ObjectiveTo prepare specific anti- Fusarium yolk immunoglobulin (IgY) and investigate its tolerance to temperature and pH and verify its antifungal effect against Fusarium.

MethodsEighteen 22-week-old Leghorn laying hens were selected and randomized into negative control group and experimental group according to the random number table method, with 9 hens in each group.The 2×10 ⁷ colony forming units (CFU)/ml suspension of inactivated hyphae of Fusarium and Freund complete adjuvant was mixed in a 1∶1 ratio and emulsified.The hens in the experimental group were injected with 1 ml of the mixture to immunize and received 1 ml of Freund incomplete adjuvant as booster injection at two weeks after the initial injection.The egg yolk was collected from the 5th to 16th week after immunization.Specific anti- Fusarium IgY protein was prepared by salting out method using ammonium sulfate.The obtained protein solution was put into a freeze dryer and made into freeze-dried powder stored at 4 ℃.The hens in the negative control group were injected with 0.9% sodium chloride to prepare the non-specific antibody as the negative control.Bradford method was used to determine the concentration of specific egg IgY protein and the indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure its titer.The 1×10 ⁵ CFU/ml and 1×10 ³ CFU/ml Fusarium suspension were cultured with different concentrations of IgY and phosphate buffered saline (PBS) for 4 days, and the absorbance value at 600 nm was measured.The co-incubated PBS/negative IgY with Fusarium solution was set as blank control/negative control accordingly.The concentration-killing curve of anti- Fusarium IgY against Fusarium was obtained.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 7.4, and the diluted specific IgY solution was placed into the water bath for 30 minutes at 30, 40, 50, 60, 70, 80, 90 ℃, respectively, and was cooled down to room temperature.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, pH 12, respectively, and the diluted specific IgY solution was placed at 4 ℃ for one hour.The activity of diluted specific IgY solution by different methods was measured by indirect ELISA, and the tolerance of IgY to various temperatures and pH was evaluated.Twelve 8-week-old SPF female C57BL/6 mice were selected and randomized into the PBS control group and specific IgY treatment group according to the random table method, with 6 mice in each group.The right eyes of the 12 mice were infected with Fusarium to establish mice model of fungal keratitis.One day after modeling, 200 mg/ml of anti- Fusarium IgY was dropped to the right eyes of mice in the specific IgY treatment group, and PBS was dropped to the right eyes of mice in the PBS control group.The corneas of mice in the two groups were observed under the slit lamp microscope at 1, 3 and 5 days following modeling, and the corneal ulcer was scored according to the grading scale for inflammation score.The use and care of experimental animals followed the Association for Research in Vision and Ophthalmology statement.This study protocol was approved by an Ethics Committee of The Affiliated Hospital of Qingdao University (No.QYFYWZLL26168).

ResultsThe IgY protein concentration from the 5th to 16th week after immunization was 1.57, 2.89, 24.98, 25.09, 23.89, 25.78, 21.57, 21.37, 18.98, 15.78, 14.67, 12.67 mg/ml, respectively.The titer of IgY was increased from the 5th week, and it reached the highest titer 1∶10 000 at the 7th week, which could be maintained until the 12th week after immunization before it dropped gradually.The concentration-killing curve showed that compared with the blank control group and negative control group, Fusarium grew slowly in the specific IgY treatment group.The specific IgY with a titer greater than 1∶10 000 had thermal stability below 60 ℃.The activity of specific IgY was highest at pH 4 to 6, which could be maintained above 70% at pH 3 to 9 and was further reduced with the decrease or increase of pH.At 1, 3 and 5 days after Fusarium infection, the inflammation scores were 3.50±0.55, 7.33±0.82, 4.00±0.63 in the PBS control group, and 3.33±0.82, 4.17±0.75, 2.50±0.55 in the specific IgY treatment group.There was a statistically significant overall difference in inflammation scores at various time points between the two groups ( F _group=247.35, P<0.05; F _time=23.19, P<0.05). At 3 and 5 days after Fusarium infection, there was a smaller ulcer area and decreased inflammation scores in the specific IgY treatment group compared with the PBS control group, and the differences were statistically significant (all at P<0.05).

ConclusionsThe high titer specific IgY can be successfully prepared by salting out method using ammonium sulfate, which is with high stability, tolerance to temperature and pH.Moreover, it can alleviate the severity of corneal ulcers and reduce inflammation scores in the mouse model of fungal keratitis.