STEM I: Independent Research Project

In STEM I, students complete approximately six-month-long independent research projects. We began researching and brainstorming ideas and topics of interest during the summer prior to junior year. We continued our research and idea development into the beginning of the school year by reading scientific literature and discussing with our peers. Once our ideas were finalized, we were able to begin experimenting and gathering results. After analyzing these results, we were able to come to conclusions to answer our researchable questions.

On top of our individual projects, we also learned and studied important topics in the scientific, engineering, and research fields, including statistics and science and technical writing.

Production of Anti-Fel d 1 Chicken Antibodies Through Natural Exposure to Cats

The goal of this project was to determine if levels of anti-Fel d 1 IgY in egg yolks from chickens naturally exposed to cats were significant enough to reduce active Fel d 1 in cat dander.

Abstract

Allergic sensitization to cats in humans is the most common animal origin allergy, with the major cat allergen being Fel d 1. In past studies, chicken anti-Fel d 1 IgY was found effective at neutralizing the allergen and these antibodies can be found in egg yolks and are produced post-inoculation of chickens. The goal of this study was to determine if chickens naturally exposed to cats can produce Fel d 1 specific IgY that significantly decreases active Fel d 1 levels without inoculation. Eggs from chickens with outdoor cat exposure and indoor cat exposure were collected and total IgY antibodies were purified. Active Fel d 1 was obtained by brushing the hair of three different cats and extracting the allergen in water. Baseline Fel d 1 levels and active Fel d 1 levels after being mixed with the purified IgY were determined using a Fel d 1 ELISA. The data from two of the cats were omitted as their values exceeded those of the standard curve. The results of the other cat were used to determine that the anti-Fel d 1 levels were not great enough to significantly reduce active Fel d 1. However, a decrease of 131.47 ng/ml after treatment with IgY from chickens with exposure to outdoor cats was observed. In further studies, antibodies produced through natural interactions with cats can continue to be examined and adapted. A need remains for a comprehensive and effective solution to the global problem of cat allergies that is ethical and safe for all species.

Graphical Abstract

Graphical Abstract

Research Proposal & Literature Review

The project proposal for this project and a literature review of previous research in the cat allergy field can be found here: STEM Subpage.

Researchable Question

Can chickens naturally exposed to cats in a non-controlled environment produce anti-Fel d 1 IgY without inoculation?

Hypothesis

If chickens are exposed to Fel d 1 or to cats through natural exposure, then anti-Fel d 1 IgY will be found in the chickens’ eggs because the chickens will produce specific antibodies in response to environmental Fel d 1 to provide immunity to their offspring.

Background

Background

Sensitivity to pets, including Felis catus, domestic cats, and Canis lupus familiaris, domestic dogs, is typically the second most prevalent allergy in any given region (Dance, 2020). Human allergic sensitization to cats is the most common animal origin allergy, posing as a global problem that affects approximately one in five adults, with symptoms that include asthma, allergic rhinitis, and allergic conjunctivitis (Satyaraj et al., 2019a). Additionally, allergic sensitization is the most common reason given by cat owners for returning cats to shelters, as allergists recommend removing the cat from the home or environment as the most effective solution. However, many cat owners form a bond with their cats and view relinquishing them to shelters as an unacceptable option. If there were more accessible allergy treatments with fewer potential side effects, more prospective cat owners may be able to adopt animals in need.

While there are various cat allergens, Felis domesticus allergen 1, Fel d 1, has been identified as the major cat allergen (Thoms et al., 2019). Fel d 1 is a small protein produced by all cats, primarily in the cat’s salivary, sebaceous, and lacrimal glands. Studies suggested a pheromone or chemical signaling role, though the biological function of Fel d 1 remains unknown (Satyaraj et al., 2019a). This allergen is easily spread via the cat’s hair and saliva. Its small size and ability to adhere to surfaces and materials allows it to infiltrate environments that do not contain cats.

Cat allergy treatments and causes have been examined for decades, and a variety of solutions have been proposed or employed. Treatments include immunotherapy, frequent cat bathing, topical solutions, and a specialized cat diet. Currently, none of these solutions are completely effective and/or have the risk of adverse effects.

Immunoglobulin Y (IgY) is an antibody produced by Gallus gallus domesticus, or chickens, and these antibodies can be found concentrated in chicken egg yolks to provide immunity to chicken offspring (Satyaraj et al., 2019b). Egg components and IgY have been used to deliver active antibodies through diet in human and veterinary medicine for decades (Satyaraj, personal communication, September 22, 2021). IgY from chicken eggs can offer a potential alternative to antibodies obtained through the invasive extraction of mammalian serum. Using anti-Fel d 1 IgY as a cat allergy treatment does not attempt to alter Fel d 1 production, only neutralize the allergen before it becomes airborne. The IgY is able to bind to the Fel d 1 before IgE, therefore blocking the allergic response and number of active allergens.

