Immune effect evaluation method of fowl adenovirus 11 vaccine

Abstract

The invention discloses an immune effect evaluation method of an avian adenovirus 11 vaccine, and belongs to the technical field of vaccine evaluation. According to the method, immune antibodies are obtained after breeding hens are used for immunizing the fowl adenovirus 11 vaccine, then the immune antibodies are transmitted to fertilized eggs and hatched offspring chicks through passive immunization, the offspring chicks passively obtain maternal antibodies, the offspring chicks are used for counteracting toxic substances, and the fowl adenovirus 11 vaccine is obtained. Meanwhile, the death condition and / or clinical symptoms of the immune offspring chicks after challenge are / is taken as an index, or the death condition and / or inclusion body hepatitis symptoms after dissection of the immune offspring chicks after challenge are / is taken as an index, or the virus load in liver tissues of the immune offspring chicks after challenge is taken as an index; the method can be used for efficiently, quickly and accurately evaluating the fowl adenovirus 11 type vaccine. Therefore, the method for evaluating the immune effect of the fowl adenovirus type 11 vaccine has a relatively good application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine evaluation, and in particular relates to a method for evaluating the immune effect of an avian adenovirus type 11 vaccine. Background Art

[0002] Avian adenovirus (FAdV) belongs to the Adenoviridae family and the genus Avian Adenovirus. It is highly prevalent in poultry, including chickens and ducks, and is a common cause of avian infectious diseases worldwide. Based on serum cross-neutralization tests, it is divided into 12 serotypes (1-7, 8a, 8b, 9-11). Inclusion body hepatitis (IBH) is a disease caused by FAdV infection in chickens, primarily caused by FAdV-2, FAdV-11, FAdV-8a, and FAdV-8b. It typically occurs in chickens aged 3-5 weeks, with a mortality rate as high as 30%. Necropsy findings include liver enlargement, jaundice, white necrotic foci on the surface, and a friable texture. Epidemiological surveys have revealed that in recent years, FAdV-11 has been the primary serotype responsible for IBH, accounting for over 39% of cases and making it the predominant serotype.

[0003] Clinically, FAdV-11 can be isolated from chickens of all ages, suggesting widespread infection. However, in laboratory experiments using FAdV-11 to infect SPF chickens, the age of the chickens plays a decisive role in the infection: older chickens are neither effectively infected with FAdV-11 nor develop IBH. This may be related to the more complex clinical conditions of chickens (e.g., immunosuppression, immunodeficiency, malnutrition, mixed infections, etc.). The low susceptibility and pathogenicity of FAdV-11 to 7-day-old and 14-day-old SPF chickens in laboratory environments will cause the following problems: when 0-1-day-old chicks are immunized with avian adenovirus type 11 vaccine, according to the rules of antibody production, high specific antibodies can only be produced at 7-14 days of age (immunological principles believe that the incubation period of the response induced by primary immunization (vaccine immunization) is long, and the highest antibody level can often be produced 21-28 days after immunization). At this time, regardless of whether the chickens are vaccinated or not, they cannot be effectively infected with the virus, resulting in the inability to obtain effective conclusions when evaluating the FAdV-11 vaccine for virus attack.

[0004] Although clinically, the FAdV-11 carrier rate in chickens is high, with a mortality rate of about 25%, and they also show obvious symptoms of inclusion body hepatitis and high pathogenicity (which may be related to factors such as immunosuppression, immunodeficiency, malnutrition, and mixed infection); however, in a laboratory environment, when SPF breeder chickens are vaccinated with FAdV-11, the pathogenicity fluctuates, resulting in different degrees of disease, unclear clinical symptoms, poor experimental repeatability, and the inability to evaluate the effectiveness of the avian adenovirus type 11 vaccine. Summary of the Invention

[0005] The present invention aims to provide a method for evaluating the immune efficacy of avian adenovirus type 11 vaccines. This method addresses the problems of existing FAdV-11 vaccinations in SPF breeder chickens, which can lead to fluctuating pathogenicity, varying degrees of disease severity, ambiguous clinical symptoms, poor test reproducibility, and an inability to evaluate the effectiveness of avian adenovirus type 11 vaccines.

