO2And 83.2% N2Culturing in the three-air culture box, changing the culture solution for LI culture after 3h, and continuously culturing for 7d, wherein the culture solution is changed every 2-3 d;
3) passage and cryopreservation of lawsonia intracellularis
After the lawsonia intracellularis is cultured in McCoy cells for 7d, discarding the old culture solution, adding 0.1% KCI, and acting at 37 ℃ for 10 min; discarding 0.1% of KCI, adding 4mL of DMEM culture solution containing 10% of FBS, scraping cells, uniformly blowing by a Pasteur pipette, transferring the cell suspension into a 50mL centrifuge tube, repeatedly blowing by a No. 20 sterile syringe needle for about 10 times to fully crack the cells, centrifuging at room temperature for 5min, repeating for 3 times, taking part of supernatant to add to a fresh McCoy cell monolayer with the cell density of about 30%, and then placing in a three-gas culture box to continue to carry out next-generation culture; adding DMSO with final concentration of 10% into the rest supernatant, mixing well, and packaging in freezing tube at 1 mL/tube, and storing in refrigerator at-80 deg.C.
Example 3: identification of Lawsonia intracellularis
3.1 Transmission Electron microscopy of the morphology of the strains of Lawsonia intracellularis in McCoy cells
Ultrathin sections of the samples obtained by inoculating the lawsonia intracellularis obtained in example 2 into McCoy cells for 3 hours are prepared, and the samples are stained with phosphotungstic acid and then the McCoy cytoplasm is observed under a transmission electron microscope to see whether bacteria conforming to the morphological characteristics of LI are contained in the McCoy cytoplasm; as a result, as shown in FIG. 1, rod-shaped and comma-shaped bacteria were observed in the cytoplasm of McCoy cells, consistent with the morphological characteristics of Lawsonia intracellularis.
3.2 general PCR identification
The LI aspA gene (F: GCTGTGGATTGGGAGAAATC; R: CAAGTTGACCAGCCTCTGC) was synthesized according to literature reports in this experiment, and the amplification sequence was 167 bp. The bacterial genomic DNA of LJS19051 strain obtained in example 2 was extracted using a bacterial genomic DNA extraction kit, and LI aspA gene was sequentially PCR-amplified using the same as a template, while lawsonia intracellularis genomic DNA in blingular ileitis attenuated seedlings was used as a negative control. The PCR reaction system (25. mu.L) was: PCR Mix 12.5. mu.L, upstream and downstream primers 0.5. mu.L each, template 1.0. mu.L, ddH2O10.5. mu.L. The PCR cycling conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 50s, extension at 72 ℃ for 60s, and extension at 72 ℃ for 10min after 30 cycles. Carrying out agarose gel electrophoresis on the PCR product, taking a picture of a gel imaging system to observe the result, and simultaneously setting ddH in the experiment2Negative control with O instead of template. As shown in FIG. 2, a fragment corresponding to the expected result of the LI aspA gene was amplified using genomic DNA extracted from the L.intracellularis strain isolated and cultured in example 2 as a template, and the sequence result showed that the homology of the gene fragment to the LI aspA gene was 100%, and the isolated strain was preliminarily determined to be L.intracellularis and named LJS 19051.
3.3 Indirect immunofluorescence identification
McCoy cell concentration was adjusted to 1X 10 with 10% FBS-containing HDMEM complete medium5Inoculating to 24-well cell culture containing cell slideCulturing at 500 μ L/well, 37 deg.C and 5% CO2Culturing in an incubator until the cell density is about 30%, discarding the cell culture solution, adding 500 μ L of LJS19051 bacterial solution (diluted by 10 times with the culture solution for LI inoculation) obtained in example 2 into each hole, setting a negative control and a positive control, culturing in a three-gas culture incubator for 3h, replacing the culture solution for LI culture, continuously culturing for 9h and 21h, respectively, discarding the culture solution, washing with PBS for 3 times and 5 min/time, adding 500 μ L of methanol pre-cooled at 20 ℃ into each hole, fixing at room temperature for 10min, discarding the fixing solution, and washing with PBS for 3 times; adding a 1: 200-time diluted rat anti-lawsonia intracellularis Omp2 protein monoclonal antibody (a diluent is PBS) into each hole, incubating for 60min in a 37 ℃ wet box, washing for 5 times and 5 min/time with PBS, sequentially adding 1: 1000-time diluted FITC-labeled goat anti-rat fluorescent secondary antibody (a diluent is PBS) into each hole, incubating for 60min in a 37 ℃ wet box, washing for 5 times and 5 min/time with PBS, adding 100 mu LDAPI staining solution into each hole, reacting for 15min in a dark place at room temperature, washing for 5 times and 5 min/time with PBS, sealing with a sealing agent, taking out a slide, airing, placing on a glass slide, and observing the result with a laser confocal microscope. The results showed that when the cell culture inoculated with the bacteria was cultured for 12h, small green fluorescent particles in a comma-like distribution were visible in the cytoplasm (see FIG. 3A), indicating that the bacteria had entered the cells; when the action time is prolonged to 24h, the green fluorescence in cytoplasm is gradually increased, and the bacteria are gathered into clusters and distributed around the cell nucleus (see figure 3B); as shown in FIG. 3C, specific green fluorescence was also observed in cytoplasm when the positive control group cells were cultured for 24 h; while cells not inoculated with LJS19051 showed no green fluorescence (see fig. 3D), it was further determined that isolate strain LJS19051 was lawsonia intracellularis.
