Vertical Transmission And Pathogenicity Of H9N2 Avian Influenza Virus In Breeder Goose

Avian influenza virus(AIV), a kind of single-strand negative virus, belongs to Orthomyxoviridae family. The virus was firstly discovered in Italy, then many countries and areas reported the virus successively. Since the first isolation from chicken was reported in Guangdong province in China in 1994, H9N2 has became prevalent. Although H9N2 AIV are indentified as low pathogenic avian influenza viruses(LPAIVS), they can cause respiratory symptoms and egg drop symptoms in poultry, leading to huge economic losses to poultry industry in China. H9N2 avian influenza virus mainly through feed, water, air, utensils, etc. horizontal transmission by direct or indirect contact. But in actual production, breeding poultry which is infected by H9N2 avian influenza virus show egg-dropping, hatching rate of eggs decreased, dead embryo increased, and the young bird of early mortality is high. We hypothesized that H9N2 avian influenza virus can pass through the poultry, eggs, embryos to infect the young bird by vertical infection way which is the cause of this phenomenon. In the incubation process, the virus, massive proliferation in eggs, caused the embryo infecting virus or death, poultry due to virus show early high mortality. This study is about whether the H9N2 AIV can be transmitted vertically in the geese and the pathogenicity of H9N2 AIV to the breeder geese, which has a great significance to virology, veterinary research and public health. 1. Detection of H9N2 AIV from eggs, embryos and chicksBetween 2014 and 2015, eggs, poultry embryos and chicks were collected from part areas of our country. We collected vitelline membrane and lung or brain samples to test the H9N2 AIV, the detection rates of H9N2 AIV in eggs, dead embryos and chicks were 33.3%(22/66)?27%(28/103)and 43%(103/241)respectively. 2. Establishment of artificial infection model of H9N2 AIV360-day-old breeder geese which were negative with H9N2 AIV were divided into 4 groups(A/B/C/D). Every group was comprised of 18 female geese and 3 male geese, Group A/B and C were inoculated 2.8 m L H9N2 avian influenza virus(ELD50=10-7.5/0.2 m L) by intravenous(i.v.)?intranasal instillation and throat inoculation way respectively. All groups were bred separately. During the period of the experiment, eggs of every group were collected and incubated, meanwhile we recorded the basic situation of hatching and fertilization timely, and collected vitelline membrane and lung or brain samples. All samples collected were treated normally and inoculated into SPF chicken embryos for virus isolation and detection by nested RT-PCR later. As a result, cutaneous hemorrhage, malabsorption and thin in yolk, and hemorrhage in liver and heart was found. Moreover, edema and hemorrhage in neck, hyperemia in brain were found in several dead embryos. Virus detection results show that, H9N2 avian influenza virus detection rates of vitelline membrane and embryos for three groups were 80%(4/5) and 43%(3/7)?25%(1/4) and 44%(4/9)?33%(1/3) and 42%(3/7), respectively. Eggs of group D were negative with the detection of H9N2 avian influenza virus. HA gene of the newly isolated virus was amplified and sequenced, and the homology of nucleic acid with the original strain was 99.0%~100%. In a word, H9N2 avian influenza virus was detected in eggs, embryos and chicks of infected breeder goose, indicating that H9N2 avian influenza virus could pass on from infected goose to offspring via eggs. In other words, H9N2 avian influenza virus can transmit vertically in goose. 3. Pathogenicity of H9N2 avian influenza virus in the Breeder GooseAfter infection, three geese were selected randomly every three days to collect blood for the serum, which was used for detecting Goose Serum antibody level by HA-HI test, and detection IFN-? and IL-4 level by using cytokine detection reagent kit. The RT-PCR method was used for detecting the H9N2 AIV on cloacal swab. Every three days two geese were killed to collect liver, brain, spleen, lung, trachea, vitelline membrane, fallopian tube samples and e.g., which were made into pathological sections. The experiment results showed that, sand shell eggs were increased, following with loose stools and mild respiratory symptoms after virus infection. Antibody level tests showed that, the antibody level of group A was rised fastest and maintained for a long time, while group C was increased slowly. The cell factor levels revealed that:levels of IFN-? and IL-4 fall from 1dpi until 13 dpi, suggesting that the virus affects the immune system. Levels of antibody and cell factor were stable in control group. The H9N2 AIV of the three groups was detected positive in swab from 4 dpi?6 dpi?8 dpi respectively till the end of experiment. The results of the anatomy show that: after infection, the changes of goose were hemorrhage in lungs, ovary, fallopian tube and testis atrophic, spleen edema; Congestion and hemorrhage in liver; necrosis in pancreas and so on. According to the above experimental results, H9N2 AIV infection mainly damage lung and ovarian e.g., and with the respiratory and egg production symptom. The goose can discharge virus via cloaca, promoting the spread of the virus.