[0006] In a first aspect, the present invention provides a method for evaluating the immune effect of an avian adenovirus type 11 vaccine, comprising the following steps: S1, immunizing female breeder chickens and male breeder chickens with an avian adenovirus type 11 vaccine, and after the immunization, combining the chickens to obtain a first fertilized egg; S2, hatching the first fertilized egg to obtain immune offspring chicks, and challenging the immune offspring chicks with avian adenovirus type 11, using the death and / or clinical symptoms of the immune offspring chicks after the challenge as indicators, or using the death and / or inclusion body hepatitis symptoms of the immune offspring chicks after the challenge as indicators, or The immune effect of avian adenovirus type 11 vaccine was evaluated using the viral load in the liver tissue of the immune offspring chicks after the challenge as an indicator; the immune effect of avian adenovirus type 11 vaccine was evaluated using the mortality and / or clinical symptoms of the immune offspring chicks after the challenge as indicators, specifically including: if there was death among the immune offspring chicks after the challenge, the avian adenovirus type 11 vaccine was judged to be unqualified; if there was no death among the immune offspring chicks after the challenge, or there was no death but clinical symptoms appeared, the avian adenovirus type 11 vaccine was judged to be qualified; clinical symptoms included lethargy and small body size.

[0007] In the present invention, the inventors have discovered that by immunizing breeder chickens with avian adenovirus type 11 vaccine to obtain immune antibodies, and then transferring the immune antibodies to fertilized eggs and their hatched offspring chicks through passive immunization, the offspring chicks can passively obtain maternal antibodies, and the offspring chicks are used for virus attack. At the same time, the mortality and / or clinical symptoms of the immune offspring chicks after virus attack are used as indicators, so that the avian adenovirus type 11 vaccine can be evaluated efficiently, quickly and accurately. This solves the technical difficulties in the prior art of directly using breeder chickens for virus attack, which have the problems of fluctuating pathogenicity, different degrees of disease onset, unclear clinical symptoms, poor experimental repeatability, and inability to evaluate the effectiveness of the avian adenovirus type 11 vaccine.

[0008] In some embodiments, in step S1, the ratio of female breeder hens to male breeder hens is (3-5):1, preferably 4:1.

[0009] It is understandable that the ratio of female breeder hens to male breeder hens can be routinely adjusted according to actual usage needs, as long as they can produce eggs efficiently.

[0010] In some embodiments, in step S1, both the female breeder hens and the male breeder hens are SPF breeder hens.

[0011] In some embodiments, in step S1, the first fertilized eggs include first fertilized eggs that are caged for 5-10 weeks.

[0012] In the present invention, the inventors further discovered that the immune offspring chicks hatched from the first fertilized eggs that were caged together for 5-10 weeks have higher immune antibodies, which facilitates a more accurate evaluation of the immune effect of the avian adenovirus type 11 vaccine after subsequent challenge.

[0013] In some embodiments, in step S2, immunizing the offspring chicks includes immunizing the offspring chicks at one day of age.

[0014] In the present invention, the inventors further discovered that after using one-day-old immune offspring chicks for virus attack, 100% of the chicks died, the chicks became seriously ill, the symptoms of inclusion body hepatitis were obvious and severe, and the viral load was high. Therefore, the one-day-old immune offspring chicks are relatively stable targets for virus attack, and can more accurately evaluate the immune effect of avian adenovirus type 11 vaccine.

[0015] In some embodiments, in step S2, the route of challenge includes intramuscular injection.

[0016] In the present invention, the inventors further discovered that intramuscular injection for attacking the virus has the advantages of high pathogenicity, little difference in individual disease severity, clear symptoms, and high reproducibility of results. Therefore, intramuscular injection is a better way to attack the virus and can more accurately evaluate the immune effect of avian adenovirus type 11 vaccine.

[0017] In some embodiments, in step S2, the dose of the challenge includes an absolute lethal dose, and the absolute lethal dose is at least 3×10 7 TCID 50 / chicken.

[0018] In the present invention, the inventors first determined the titer of fowl adenovirus 11 (FAdV-11) using LMH cells, and then used one-day-old blank SPF chickens to challenge the virus by intramuscular injection of different doses of FAdV-11. The virus dose at which FAdV-11 caused all death of the one-day-old blank SPF chickens was determined. This dose was the absolute lethal dose; using this absolute lethal dose for challenge can more accurately evaluate the immune effect of the fowl adenovirus type 11 vaccine.

[0019] In some embodiments, in step S2, the immune effect of the avian adenovirus type 11 vaccine is evaluated based on the death of the immune offspring chicks after the challenge and / or the symptoms of inclusion body hepatitis after autopsy, specifically including: if there is death among the immune offspring chicks after the challenge, the avian adenovirus type 11 vaccine is judged to be unqualified; if there is no death among the immune offspring chicks after the challenge, and symptoms of inclusion body hepatitis are found after autopsy, the avian adenovirus type 11 vaccine is judged to be unqualified; if there is no death among the immune offspring chicks after the challenge, and symptoms of inclusion body hepatitis are found after autopsy, the avian adenovirus type 11 vaccine is judged to be qualified.

[0020] In the present invention, the inventors further discovered that the use of the mortality of immune offspring chickens after infection and / or the symptoms of inclusion body hepatitis after autopsy as indicators can more efficiently and accurately evaluate the immune effect of avian adenovirus type 11 vaccine.