3.4 peroxidase Single layer assay identification
McCoy cell concentration was adjusted to 1X 10 with 10% FBS-containing HDMEM complete medium5one/mL, 100. mu.L/well inoculated in 96-well cell culture plates at 37 ℃ with 5% CO2When the cells were cultured in the incubator to a cell density of about 30%, the culture medium was discarded, 100. mu.L of the bacterial suspension (obtained by diluting the bacteria LJS19051 isolated in example 2 and the culture medium for LI inoculation at a ratio of 1: 10) was added to each well, and a negative control and a positive control were simultaneously set in the experiment, and the cells were cultured in a three-gas incubator for 3 hours and then replaced with the culture medium for LI inoculationContinuously culturing for 7d, and replacing the culture solution every 2-3 d; 7d, discarding the culture solution, washing with PBS for 3 times, and patting to dry; adding 100 μ L of 4% paraformaldehyde into each well, fixing at room temperature for 10min, discarding the fixing solution, washing with PBS for 3 times and 5 min/time; adding 0.5% Triton-X100 into each well, acting at room temperature for 10min, washing with PBS for 3 times, adding 1:100 times diluted rabbit anti-Lawsonia intracellularis Omp2 protein polyclonal antibody (the diluent contains 0.05% Tween-20 PBS), incubating in 37 deg.C wet box for 60min, washing with PBST for 5 times, 5 min/time, sequentially adding 1:2000 times diluted HPR labeled goat anti-rabbit enzyme labeled secondary antibody (the diluent contains 0.05% Tween-20 PBS) into each well, incubating in 37 deg.C wet box for 60min, washing with PBST for 5 times, 5 min/time, adding 100 μ L AEC developer into each well, reacting at 37 deg.C in dark for 15min, discarding, and sterilizing ddH2Terminating the reaction by O; washing with PBS for 3 times and 5 min/time, adding 100 μ L hematoxylin per well for 1min, washing with PBS for 3 times and 5 min/time, and observing the result under inverted microscope. As a result, as shown in FIGS. 4A-B, the McCoy cell culture inoculated with isolate LJS19051 exhibited specific red fluorescence in its cytoplasm; specific red fluorescence was also present in the cytoplasm of McCoy cells, a positive control group for LI in the co-vaccination (fig. 4C); while cells of uninfected bacteria did not show red fluorescence (see FIG. 4B), the above results indicated that isolate LJS19051 is Lawsonia intracellularis.
In conclusion, the method for separating and culturing lawsonia intracellularis provided by the invention has simple operation process, greatly improves the success rate of separating bacteria, and provides important basis for timely prevention, diagnosis and prevention and control of PPE.
Example 4 replication of isolated strain LJS19051 in mice
4.1 cultivation of isolate LJS19051
The culture of LJS19051 was carried out as in example 2, harvested after 7d, and then adjusted to 1X 10 bacteria concentration by HDMEM culture medium8one/mL for use.
4.2 grouping of mice
20 SPF-grade female ICR mice of 4 weeks old are selected, fed with antibiotic-free feed for 7 days and then randomly divided into 2 groups (test group and control group). Before the test, the Lawsonia intracellularis antigen antibody in the experimental mouse excrement and serum is detected, and after the results are negative, the artificial infection test is carried out.
