CN113122509A - Infectious laryngotracheitis virus virulent strain and application thereof - Google Patents
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Abstract
The invention relates to separation and identification of infectious laryngotracheitis virus (ILTV) and establishment of an animal infection model, and belongs to the technical field of microorganisms. A sick chicken suspected to be infected by ILTV in Fujian province is named as an ILTV WF03 strain after ILTV separation, purification and identification, and the pure culture of the strain is used for infecting SPF chickens of 4 weeks or 8 weeks old, so that the sick chicken has high pathogenicity. The virulent strain of the infectious laryngotracheitis virus has strong toxicity, typical ILTV infection clinical symptoms are caused by infecting SPF chickens of 4 weeks old or 8 weeks old, the morbidity is 95% and 100% respectively, and the mortality is 40% and 27.3% respectively. The animal infection model established can be used as a virulent strain for evaluating the effectiveness of different drugs or preventive treatment methods or testing the effectiveness of vaccines.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to separation and identification of an infectious laryngotracheitis virus and establishment of an animal infection model.
Background
Infectious laryngotracheitis virus (ILTV) belongs to infectious laryngotracheitis virus of herpesviridae and alphaherpesviridae, infection mainly causes death and/or egg laying reduction of chickens, severe infection is often manifested by dyspnea, gasping and cough with blood sample liquid and has high lethality, while mild infection is mainly manifested by various forms of mucoid tracheitis, rhinitis, conjunctivitis, unwilling activity, low mortality and the like, and no matter severe infection or mild infection brings serious loss and threat to production.
The virulence of ILTV wild strains is very different, some strains have very high morbidity and mortality, belong to virulent strains, and also have attenuated strains with slight or unobvious symptoms after infection, and because the virulence difference of the wild strains is large, the establishment of a stable animal infection model for vaccine efficacy test is very difficult. The ILTV virulent strain WF03 has high morbidity, the morbidity of SPF chickens at 4 weeks and 8 weeks is 95% and 100% respectively, the mortality of SPF chickens at 4 weeks and 27.3% respectively, the morbidity of SPF chickens mainly takes hemoptysis, death and dyspnea as main clinical symptoms, and evaluation indexes of the SPF virulent strain are easy to observe, so that the WF03 strain belongs to a virulent strain and can be used as an attacking strain for vaccine efficacy evaluation.
Disclosure of Invention
The invention aims to construct an infectious laryngotracheitis virus, and establish a virus separation, identification and animal infection model, and specifically comprises the following steps:
an infectious laryngotracheitis virus strain, which is named as ILTV WF03 strain and is preserved in China Center for Type Culture Collection (CCTCC) No. V202117 at 2.4.2021, and the China center for type culture Collection is Wuhan, Wuhan university.
The construction method of the strain comprises the following steps:
(1) collecting clinical suspected ILTV infected chickens for dying, aseptically collecting trachea and larynx tissues, adding PBS into collected pathological tissue according to the proportion of 1:5, grinding and freezing for 3 times, centrifuging at 12000 r/min for 10 min, filtering with a 0.45-micrometer filter membrane, inoculating the filtrate into SPF (specific pathogen free) chick embryos of 10 days old by an allantoic membrane way, continuously incubating for 7 days, aseptically collecting allantoic membranes, adding PBS into the allantoic membranes according to the proportion of 1:5 (mass-volume ratio g/mL), grinding and freezing for 3 times, centrifuging, taking supernatant, continuously inoculating SPF chick embryos of 10 days old by an allantoic membrane way, and observing chick embryo lesions after 2 times of replacement; the chick embryo allantoic membrane shows grey white pocks after blind passage of 3 generations, is suspected to be infected by ILTV virus, and is identified as the ILTV virus according to the diagnosis technology of chicken infectious laryngotracheitis of the agricultural industry standard NY/T556-;
(2) purifying the identified ILTV isolate in 10-day-old chick embryo by limiting dilution method, and purifying suspected ILTV virus liquid by 10-1、10-2、10-3、10-4、10-5、10-6Inoculating SPF chick embryos of 10 days old at dilution, inoculating 6 embryos at each dilution, continuously incubating for 7 days, detecting allantoic membranes, taking the positive allantoic membrane with gray white spots as positive, grinding the positive allantoic membrane with the highest dilution to prepare virus liquid, and continuously purifying for 2 times in the same way; the purified ILTV isolate was designated ILTV WF03 strain.