[0021] In some embodiments, the symptoms of inclusion body hepatitis include at least one of liver enlargement, liver hemorrhage, liver necrosis, liver yellowing, and liver friability.

[0022] It is understood that the symptoms of inclusion body hepatitis can be selected from conventional inclusion body hepatitis symptom indicators in the prior art according to actual needs, as long as they can reflect inclusion body hepatitis. In the present invention, the symptoms of inclusion body hepatitis preferably include at least one of liver enlargement, liver hemorrhage, liver necrosis, liver jaundice, and liver brittleness.

[0023] In some embodiments, in step S2, the immune effect of the avian adenovirus type 11 vaccine is evaluated using the viral load in the liver tissue of the immune offspring chickens after the challenge as an indicator and the viral load in the liver tissue of the negative control non-immune offspring chickens after the challenge as a reference, specifically comprising: if the viral load in the liver tissue of the immune offspring chickens after the challenge is less than or equal to the geometric mean and 3 times the standard deviation of the viral load in the liver tissue of the negative control non-immune offspring chickens, the avian adenovirus type 11 vaccine is determined to be qualified; if the viral load in the liver tissue of the immune offspring chickens after the challenge is less than or equal to the geometric mean and 3 times the standard deviation of the viral load in the liver tissue of the negative control non-immune offspring chickens, the avian adenovirus type 11 vaccine is determined to be qualified; If the viral load is greater than the sum of the geometric mean of the viral load in the liver tissue of the negative control non-immune offspring chicks and 2, the avian adenovirus type 11 vaccine is judged to be unqualified; wherein, the negative control non-immune offspring chicks after the virus attack are obtained by directly combining female breeder chickens and male breeder chickens to obtain a second fertilized egg; then the second fertilized egg is hatched to obtain the negative control non-immune offspring chicks, and finally the negative control non-immune offspring chicks are attacked with avian adenovirus type 11, the route of attack includes intramuscular injection, and the dose of the attack includes an absolute lethal dose, and the absolute lethal dose is at least 3×10 7 TCID 50 / chicken.

[0024] In the present invention, the inventors further discovered that the immune effect of avian adenovirus type 11 vaccine can be evaluated more efficiently and accurately by using the viral load in the liver tissue of immune offspring chickens after infection as an indicator and the viral load in the liver tissue of negative control non-immune offspring chickens after infection as a reference.

[0025] In some embodiments, the ratio of female breeder hens to male breeder hens is (3-5):1, preferably 4:1; the second fertilized eggs include second fertilized eggs that are caged for 5-10 weeks; and the negative control non-immune offspring chicks include 1-day-old negative control non-immune offspring chicks.

[0026] In the present invention, by controlling the incubation conditions of the negative control non-immune offspring chicks to be consistent with the incubation conditions of the immunized offspring chicks, it is convenient to more accurately evaluate the immune effect of the avian adenovirus type 11 vaccine.

[0027] The beneficial effects of the present invention are as follows: different from the existing technology, the present invention obtains immune antibodies after immunizing breeder chickens with avian adenovirus type 11 vaccine, and then transfers the immune antibodies to fertilized eggs and their hatched offspring chicks through passive immunization, so that the offspring chicks passively obtain maternal antibodies, and the offspring chicks are used for virus attack. At the same time, the mortality and / or clinical symptoms of the immune offspring chicks after virus attack are used as indicators, so that the avian adenovirus type 11 vaccine can be evaluated efficiently, quickly and accurately, solving the technical difficulties in the existing technology of directly using breeder chickens for virus attack, which have the problems of fluctuating pathogenicity, different degrees of disease onset, unclear clinical symptoms, poor experimental repeatability, and inability to evaluate the effectiveness of avian adenovirus type 11 vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention is a flowchart of the immune effect evaluation method of the avian adenovirus type 11 vaccine. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Experimental procedures in the examples, where specific conditions are not specified, were generally performed in accordance with conventional methods in molecular biology, including but not limited to those described in M.R. Green's Molecular Cloning: A Laboratory Manual and Robert F. Weaver's Molecular Biology, or according to the recommendations of kit and instrument manufacturers. Unless otherwise specified, reagents and biological materials used in the examples were commercially available.

[0031] See also Figure 1 , which is a flow chart of the immune efficacy evaluation method of the avian adenovirus type 11 vaccine of the present invention. Specifically, the immune efficacy evaluation method of the avian adenovirus type 11 vaccine comprises the following steps: S1, immunizing female and male breeder chickens with the avian adenovirus type 11 vaccine, and after immunization, combining the chickens to obtain first fertilized eggs; S2, hatching the first fertilized eggs to obtain immune offspring chicks, and challenging the immune offspring chicks with avian adenovirus type 11. The immune efficacy of the avian adenovirus type 11 vaccine is evaluated based on the mortality and / or clinical symptoms of the immune offspring chicks after challenge, or the mortality and / or inclusion body hepatitis symptoms of the immune offspring chicks after challenge, or the viral load in the liver tissue of the immune offspring chicks after challenge.

