Avian influenza is the most contagious disease not only in poultry, but also in humans. Avian influenza in humans occurs mainly in Southeast Asia, but no human-to-human pandemic has occurred. Meanwhile, outbreaks of avian influenza in poultry occur on a global scale and cause a large economic loss. Migration antibodies passed from mother birds via eggs are said to be an important component of the immune system that protects birds from infection. Thus, the immunity status of mother birds can determine the ability of offspring to defend against infection. In this study, we investigated the presence of anti-avian influenza virus antibody in chickens hatched on a poultry farm in Indonesia and examined the involvement of migratory antibodies in protecting against virus infection by infectious experiments of highly pathogenic avian influenza in chickens. Blood was collected from randomly selected chicks, and antibodies against avian influenza virus were evaluated in all birds. Since these young birds had no history of vaccination, the antibodies were deemed to have been transferred from the mother birds. The enzyme-linked immunosorbent assay antibody titer in each bird varied. Infection of these birds with highly pathogenic avian influenza virus A/H5N1 intra-nasally resulted in a high mortality rate in chicks with low antibody titers but a low mortality rate in chicks with high antibody titers. These findings indicate that migratory antibody prevented highly pathogenic avian influenza A/H5N1 infection in chicks, suggesting that such a preventive effect could also be expected with outdoor natural infection.
Mammals are equipped with an immune system in which antibodies from mothers transfer to children through the placenta and breast milk. These antibodies are called migratory antibodies and are responsible for protecting young children with an incomplete immune function against infection. In egg-laying animals, such as birds, migratory antibodies can be transferred from mothers to their eggs, thereby protecting their offspring. Even in domestic chickens, migratory antibodies from mothers are useful for defense against various infectious diseases, and the mother’s immune status determines the susceptibility of chicks to infection [
Highly pathogenic avian influenza is the most feared infectious disease in poultry, and in recent years, a number of infectious and death cases have been reported in humans as well, mainly in Southeast Asia [
In the present study, the presence of anti-avian influenza virus antibodies in the blood of infant chicks hatched at a poultry farm in Indonesia was assessed, and the protective effect of transferred antibody on highly pathogenic avian influenza A/H5N1 infection was verified by infection experiments.
Thirty newly-hatched chicks were collected from poultry farms selected at random in Indonesia. We confirmed that these birds had no history of vaccination with avian influenza vaccine. In addition, specific-pathogen-free (SPF) chicks were purchased and used for experiments as controls. They were reared under conditions of water and feed provided ad libitum in BSL-3 facility, and blood was sampled at 10 days of age. The serum titer of antibodies against A/H5N1 antigens was measured by an enzyme-linked immunosorbent assay (ELISA).
The antibody titer was measured by an ELISA according to our previous report [
At 10 days of age, seventy-two chicks from farm and ten SPF chicks were inoculated intranasally with a field-isolated strain of A/H5N1 virus (A/LAWANG/FKH-IPB/H5N1) at a dose of 105TCID50 per bird. This virus strain was isolated from a chicken spontaneously infected with H5N1 in a local poultry farm in the Bogor area of Indonesia. Five days after the viral inoculation, the lethality of infected birds was scored as the percentage of dead birds. The surviving chickens were sacrificed by administration of overdose pentobarbital sodium solution. The tracheae and lungs of subjects were removed and immersed in 10% neutral buffered formalin for further histopathology and immunostaining studies. All of the animal experiments were performed in accordance with the guidelines for studies with laboratory animals of the Kyoto Prefectural University Experimental Animal Committee. After completion of the experiment, all samples were completely sterilized by autoclave.
The formalin fixed samples were dehydrated with serial concentrations of ethanol and embedded with xylene and paraffin using an automatic embedding processor. The sections were then cut using a microtome and attached to slide-glasses. The sections were dried and de-paraffinized inxylene and ethanol and then stained with hematoxylin and eosin. They were next serial-dehydrated with ethanol and immersed in xylene before finally being mounted with cover-glass using Canada balsam and examined under a light microscope.