The method for establishing the SPF chicken artificial infection model by using the strain is characterized by comprising the following steps: the ILTV WF03 strain is used for performing tracheal challenge on SPF chickens of 8 weeks or 4 weeks, and clinical symptoms are continuously observed for 14 days after challenge.
The dosage of the counteracting toxic is 0.2 mL/feather, and the virus content is 104.0 EID50。
The invention has the advantages that:
according to the invention, ILTV WF03 strain is used for attacking virus of SPF chickens of 8 weeks and SPF chickens of 4 weeks, an artificial infection model is established, and the result shows that WF03 strain belongs to virulent strain, which can cause 100% morbidity and 27.3% mortality of SPF chickens of 8 weeks; can result in 95% morbidity and 40% mortality in SPF chickens at 4 weeks of age.
Drawings
FIG. 1 shows pathologic changes of allantoic membrane after 7 days of inoculation of ILTV WF03 strain into chick embryo, i.e., grey white pock (upper) and normal allantoic membrane control (lower).
FIG. 2 shows the mouth opening respiration and blood stain in the corner of the mouth after the attack of toxin.
FIG. 3 shows that a large amount of fresh blood or blood clots can be seen in the trachea of the dead chicken after challenge.
Detailed Description
After aseptically collecting the trachea throats of sick chickens suspected of being infected by ILTV, 10-day-old SPF (specific pathogen free) chick embryos are inoculated by an allantoic membrane way for pathogen separation, purification and identification, an artificial infection model is established by artificially infecting SPF chickens of 4-week age or 8-week age, and the separated and identified WF03 strain is proved to belong to a virulent strain and can be used as the virulent strain for vaccine immunity efficacy test. The method comprises the following specific steps:
example 1 isolation and purification of strains, identification
Collecting clinically suspected ILTV infected chickens for dying, aseptically collecting trachea and larynx tissues, adding PBS into collected pathological tissue according to the proportion of 1:5, grinding, freezing and thawing for 3 times, centrifuging at 12000 r/min for 10 min, filtering with a 0.45-micron filter membrane, inoculating the filtrate into SPF (specific pathogen free) chick embryos of 10 days old by an allantoic membrane way, continuously incubating for 7 days, aseptically collecting allantoic membranes, adding PBS into the allantoic membranes according to the proportion of 1:5 (mass-volume ratio g/mL), grinding, freezing and thawing for 3 times, centrifuging, taking supernatant, continuously inoculating SPF chick embryos of 10 days old by an allantoic membrane way, and observing chick embryo lesions after 2 times of replacement. The chick embryo allantoic membrane shows grey white variola after blind passage 3 (figure 1), is suspected to be infected by ILTV virus, and is identified as ILTV virus according to the diagnosis technology of infectious laryngotracheitis of chicken in accordance with the agricultural industry standard NY/T556 and 2002 of the people's republic of China.
Purifying the identified ILTV isolate in 10-day-old chick embryo by limiting dilution method, and purifying suspected ILTV virus liquid by 10-1、10-2、10-3、10-4、10-5、10-6Inoculating SPF chick embryos of 10 days old at dilution, inoculating 6 embryos at each dilution, continuously incubating for 7 days, detecting allantoic membranes, taking the positive allantoic membrane with gray white spots as positive, grinding the positive allantoic membrane with the highest dilution to prepare virus liquid, and continuously purifying for 2 times in the same way. The purified ILTV isolate was designated as WF03 strain, allantoic membranes were harvested by scale-up culture in 10-day-old SPF chick embryos to prepare virus solutions, which were designated as P1 generations, and sequentially passaged to prepare virus solutions of different generations, designated as P2 generations, P3 generations, and so on.