[0032] In the present invention, the avian adenovirus type 11 vaccine can be conventionally selected according to actual use needs. For example, it can be a vaccine reported in the prior art, or it can be a recombinant avian adenovirus type 11 fiber protein subunit vaccine. The recombinant avian adenovirus type 11 fiber protein subunit vaccine is prepared as follows: The recombinant avian adenovirus type 11 fiber protein was filtered through a 0.22μm microporous membrane and diluted with sterile PBS. 2% Tween-80 was added to create an aqueous phase. This phase was then mixed with a white oil adjuvant in a 1:3 volume ratio. The mixture was sheared using a high-speed shear at 20,000 rpm for 3 minutes, followed by a 2-minute pause, and repeated three times. A small amount of the emulsified vaccine was dripped into clean, cold water. Complete emulsification was indicated if all but the first drop did not immediately disperse. A 2mL portion of the fully emulsified vaccine was centrifuged at 3,000 rpm for 15 minutes. No delamination was observed, indicating good vaccine stability. The final vaccine concentration was 50μg / mL.

[0033] Among them, the recombinant fowl adenovirus type 11 fiber protein is obtained by PCR amplifying the gene sequence of the nucleotide sequence shown in SEQ ID NO: 2, connecting it with pET28a, and transforming it into Escherichia coli BL21 (DE3). Positive recombinant bacteria are obtained by screening, and the positive recombinant bacteria are subjected to lactose induction expression. After purification, the recombinant fowl adenovirus type 11 fiber protein is obtained, and its amino acid sequence is shown in SEQ ID NO: 1.

[0034] Example 1 Pathogenicity of Avian Adenovirus Type 11 in SPF Chickens by Challenge Route 1) Animal challenge plan The purchased SPF eggs were incubated in a dedicated incubator. The 60 one-day-old chicks that were successfully hatched were randomly divided into three challenge groups (groups A to C) and three negative control groups (groups D to F), with 10 chicks in each group. Groups A to C were challenged with 200 μL (10 7 TCID 50 The challenge experiment design is shown in Table 1.

[0035] Table 1 Virus challenge plan for one-day-old SPF chickens

[0036] 2) Analysis of the pathogenicity of the virus Chickens in each group were clinically observed for one week after challenge, focusing on signs of lethargy and undersized size. Any deaths were immediately necropsied. All birds were necropsied seven days after challenge to examine for signs of inclusion body hepatitis (swelling, hemorrhage, necrosis, yellowing, and brittleness). Analysis of challenge pathogenicity is shown in Table 2.

[0037] Table 2 Pathogenicity of avian adenovirus type 11 to SPF chickens by challenge route

[0038] As can be seen from Table 2, the route of attack is 1 / 2 eye drops + 1 / 2 nasal drops or 1 / 5 eye drops + 4 / 5 nasal drops, which has certain pathogenicity to 1-day-old SPF chicks, but the pathogenicity is moderate, the individual disease severity varies greatly, the symptoms are unclear, and the reproducibility of the results is low; while the same dose is used for intramuscular injection, the pathogenicity is higher, the individual disease severity varies little, the symptoms are clear, and the reproducibility of the results is high.

[0039] Therefore, the intramuscular injection is selected as the route for attacking the poison in the present invention.

[0040] Example 2 Pathogenicity of Avian Adenovirus Type 11 to SPF Chickens of Different Ages 1) Animal experiments The SPF eggs were incubated in a dedicated incubator, and the 80 chicks that successfully hatched were randomly divided into four challenge groups (groups A to D, 10 chicks each) and one negative control group (Control, 40 chicks). The challenge groups A to D were injected intramuscularly with FAdV-11 virus solution at 1, 7, 14, and 21 days of age, respectively, with a dose of 200 μL (10 7 TCID 50 Ten chickens in the negative control group were injected with phosphate buffered saline at 1, 7, 14, and 21 days of age. The challenge experiment design is shown in Table 3.

[0041] Table 3 Virus challenge scheme for SPF chickens at different ages

[0042] 2) Post-challenge mortality, clinical symptom monitoring, and hepatitis testing The survival of chickens was counted for 7 consecutive days after the infection, and the changes in animal spirits and body shape were monitored. If any dead chickens were found, an autopsy was performed immediately. All chickens were autopsied on the 7th day after the infection to check for the occurrence of inclusion body hepatitis.

[0043] 3) Absolute fluorescence quantitative PCR detection of viral load The chicken livers were taken after autopsy and total DNA was extracted.

