In hydrallantois, accumulation of
allantoic fluid was rapid due to placental abnormalities and possible interference with sodium metabolism at cellular level (Jackson, 1980).
This observation is interesting, because some authors recommend testing the
allantoic fluid for hemagglutination activity only from dead embryos.
Hemagglutination (HA) test is used to harvest
allantoic fluid from embryonating chicken eggs for hemagglutinating agents, such as type A influenza (Killian, 2008).
All of these samples were inoculated into the allantoic cavity of 9 to 11 day old chicken embryos and the
allantoic fluids were harvested after 48 hrs.
After 18 hrs of dilation therapy, cervix was properly dilated and allantoic cavity (water bag) ruptured and about 45-50 liters of
allantoic fluid escaped out from birth canal.
The total RNA was extracted from the
allantoic fluid using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's protocol.
The
allantoic fluid tested negative through hemagglutination test and PCR for common duck viruses, including avian influenza virus, Newcastle disease virus and duck herpesvirus 1.
The chicken embryo died within 2-5 days post inoculation and showing IBV specific lesions were harvested
allantoic fluid and 50% chicken infectious dose (EID50) was calculated 103 EID50 by inoculation of serial 10 fold dilution of virus in 10-day-old SPF embryonated chicken eggs.
Rupture of fetal membranes resulted in 90-100 liters of amber colored
allantoic fluid gushing from uterus which confirmed our diagnosis as hydroallantois.
The virus identification from the positive
allantoic fluid was done through hemagglutination inhibition test and the confirmation was done through RT-PCR (Siddique et al., 2008).
Fresh infective
allantoic fluid obtained after passaging the AIV H9N2 in embryonated eggs with a HA titer greater than 1/16 was diluted 1/10 in sterile isotonic saline.
Allantoic fluid of the 6 cultured specimens was then shipped to the University of Florida for confirmation testing and sequencing.