Taking out the virus solution from-70 deg.C, thawing to room temperature, adding 0.5 mL into corresponding 4.5 mL PBS, sequentially diluting to 10 times of the volume-66 virus solutions of each dilution were inoculated to 10 days oldTaking another 6 SPF chick embryos as negative control, putting the chick embryos into an incubator at 37 ℃ for further incubation for 7 days, marking the glottis with grey white spots appearing on the allantoic membrane as positive, and calculating the content EID of the virus according to the positive number and the Reed-Muench method50And/ml. The virus content of P1, P2 and P3 generations is determined as follows: 105.4 EID50/ml、105.8 EID50/ml、105.5 EID50/ml。
Example 2 culture Properties of ILTV WF03 Strain
Culturing ILTV WF03 strain needs to be carried out on SPF chick embryos of 9-10 days old, inoculation is carried out by an allantoic membrane approach, the virus content in virus liquid prepared by harvesting the allantoic membrane is highest, and large blocks of off-white lesions or a large amount of off-white pocks appear on the allantoic membrane 7 days after inoculation of the chick embryos. The virus is detected according to a method for detecting bacteria-free, mycoplasma-free and exogenous virus in Chinese veterinary pharmacopoeia (2015 edition), has no bacteria, mycoplasma and exogenous virus pollution, and is pure ILTV virus.
Example 3 establishment of Artificial infection model of ILTV WF03 Strain on 8-week-old SPF Chicken
The 22-feather 8-week SPF chickens are divided into 2 groups, wherein one group is subjected to trachea challenge by using infectious laryngotracheitis virulent WF03 strain, the challenge dose is 0.2 mL/feather, the virus content is 104.0 EID50The other group was used as a negative control group, and clinical symptoms were observed for 14 consecutive days after challenge.
The results show that: the morbidity of the WF03 strain after challenge is 100 percent (11/11), the mortality is 27.3 percent (3/11), and the negative control group has no specific clinical symptoms. Clinical symptoms appeared in 2 days after challenge of WF03 strain, the duration was over 3 days and the appetite was abolished, 3 deaths were observed at day 5, with moderate to severe asthma (11/11) (FIG. 2), hemoptysis (7/11) (FIG. 2), moderate to severe dyspnea (7/11) and flail head (10/11) as the main clinical symptoms. The dead chicken on day 5 was dissected to find that there was a lot of blood or blood clots in the trachea (fig. 3), and the remaining live chickens had no clinical symptoms by day 9 after challenge. Therefore, WF03 strain belongs to a virulent strain, and can cause 100% morbidity and 27.3% mortality of SPF chickens at 8 weeks of age.
Example 4 establishment of Artificial infection model of ILTV WF03 Strain on 4-week-old SPF Chicken
Dividing 30-feather 4-week SPF chickens into 2 groups, wherein one group of 20 feathers is subjected to tracheal challenge by using infectious laryngotracheitis virulent WF03 strain, the challenge dose is 0.2 mL/feather, the virus content is 104.0 EID50And the other group of 10 feather non-offensive toxin is used as a negative control group, and clinical symptoms are observed for 14 days after offensive toxin.
The results show that: after challenge, the morbidity of the WF03 strain is 95 percent (19/20), the mortality of the WF03 strain is 40 percent (8/20), and specific clinical symptoms do not occur in a negative control group. Clinical symptoms appear in succession 2 days after challenge of WF03 strain, the duration exceeds 2 days, the appetite is abolished, hemoptysis (15/20), death (8/20) and dyspnea (5/20) are used as main clinical symptoms, and the death occurs 2-5 days after challenge, wherein 3-feather chickens die acutely and do not have symptoms such as hemoptysis and dyspnea. Dead chickens were dissected and found to have large amounts of blood or blood clots in the trachea. The remaining surviving chickens had clinical symptoms disappeared from day 6 after challenge. Therefore, WF03 strain belongs to a virulent strain, and can cause 95% morbidity and 40% mortality of SPF chickens at 4 weeks of age.