[0044] A pair of fluorescent quantitative PCR primers was synthesized based on the FAdV-11 sequence: the forward primer sequence was FAdV-11 RT F1 (5'-GGTTACAGACAAGCATTCAGG-3' (SEQ ID NO:3)), and the reverse primer sequence was FAdV-11 RT R1 (5'-TCAGAGACAGACCGTTAGATGA-3' (SEQ ID NO:4)). Total DNA containing the FAdV-11 genome (extracted from chicken liver tissue infected with avian adenovirus type 11 (FAdV-11)) was used as a template, and a DNA fragment of approximately 150 bp was amplified using FAdV-11 RT F1 / FAdV-11 RTR1. After gel purification, the fragment was subcloned into the pTOPOT vector and transformed into DH5α cells. Correct clones were identified by PCR, and plasmids were extracted and their concentrations were determined using an ultramicrospectrophotometer. This plasmid served as a standard for absolute quantification. The copy number of the extracted plasmid was calculated according to the copy number calculation formula: copy number (copy / μL) = Avogadro constant (copy / mol) × plasmid concentration (ng / μL) × 10 -9 / [vector fragment length (bp) + fragment size (bp)] × 660, the Avogadro constant is 6.02 × 10 23 .

[0045] The standard plasmid was diluted in a 10-fold gradient to obtain 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 A series of standards with 100 copies / μL were prepared, with three replicates for each standard. Absolute fluorescence quantitative PCR was performed using the optimized reaction system (Table 4) and program. A standard curve was drawn with the corresponding Ct value as the ordinate and the logarithm of the gradient-prepared standard concentration as the abscissa.

[0046] Table 4 Absolute fluorescence quantitative PCR amplification reaction system

[0047] The reaction procedure was as follows: first step, pre-denaturation at 95°C for 1 min; second step, denaturation at 95°C for 10 s, annealing at 52°C for 20 s, extension at 72°C for 30 s, 40 cycles; third step, melting curve analysis, 95°C for 10 s, 70°C-95°C, 0.5°C / cycle.

[0048] Fluorescence quantitative detection was performed according to the above-mentioned absolute fluorescence quantitative method and standard curve. Three replicates were set for each sample, and the final results were expressed as the geometric mean ± standard deviation of Log10 DNA copies per mg of liver tissue.

[0049] 4) Analysis of pathogenicity of the attack virus Chickens in each group were observed clinically for one week after challenge, primarily for signs of lethargy and thinness. Any deaths were immediately necropsied. All birds were necropsied seven days after challenge to examine for signs of inclusion body hepatitis (swelling, hemorrhage, necrosis, yellowing, and brittleness). Viral load was measured using absolute fluorescence quantitative PCR. Analysis of challenge pathogenicity is shown in Table 5.

[0050] Table 5 Pathogenicity of avian adenovirus type 11 to SPF chickens of different ages

[0051] As can be seen in Table 5, avian adenovirus type 11 is pathogenic to young SPF chicks, and the pathogenicity is age-related, with the younger the chick, the stronger the pathogenicity. When avian adenovirus type 11 was inoculated into one-day-old chicks, 100% of the chicks died, and they became severely ill, with obvious and severe symptoms of inclusion body hepatitis and high viral loads. However, when inoculated into seven-day-old chicks, only a small number of chicks became ill and developed inclusion body hepatitis, and the viral load was significantly lower than that of the one-day-old challenge, indicating lower pathogenicity. Furthermore, when inoculated into 14- and 21-day-old chicks, no disease occurred, and the viral load was also low, indicating very low pathogenicity.

[0052] Therefore, in the present invention, one-day-old SPF chicks were used for challenge.

[0053] Example 3 Absolute lethal dose of avian adenovirus type 11 for one-day-old SPF chickens The viral titer of FAdV-11 was determined using LMH cells. The viral solution was diluted to 1×10 8 TCID 50 / 0.1ml、3×10 7 TCID 50 / 0.1ml, 1×10 7 TCID 50 / 0.1ml、3×10 6 TCID 50 / 0.1ml, 1×10 6 TCID 50 / 0.1ml、3×10 5 TCID 50 / 0.1ml, 1×10 5 TCID 50 / 0.1ml, a total of 7 gradients.

[0054] For each gradient, 0.1 ml of the virus solution was inoculated intramuscularly into one-day-old SPF chickens, with 10 chickens per gradient. A control group was established, which was injected with 0.1 ml of PBS. The survival of the chickens was monitored daily. By day 7 after challenge, the mortality rate of the experimental animals in each gradient was: 10 / 10 (1×10 8 TCID 50 / chicken); 10 / 10 (3×10 7 TCID 50 / chicken); 9 / 10 (1×10 7 TCID 50 / chicken); 5 / 10 (3×10 6 TCID 50 / chicken); 3 / 10 (1×10 6 TCID 50 / chicken); 1 / 10 (3×10 5TCID 50 / chicken); 0 / 10 (1×10 5 TCID 50 / chicken); 0 / 10 (control group). Based on this result, it can be seen that the 7 TCID 50 The challenge dose of the drug to chickens can cause the death of all one-day-old SPF chickens within a 7-day challenge period.