The chick organs fixed in buffered formalin were washed in PBS, cut into 5-mm cubes, and soaked in 30% sucrose in PBS overnight. The pieces were mounted in a compound, frozen, and cut into 20-μm sections with a cryostat. The frozen sections were attached to glass slides and air-dried at room temperature. After being washed in PBS, the samples were incubated with an FITC conjugated ostrich IgY against A/H5N1 viruses (1:1000) at 4˚C overnight [
The immunoreactivity of subject sera against A/H5N1 virus was estimated by ELISA. The titers for A/H5N1 varied among the chicks, as shown in
The experimental challenge in SPF chickens (10 days old) with A/H5N1 caused high lethality among the birds (100%) within 5 days after infection, indicating that this viral strain isolated from poultry farm was a highly virulent type (
difference was observed in the fatality rate of the infected chicks purchased from the poultry farm. The mortality rate due to virus infection was quite high in chicks with low antibody titers in blood, whereas the birds with high serum antibody titers showed low mortality. Furthermore, 100% of birds with an ELISA value of 102,400 survived despite virus infection. We therefore concluded that viral infection could be suppressed in individuals with high transitional antibody titers.
The histopathological study of the infected SPF chickens showed the typical avian flu findings of acute inflammation accompanied by heterophilic infiltration, hemorrhaging, edema and severe congestion in various organs, mainly in the pulmonary tissues, including the trachea and lung (
Viral antigens were found in the tracheal epithelial cells and pulmonary tissues of dead chickens or infected chickens with low ELISA values (≤25,600). In contrast, viral antigens were scarcely seen in the pulmonary tissues of in surviving chicks showing ELISA values exceeding 51,200 (
The present study confirmed that antibodies against avian influenza virus A/H5N1 existed in chicks from a poultry farm in Indonesia. Since these birds had no history of vaccination, we concluded that they had migratory antibodies obtained from their mother. However, since the mother’s vaccination history was unknown, we could not confirm whether the migratory antibody was the influence of vaccination of the mother bird or had been acquired by natural infection. Given that the antibody titer against the A/H5N1 virus varied greatly among the birds, we predicted that susceptibility to avian influenza infection would vary among animals at chicken farms in Indonesia.
The A/H5N1 virus used for infection experiments on conventional chicks was a field isolate from Indonesia and proved to be highly pathogenic to chickens, as the infected SPF chicken shows a 100% mortality rate. Regarding the conventional chicks obtained from farms, a high mortality rate was also observed in individuals with low antibody ELISA values. In contrast, the mortality rate drastically
decreased in chicks with an antibody titer of ≥51,200, and none with values exceeding 102,400 died. These findings indicate that the antibody transferred from the mother had neutralizing activities against A/H5N1 virus infections and that the birds with an antibody titer above a certain level were resistant to death from avian influenza. At present, there are benefits and drawbacks for employing influenza vaccines in domestic fowls: 1) in avian influenza-free countries, it is difficult to diagnose natural infections; 2) the virus resides in the poultry body; and 3) the virus may mutate and become able to infect pigs or humans, causing a pandemic of a novel influenzas train [
Our findings here suggest that the transfer of antibodies from mother birds to their chicks may be a way to prevent avian influenza infection in the offspring. Maternal antibodies migration to chicks is considered effective for preventing virus infection, but the development of a good-quality vaccine and improvements in existing vaccination programs are required.
We thank Miss Akari Iki and Mrs. Hatsuki Matsunaga at Kyoto Prefecture University for their valuable technical support.
The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.
Adachi, K., Suryaman, G.K., Soejoedono, R.D., Handharyani, E. and Tsukamoto, Y. (2018) Suppression of Highly Pathogenic Avian Influenza A/H5N1 Infection Using Migratory Antibody Passed from Mother to Chick. World Journal of Vaccines, 8, 89-97. https://doi.org/10.4236/wjv.2018.84008