Immunizing commercial chicken infectious laryngotracheitis recombinant fowlpox virus bigeminal live vaccine 1 feather of 20-feather 1-day-old SPF chicken per feather, immunizing for 28 days, and performing tracheal challenge on the immune vaccine together with other 20-feather non-immune chickens (positive challenge group) by using infectious laryngotracheitis virulent WF03 strain, wherein the challenge dose is 0.2 mL/feather, and the virus content is 104.0 EID50In addition, 10 feather chickens which do not attack the toxin are used as a negative control group, and clinical symptoms are observed continuously for 14 days after the attack of the toxin.
The results show that: the morbidity of the positive challenge group is 95% (19/20), the mortality is 40%, the main manifestations are hemoptysis and dyspnea, the immune chicken does not show any clinical symptoms and pathological changes, the protection rate is 100%, and the negative control chicken shows normal.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
1. An infectious laryngotracheitis virus strain is named as infectious laryngotracheitis virus ILTV WF03 strain, which is preserved in China center for type culture Collection (CCTCC NO: V202117) at 2 months and 4 days of 2021.
2. A method for constructing the strain as claimed in claim 1, which comprises the following steps:
(1) taking clinically suspected ILTV infected chickens to die, aseptically collecting trachea and larynx tissues, adding PBS into collected pathological tissue according to the proportion of 1:5, grinding, freezing and thawing for 3 times, centrifuging at 12000 r/min for 10 min, filtering with a 0.45-micrometer filter membrane, inoculating the filtrate into SPF (specific pathogen free) chick embryos of 10 days old by an allantoic membrane way, continuously incubating for 7 days, aseptically obtaining allantoic membranes, adding PBS into the allantoic membranes according to the proportion of 1:5 g/mL, grinding, freezing and thawing for 3 times, centrifuging, taking supernatant, continuously inoculating SPF chick embryos of 10 days old by the allantoic membrane way, and observing the pathological changes of the chick embryos after 2 times of replacement; the chick embryo allantoic membrane shows grey white pocks after blind passage of 3 generations, is suspected to be infected by ILTV virus, and is identified as the ILTV virus according to the diagnosis technology of chicken infectious laryngotracheitis of the agricultural industry standard NY/T556-;
(2) purifying the identified ILTV isolate in 10-day-old chick embryo by limiting dilution method, diluting suspected ILTV virus liquid with PBS 10 times in series, and then diluting according to 10 times-1、10-2、10-3、10-4、10-5、10-6Inoculating SPF chick embryos of 10 days old at dilution, inoculating 6 embryos at each dilution, continuously incubating for 7 days, detecting allantoic membranes, taking the positive allantoic membrane with gray white spots as positive, grinding the positive allantoic membrane with the highest dilution to prepare virus liquid, and continuously purifying for 2 times in the same way; the purified ILTV isolate was designated ILTV WF03 strain.
3. Method for establishing an artificial infection model of SPF-chickens using the strain of claim 1, comprising the steps of: the ILTV WF03 strain is used for performing tracheal challenge on SPF chickens of 4 weeks and 8 weeks, clinical symptoms are continuously observed for 14 days after challenge, and the morbidity of the SPF chickens is over 80 percent.
4. The method of claim 3, wherein the challenge dose is 0.2 mL/plume and the virus content is 104.0 EID50。
5. Use of the infection model constructed by the method of claim 3 to evaluate the efficacy of a vaccine.
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| CN113652406A (en) * | 2021-09-01 | 2021-11-16 | 青岛易邦生物工程有限公司 | Recombinant virus strain for avian infectious laryngotracheitis and application thereof |
| CN118319960A (en) * | 2024-06-12 | 2024-07-12 | 华南农业大学 | Method for constructing animal model of long-acting wind-cold lung disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113652406A (en) * | 2021-09-01 | 2021-11-16 | 青岛易邦生物工程有限公司 | Recombinant virus strain for avian infectious laryngotracheitis and application thereof |
| CN118319960A (en) * | 2024-06-12 | 2024-07-12 | 华南农业大学 | Method for constructing animal model of long-acting wind-cold lung disease |
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