[0055] Therefore, the absolute lethal dose of avian adenovirus type 11 for one-day-old SPF chickens is 3×10 7 TCID 50 / chicken; Subsequent challenge regimens used 1-day-old SPF chickens, with a challenge dose of 3×10 7 TCID 50 / chickens, the monitoring period after infection was 7 days.

[0056] Example 4 Challenge mortality and clinical symptoms as indicators for evaluating the efficacy of avian adenovirus type 11 vaccines 140-day-old SPF breeder chickens are vaccinated with avian adenovirus type 11 vaccine, followed by a booster vaccination two weeks after the initial vaccination. Following vaccination, the chicks are caged together at a male-to-female ratio of 4:1. Fertilized eggs from cages 5-10 weeks after caged together are harvested and incubated. Eggs from unvaccinated SPF breeder chickens are also incubated simultaneously. Hatched chicks are transferred to isolators for rearing.

[0057] One-day-old chicks (immune offspring chicks) were injected intramuscularly with FAdV-11 for challenge with a dose of 3×10 7 TCID 50 / chicken; at the same time, a challenge control (non-immune offspring chicks challenged with poison) and a negative control (non-immune offspring chicks not challenged with poison) were set up.

[0058] After the challenge, the chicks were monitored daily for survival and morbidity for a total of 7 days. The specific vaccine evaluation is as follows: If the immunized offspring chicks have no clinical symptoms (the chickens are in normal spirit and body shape) and no deaths occur, the immunization effect of the avian adenovirus type 11 vaccine is qualified; if the immunized offspring chicks die, the immunization effect of the avian adenovirus type 11 vaccine is unqualified.

[0059] Among them, clinical symptoms refer to the symptoms of lethargy and thin body in chicks; death refers to specific death, which usually occurs 4-7 days after infection. Non-specific death that occurs within 48 hours of infection is not included.

[0060] Specifically, if there is no death among the immunized offspring chicks within 7 days of infection, and their body shape and mental state are consistent with those of the negative control (non-immunized offspring chicks not infected), then the immune effect of the avian adenovirus type 11 vaccine is qualified and has complete protection against infection.

[0061] If there is no death in the immunized offspring chicks within 7 days of infection, and compared with the negative control (non-immunized offspring chicks not infected), the clinical characteristics of lethargy and smaller body size are shown, then the immune effect of the avian adenovirus type 11 vaccine is qualified, but the antibody level has not yet reached the level that prevents the immunized offspring chicks from developing clinical symptoms.

[0062] In this example, the results of the immune evaluation of avian adenovirus type 11 vaccine are shown in Table 6.

[0063] Table 6 Results of immune evaluation of avian adenovirus type 11 vaccine

[0064] As can be seen from Table 6, all chickens in the challenge control group (non-immune offspring chickens challenged with virus) died; the chickens in the negative control group (non-immune offspring chickens not challenged with virus) did not die and their body shape and mental state were normal; no chickens in the vaccine group (immune offspring chickens) died and their body shape and mental state were normal; the results showed that the avian adenovirus type 11 vaccine was effective.

[0065] Example 5 Inclusion body hepatitis after challenge as an indicator for evaluating the efficacy of avian adenovirus type 11 vaccine 140-day-old SPF breeder chickens are vaccinated with avian adenovirus type 11 vaccine, followed by a booster vaccination two weeks after the initial vaccination. Following vaccination, the chicks are caged together at a male-to-female ratio of 4:1. Fertilized eggs from cages 5-10 weeks after caged together are harvested and incubated. Eggs from unvaccinated SPF breeder chickens are also incubated simultaneously. Hatched chicks are transferred to isolators for rearing.

[0066] One-day-old chicks (immune offspring chicks) were injected intramuscularly with FAdV-11 for challenge with a dose of 3×10 7 TCID 50 / chicken; at the same time, a challenge control (non-immune offspring chicks challenged with poison) and a negative control (non-immune offspring chicks not challenged with poison) were set up.

[0067] After the challenge, chick mortality was continuously monitored daily. If any chick died, an autopsy was performed immediately. All chicks were autopsied on day 7 after the challenge. The livers were observed for typical symptoms of inclusion body hepatitis, including swelling, bleeding, necrosis, yellowing, and brittleness. Specific vaccine evaluations were as follows: If no chickens die and no inclusion body hepatitis occurs in the immunized offspring chicks after being challenged with the virus, the immune effect of the avian adenovirus type 11 vaccine is qualified and it has complete protection against the virus.