4.3 mice Artificial infection test
The test group mice were inoculated with 500. mu.L/mouse of the isolated strain LJS19051 obtained from 4.1 by gavage; the control mice were drenched with equal volume of HDMEM culture solution, the experimental period was 35d, and the health status of the mice was observed every day. Feces and blood were collected from each group of mice at 7d, 14d, 21d, 28d and 35d post challenge, respectively. Extracting fecal genome DNA by using a fecal genome DNA extraction kit, and carrying out PCR amplification on an LI 16S rRNA gene with a target fragment of 846bp by using an LI 16S rRNA gene specific identification primer (LI 16S rRNA-F: CTTCGGGATGAGTAAAGTG; LI 16S rRNA-R: CTTTGAGTTTCAGCCTTGC); detecting antibody titer in serum of each group of mice at different time points after challenge by an indirect ELISA method; meanwhile, mice are sacrificed by cervical dislocation at 21d after challenge, intestinal changes of the mice in each group are observed after necropsy, and recording and photographing are performed. Aseptically taking at least 2 mouse ileum, soaking 2 parts of ileum sample in formalin solution, embedding 1 part of ileum sample in a conventional method, preparing paraffin sections, performing microscopic examination after H & E staining, and observing results; and in the other 1 part, an LI monoclonal antibody is used, a commercial immunohistochemical detection kit is adopted, immunohistochemical staining of tissue sections is carried out according to instructions, and LI antigen in ileum tissue cells of 21d mice after challenge is detected. The results showed that the mice in the test group showed mild diarrhea at 21d after challenge, and the feces were soft, enlarged and moist (fig. 5A) compared to the control group; the results of the autopsy showed that the mice in the test group showed slight bleeding in the ileum 21d after challenge (fig. 5B), which is consistent with the hemorrhagic enteritis characteristics of lawsonia intracellularis reported in the literature. Subsequently, genomic DNA was extracted from the feces of each of the groups 7d, 14d, 21d, 28d and 35d of mice after challenge using a fecal genomic DNA extraction kit, and LI 16S rRNA gene was PCR-amplified using LI-specific primers using this as a template. As shown in FIG. 6, in comparison with the control group (lane 7), LI 16S rRNA gene was detected from the feces of mice in the experimental group at several time points (lanes 2 to 6) except that LI 16S rRNA gene was not detected from the feces of mice in the experimental group at 7d (lane 1), indicating that the isolate strain LJS19051 successfully replicated in mice and was excreted through feces. Then, the indirect ELISA method is adopted to measure the LI antibody titer in the serum of each group of mice at different time points after the challenge, the detection result is shown in Table 1, 14 days after the challenge, the LI antibody in the serum of the mice starts to turn positive, and the LI antibody titer is highest 21 days after the challenge; furthermore, at 35d after challenge, LI antibodies were still detectable from the sera of the test mice, while the sera of the control mice were still negative. The results show that the lawsonia intracellularis isolate strain LJS19051 obtained in the research is successfully replicated in mice, toxin is expelled outwards through excrement, and the 14d antibody after toxin attack turns positive, which is consistent with the toxin attack model result of lawsonia intracellularis on pigs reported in the literature. Finally, the ileum of each group of mice fixed in formalin at 21d after challenge was subjected to H & E staining and IHC test, respectively. The H & E results indicate that intestinal villi shed in the ileal samples of the test mice (fig. 7A), which is characterized by typical ileitis lesions, while the control mice had intact intestines (fig. 7B). In the IHC test using LI Omp2 monoclonal antibody on the ileal crypt epithelial cells of mice in the test group, L.intracellularis was detected, which is consistent with the results reported in the literature that LI is present in intestinal crypt epithelial cells (FIG. 8A), whereas LI is not detected in intestinal crypt cells of control group mice (FIG. 8B).
TABLE 1
The PBS buffer in the above examples was obtained by the following steps:
taking 3.63g of disodium hydrogen phosphate dodecahydrate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride and 8g of sodium chloride; mixing the above components, dissolving in 1000mL ultrapure water, adjusting pH to 7.4-7.6 with 0.1M HCI, filtering with 0.22 μ M filter membrane, and storing at 4 deg.C.
The SPG solution in the above example was obtained by the following steps:
dissolving 74.556g sucrose, 0.5168g potassium dihydrogen phosphate, 1.2528g dipotassium hydrogen phosphate and 0.9065g potassium glutamate in 1000ml ultrapure water, adjusting pH to 7.0, filtering with 0.22 μm filter membrane, and storing at room temperature for use.
The complete culture solution of McCoy cells in the above examples was obtained by the following steps:
HDMEM culture solution 90V/V%
Fetal bovine serum 10V/V%
The culture solution for LI inoculation in the above example was obtained by the following steps:
the LI culture medium in the above examples was obtained by the following steps:
the above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalent changes may be made in the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and the technical solutions of the present invention should be covered in the claims of the present invention.
