| Duck Hepatitis A Virus type 3(DHAV-3) is one of the most serious pathogens of ducklings since high morbidity and mortality of ducklings infected with this disease were recorded. Establishing the immunohistochemistry(IHC) detection of this diease so that provide a new laboratory diagnosis of duck hepatitis a virus type 3. Antigen localization and distribution in infect ducklings can be learned with the help of IHC detection, so as to study the pathogenic mechanism of DHAV-3 and provide a basis for retrospective diagnosis of this disease.1. Establishing the immunohistochemistry detection of duck hepatitis a virus type 3Propagating the virus in the allantoic sac of 10-day-old duck embryos.Collecting allantoic fluid of the embryos with obvious pathological changes that had died between 36h and 72h after innoculation. Purifying DHAV-3 antigen with three steps:Centrifugating allantoic fluid with different rotary speeds, Mixing allantoic fluid with chloroform, Ultracentrifugating allantoic fluid. Immunizing rabbits with purified DHAV-3 to gain the rabbit anti DHAV-3 serum, so as to exact IgG of the serum.Using the rabbit anti DHAV-3 IgG to establish and optimize the immunohistochemistry detection of duck hepatitis a virus type 3.These diagnosis can specificly detect DHAV-3 in the tissues and organs of infected ducklings. Negative results can be shown when the detection are applied to ducks dead of duck viral enteritis, riemerella anatipestifer, escherichia coli, salmonella, pasteurella multocida, which can prove the specific of this detection.2. Construction and detection of the DHAV-3 duckling pathological model by different artificial infection routes.Numbering the 300 1-day-old ducklings that had no maternal antibody.Dividing them into 4 groups with a random number table. Artificial infections were designed as follows: oral, nasal drops, intramuscular injection, each of the three groups contained 90 ducklings. The rest 30 ducklings belonged to the control group. The ducklings were infected with 0.4ml of duck liver homogenate which came from the dead ducklings of DHAV-3 infection at 3-day-old. The control group were injected the same dosage of sodium chloride physiological solution. At intervals of 1,2,6,12,24,36,48 72,96,120,144,192,240, 2352(two weeks) hours post infection, six ducklings of the experimentally infected groups while two of the control group were taken randomly from the four groups and killed to necropsiing.Collecting samples from 16 tissues and organs namely heart, liver, spleen, lung, kidney, cerebrum, pancreas, duodenum, jejunum, ileum, cecum, rectum, bursa of fabricius, harderian gland, thymus, muscle as well as the deadklings of control group.Using 4% para formaldehyde to complete the fixation. Number of the dead ducklings and their death time were recorded.Death of duckling first appeared in the intramuscular injection group between 24～26hpi. The oral and nasal drops group appeared similarily on the discovery of dead ducklings. Both two groups between 36-38hpi. All of the three groups reach the death peak at about 48hpi and then presented a decreasing trend.Besides, the death number of the intramuscular injection group showed a trend of normal distribution. Ending time of the oral group, the nasal drops group and the intramuscular injection group were 144hpi,120hpi and 192hpi respectively. Results indicated that the nasal drops group showed the highest motality whlie the intramuscular injection group showed the lowest, between them was the oral group and no ducklings had died in control group.Wide distributed of the virus antigen can be detected from 16 tissues and organs of ducklings which died from DHAV-3 infection. Localization and distribution of the virus which infected by different artificial routes indicated that the target organs of DHAV-3 infection were liver, spleen, kidney and intestines. Antigen staining of these argans gradually enhanced as time went by and reached to the peak at about 72hpi..Immune organs of bursa of Fabricius, Harderian gland and thymus showed an alternately antigen staining during the experiment. The rest of the tissues and organs showed weak or moderate antigen staining.3. Localization and distribution of the virus in DHAV-3 infected ducklings which came from different hours post infectionThe results of 1,2,4,6,12,24,36,48,72,96,120,144 and 192 hours post infection were as follows:(1)The oral group:Duodenum, jejunum, ileum, rectum, bursa of Fabricius, Harderian gland and thymus showed positive staining after 1h inoculation while heart, liver, spleen, kidney and cecum showed positive siaining after 4h inoculation. Pancreas and lung showed positive staining after 6h inoculation while musle and cerebrum were 24h and 36h respetively.(2)The intramuscular injection group:Duodenum, jejunum, ileum, cecum, rectum, bursa of Fabric ius, Harderian gland, thymus and muscle showed positive staining after 1h inoculation while liver and kidney showed positive staining after 2h inoculation. Heart, spleen and lung showed positive staining after 4h inoculation while pancreas and cerebrum were 12h and 36h respetively.(3)The nasal drops group:All the tissues and organs showed to be positive after 1h inoculation except heart, bursa of Fabricius and muscle. Heart and bursa of Fabricius showed to be positive after 2h inoculation while muscle was 4h.To conclude, immunohistochemistry detection of DHAV-3 which were established in my research can provide a basis for retrospective diagnosis of this disease. It can also localize the antigen in the subcellular level as well as diagnose DHAV-3.Artificial infection routes like oral, nasal drops, intramuscular injection can build the experimental model of DHAV-3 and lead to the death of ducklings.Death peak of ducklings infected with DHAV-3 were between 36hpi and 72hpi among the whole three group and the nasal drops group had the highest mortality.Distribution of DHAV-3 in infected ducklings provide useful data for the pathogenic mechanism research of the disease. |
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