[0068] If no chickens die after being infected with the virus but obvious symptoms of inclusion body hepatitis appear in the vaccinated offspring, the immune effect of the avian adenovirus type 11 vaccine is unsatisfactory.

[0069] If the vaccinated offspring chicks die after being infected with the virus, the immune effect of the avian adenovirus type 11 vaccine is unsatisfactory.

[0070] In this example, the results of the immune evaluation of avian adenovirus type 11 vaccine are shown in Table 7.

[0071] Table 7 Results of immune evaluation of avian adenovirus type 11 vaccine

[0072] As can be seen from Table 7, all the chickens in the challenge control group (non-immune offspring chickens challenged with the virus) died, and after autopsy, it was found that the chicken livers showed typical symptoms of inclusion body hepatitis, such as swelling, bleeding, necrosis, yellowing, and brittleness; no chickens in the negative control group (non-immune offspring chickens not challenged with the virus) died, and no chickens in the vaccine group (immune offspring chickens) died, and no chickens showed symptoms of inclusion body hepatitis; the results showed that the avian adenovirus type 11 vaccine was effective.

[0073] Example 6 Post-challenge viral load as an indicator for evaluating the efficacy of avian adenovirus type 11 vaccine 140-day-old SPF breeder chickens are vaccinated with avian adenovirus type 11 vaccine, followed by a booster vaccination two weeks after the initial vaccination. Following vaccination, the chicks are caged together at a male-to-female ratio of 4:1. Fertilized eggs from cages 5-10 weeks after caged together are harvested and incubated. Eggs from unvaccinated SPF breeder chickens are also incubated simultaneously. Hatched chicks are transferred to isolators for rearing.

[0074] One-day-old chicks (immune offspring chicks) were injected intramuscularly with FAdV-11 for challenge with a dose of 3×10 7 TCID 50 / chicken; at the same time, a challenge control (non-immune offspring chicks challenged with poison) and a negative control (non-immune offspring chicks not challenged with poison) were set up.

[0075] After the challenge, the mortality of the chicks was continuously monitored daily. If a chicken died, the chicken liver was taken to extract total DNA. On the 7th day after the challenge, all chickens were euthanized and the chicken liver was used to extract total DNA. The viral load of FAdV-11 in the chicken liver was determined and calculated using the method described in Example 2, and the results are expressed as the Log10 DNA copy number per mg of chicken liver. The geometric mean (G) of the DNA copy number per mg of liver tissue in the negative control group was calculated. 阴性对照 ) and standard deviation (SD). Specific vaccine evaluations are as follows: If the viral load in the liver of the immunized offspring chicks after challenge is less than or equal to the sum of the geometric mean and three times the standard deviation of the DNA copy number in the negative control group (≤G阴性对照 +3×SD), the avian adenovirus type 11 vaccine has a qualified immune effect and has complete protection against virus attack, and can completely protect against FAdV-11 infection.

[0076] If the viral load in the liver of the immunized chicks after challenge is greater than the geometric mean plus three times the standard deviation of the DNA copy number in the negative control group (>G 阴性对照 +3×SD), and not higher than the sum of the geometric mean of DNA copy number in the negative control group and 2 (≤G 阴性对照 +2), the avian adenovirus type 11 vaccine has a qualified immune effect and can effectively reduce the viral load after the virus is challenged.

[0077] If the viral load in the liver of the immunized chicks after challenge is greater than the sum of the geometric mean of the DNA copy number in the negative control group and 2 (>G 阴性对照 +2), the immune effect of the avian adenovirus type 11 vaccine is unsatisfactory.

[0078] In this example, the geometric mean of the viral load of chicken liver in the negative control (non-immune offspring chicks not challenged with the virus) was 2.04 Log10 DNA copies / mg, with a standard deviation of 0.26; the viral load of chicken liver in the challenge control (non-immune offspring chicks challenged with the virus) was 8.75 Log10 DNA copies / mg, with a standard deviation of 0.55; the viral load of each animal in the vaccine group (immune offspring chicks) was less than 2.82 Log10 DNA copies / mg; the results showed that the immune effect of the avian adenovirus type 11 vaccine was satisfactory.

[0079] In summary, the present invention obtains immune antibodies by immunizing breeder chickens with avian adenovirus type 11 vaccine, and then transfers the immune antibodies to fertilized eggs and their hatched offspring chicks through passive immunization, so that the offspring chicks passively obtain maternal antibodies, and the offspring chicks are challenged with the virus. At the same time, the mortality and / or clinical symptoms of the immune offspring chicks after the challenge are used as indicators, or the mortality and / or inclusion body hepatitis symptoms after autopsy of the immune offspring chicks after the challenge are used as indicators, or the viral load in the liver tissue of the immune offspring chicks after the challenge is used as an indicator, and the viral load in the liver tissue of the negative control non-immune offspring chicks after the challenge is used as a reference, so that the avian adenovirus type 11 vaccine can be evaluated efficiently, quickly and accurately.