Claims (10)
1. A method for separating and culturing Lawsonia intracellularis is characterized by comprising the following steps:
step 1: preparing lawsonia intracellularis isolate;
step 2: subculturing McCoy cells;
and step 3: diluting the lawsonia intracellularis isolate obtained in the step 1 and the lawsonia intracellularis inoculation culture solution according to a ratio of 1:10, adding the diluted lawsonia intracellularis isolate and the diluted lawsonia intracellularis inoculation culture solution into McCoy cells for culture, replacing the diluted lawsonia intracellularis inoculation culture solution after 3 hours, continuously culturing for 7 days, replacing the culture solution every 2-3 days, harvesting bacteria after 7 days, inoculating the bacteria to fresh McCoy cells, and continuing to perform next-generation culture;
and 4, step 4: carrying out passage and cryopreservation on lawsonia intracellularis;
and 5: identifying lawsonia intracellularis;
the lawsonia intracellularis inoculation culture solution comprises the following components in percentage by total volume:
the culture solution for culturing the lawsonia intracellularis comprises the following components in terms of the total volume of the culture solution:
% represents volume percent.
2. The method for isolating and culturing lawsonia intracellularis according to claim 1, wherein the lawsonia intracellularis isolate prepared in step 1 by the method comprising: cleaning suspected pig ileum samples with typical symptoms of PPE, longitudinally splitting an intestinal tube, scraping intestinal mucus by using a sterile glass slide into a centrifuge tube, adding pancreatin for digestion, adding SPG solution containing 10% FBS into the digested intestinal mucus, placing the diluted intestinal mucus into a tissue homogenate tube for homogenate, filtering the homogenate by using a 200-mesh cell screen, collecting filtrate into the centrifuge tube, sequentially passing through 1.2, 0.8 and 0.65 mu m filter membranes, finally adding DMSO with the final concentration of 10% into the filtrate, and subpackaging the filtrate into an EP tube and preserving in a refrigerator at-80 ℃.
3. The method for separating and culturing lawsonia intracellularis according to claim 2, wherein the McCoy cell subculture in step 2 specifically comprises:
resuscitating under sterile conditionsThe McCoy cells (see above) were transferred to a cell culture flask, and HDMEM containing 10% FBS was added thereto, and the mixture was incubated at 37 ℃ and 5% CO2Culturing in an incubator, digesting with 0.25% pancreatin after the cells grow into a monolayer, stopping digestion when a small amount of cells fall off, discarding digestive juice, adding HDMEM cell culture solution containing 10% FBS, repeatedly beating for several times until the cells are dispersed into single cells, sucking appropriate amount of cell sap into another cell bottle for subculture, and using after the cells adapt to the culture environment.
4. The method for separating and culturing Lawsonia intracellularis according to claim 1, wherein the density of the McCoy cells in step 3 is 30% to 35%, and 5% CO is at 37 ℃2The culture time in the incubator should not exceed 24 hours.
5. The method for isolating and culturing Lawsonia intracellularis of claim 1, wherein the culturing in step 3 is carried out by placing the culture in a three-gas culture chamber at 37 ℃ and 8% O2、8.8％CO2And 83.2% N2Under microaerophilic conditions.
6. The method for separating and culturing lawsonia intracellularis according to claim 1, wherein the lawsonia intracellularis inoculation culture solution in step 3 is composed of the following components:
7. the method for separating and culturing L.intracellularis according to claim 1, wherein the identification of L.intracellularis in step 5 is performed by identifying the isolated strain using transmission electron microscopy, general PCR, indirect immunofluorescence and peroxidase monolayer assay, respectively.
8. Lawsonia intracellularis obtained by the method according to any one of claims 1 to 7.
9. The lawsonia intracellularis of claim 8, which is deposited in the chinese type culture collection with a date of 21/7/2020 and a collection number of CCTCC NO: v202046.
10. Use of lawsonia intracellularis as claimed in claim 8 or 9 for the preparation of a vaccine against lawsonia intracellularis infection.
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| CN113528683A (en) * | 2021-07-09 | 2021-10-22 | 南京农业大学 | Absolute fluorescence quantitative PCR primer pair, kit and detection method for detecting lawsonia intracellularis |
| CN114836356A (en) * | 2022-06-06 | 2022-08-02 | 南京农业大学 | Lawsonia intracellularis attenuated vaccine strain, vaccine and application thereof |
| CN114836356B (en) * | 2022-06-06 | 2023-09-05 | 南京农业大学 | Lawsonia intracellularis attenuated vaccine strain, vaccine and application thereof |
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