[0080] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for evaluating the immune effect of avian adenovirus type 11 vaccine, characterized in that: The steps include: S1. Immunize female and male breeder chickens with avian adenovirus type 11 vaccine. After immunization, combine the chickens and obtain the first fertilized egg. S2. Hatching the first fertilized egg to obtain immune offspring chicks, and challenging the immune offspring chicks with avian adenovirus type 11. The immune efficacy of the avian adenovirus type 11 vaccine is evaluated using the mortality and / or clinical symptoms of the immune offspring chicks after the challenge as indicators, or using the mortality and / or inclusion body hepatitis symptoms after autopsy of the immune offspring chicks after the challenge as indicators, or using the viral load in the liver tissue of the immune offspring chicks after the challenge as an indicator. The immune effect of the avian adenovirus type 11 vaccine is evaluated using the death and / or clinical symptoms of the immunized offspring chicks after the challenge as indicators, specifically including: if the immunized offspring chicks die after the challenge, the avian adenovirus type 11 vaccine is judged to be unqualified; if the immunized offspring chicks do not die after the challenge, or do not die but develop the clinical symptoms, the avian adenovirus type 11 vaccine is judged to be qualified; The clinical symptoms include lack of energy and small body size.

2. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 1, characterized in that: In step S1, the ratio of the female breeder chickens to the male breeder chickens is (3-5):

1.

3. A method for evaluating the immune effect of the avian adenovirus type 11 vaccine according to claim 1, characterized in that: In step S1, the first fertilized eggs include first fertilized eggs that have been caged for 5-10 weeks.

4. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 1, characterized in that: In step S2, the immunized offspring chicks include 1-day-old immunized offspring chicks.

5. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 1, characterized in that: In step S2, the route of attacking the virus includes intramuscular injection.

6. The method for evaluating the immune effect of an avian adenovirus type 11 vaccine according to claim 1, wherein: In step S2, the dose of the challenge includes an absolute lethal dose, which is at least 3×10 7 TCID 50 / chicken.

7. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 1, characterized in that: In step S2, the immune effect of the avian adenovirus type 11 vaccine is evaluated based on the mortality of the immune offspring chicks after challenge and / or the symptoms of inclusion body hepatitis after autopsy, specifically including: If there is death among the immunized offspring chicks after the challenge, the avian adenovirus type 11 vaccine is judged to be unqualified; if there is no death among the immunized offspring chicks after the challenge, and symptoms of inclusion body hepatitis appear after autopsy, the avian adenovirus type 11 vaccine is judged to be unqualified; if there is no death among the immunized offspring chicks after the challenge, and no symptoms of inclusion body hepatitis appear after autopsy, the avian adenovirus type 11 vaccine is judged to be qualified.

8. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 7, characterized in that: The symptoms of inclusion body hepatitis include at least one of liver enlargement, liver hemorrhage, liver necrosis, liver jaundice, and liver brittleness.

9. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 1, characterized in that: In step S2, the immune effect of the avian adenovirus type 11 vaccine is evaluated using the viral load in the liver tissue of the immune offspring chickens after the challenge as an indicator and the viral load in the liver tissue of the negative control non-immune offspring chickens after the challenge as a reference, specifically including: If the viral load in the liver tissue of the immune offspring chickens after the challenge is less than or equal to the sum of the geometric mean and 3 times the standard deviation of the viral load in the liver tissue of the negative control non-immune offspring chickens, the avian adenovirus type 11 vaccine is judged to be qualified; if the viral load in the liver tissue of the immune offspring chickens after the challenge is greater than the sum of the geometric mean and 2 of the viral load in the liver tissue of the negative control non-immune offspring chickens, the avian adenovirus type 11 vaccine is judged to be unqualified; The negative control non-immune offspring chicks after the virus challenge are obtained by directly combining female breeder chickens and male breeder chickens to obtain a second fertilized egg; the second fertilized egg is then hatched to obtain a negative control non-immune offspring chick, and finally the negative control non-immune offspring chicks are challenged with avian adenovirus type 11, the route of the challenge includes intramuscular injection, and the dose of the challenge includes an absolute lethal dose, which is at least 3×10 7 TCID 50 / chicken.

10. The method for evaluating the immune effect of avian adenovirus type 11 vaccine according to claim 9, characterized in that: The ratio of female breeder chickens to male breeder chickens is (3-5):1; the second fertilized eggs include second fertilized eggs that are caged for 5-10 weeks; and the negative control non-immune offspring chicks include 1-day-old negative control non-immune offspring chicks.

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