In past studies utilizing IgY, the chickens were inoculated with an antigen like Fel d 1 in order to produce specific antibodies. Additionally, chickens are often kept in controlled environments and it is recommended to use a cage unit (Schade et al., 2005). While the production of specific IgY is a natural phenomenon, it is unknown if chickens can produce significant Fel d 1 specific IgY solely through natural interactions and exposure to cats in an uncontrolled environment.

Procedure

Methods

Chicken eggs were collected by the chickens’ owners and then stored at approximately 4˚C for no longer than a month. Their owners completed a survey about the chickens and their living conditions. Two groups of chickens were used. One group was from a household containing one adult outdoor cat and most of the eggs came from mixed breed chickens. The chickens lived in an outdoor fenced enclosure where some of them regularly interact with the cat. The other group of chickens was from a household containing one outdoor cat and neighbors with outdoor and indoor cats. The chickens were free-ranging and had access to their coop and the eggs came from Rhode Island Red chickens. To the owners’ knowledge, all animals from both groups were healthy.

An IgY purification kit (EggsPress IgY Purification Kit (5 yolks)) was provided by Exalpha Biologicals, Inc. This kit contains two reagents, one for removing lipids and the other for precipitating the purified IgY. In addition to the materials provided in the kit, deionized water, pipettes, phosphate buffered saline (pH 7.2), and a centrifuge were used. The kit instructions and protocols were followed; however, a couple of modifications were made due to limitations (Appendix A). The immunization protocol was omitted as the aim of this study was to examine the results without inoculation through injection. One egg from each previously described group was used. Following the acclimation of the eggs to 4-8˚C, the yolks were separated from the eggs. Then the delipidation reagent (Reagent A) was added, mixed, incubated, and centrifuged. The supernatant was collected, and the precipitating reagent (Reagent B) was added, mixed, incubated, and centrifuged. Finally, the precipitate IgY was collected, dissolved with phosphate buffered saline (PBS), and stored at 4˚C.

Fel d 1 samples were collected from the hair of three adult shorthair cats. All owners gave verbal consent and assisted in the sampling process by holding the cats. The cats were brushed primarily on the back, neck, and stomach using a new slicker brush for each cat. Hair samples were stored at room temperature in plastic bags until Fel d 1 extraction. Cats are identified as Cat 1, Cat 2, and Cat 3. Avner et al. (1997) found that significant levels of Fel d 1 could be removed from cat hair by soaking in water. Separately, hair samples from each cat were mixed with 3 ml of deionized water per 0.05 g of hair. Fel d 1 was extracted overnight at 4˚C. Solid hair particles were then removed and extracted Fel d 1 samples were stored at 4˚C until being treated with purified IgY.

3 ml of extracted Fel d 1 from Cat 1 were mixed with 3 ml of purified IgY from the egg with outdoor cat exposure. This same process was repeated with Cats 2 and 3 and with IgY from the egg with indoor cat exposure. In addition, 3 ml of extracted Fel d 1 from all three cats were mixed with 3 ml of PBS. This was done to account for the dilution caused by the purified IgY in PBS. All samples were stored overnight at 4˚C.

An enzyme-linked immunosorbent assay (ELISA) was performed to quantify sample active Fel d 1 baseline levels (mixed with PBS only) and active Fel d 1 levels after being mixed with IgY (Appendix C). A Fel d 1 ELISA (Fel d 1 ELISA 2.0 kit - Single plate (EPC-FD1-1)) was provided by Indoor Biotechnologies, Inc. In addition to the kit, micropipettes, deionized water, an Edvotek White Light Box, and the Read Plate 3.0 plugin for ImageJ were used in this procedure. Kit instructions and protocols were followed, and each dilution of all samples were completed in duplicate. Prior to using the plate reader program, an image of the ELISA plate over the illuminated Edvotek White Light Box was taken using the iPhone Camera application. The Read Plate 3.0 plugin for ImageJ was then used as the ELISA plate reader and read the absorbance at 450 nm.

A Kruskal-Wallis one-way analysis of variance test was performed to determine if there was a significant difference between different nonparametric groups. A significant difference would indicate a significant decrease in active Fel d 1 levels following treatment. The test was performed to compare the baseline levels, the levels after being mixed with IgY from chickens with exposure to outdoor cats, and the levels after being mixed with IgY from chickens with exposure to indoor cats for Cat 3 only. The test was performed on Microsoft Excel and examined at a significance level of 0.05.

Table 1

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Figure 1

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Figure 2

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Analysis

Fel d 1 Levels

The mean weight of the extracted hair for Cat 1 was 0.5097 mg, Cat 2 was 0.7225 mg, and Cat 3 was 0.2192 mg. The ELISA optical density data was fixed using the Read Plate 3.0 plugin for ImageJ. After creating an ELISA standard curve (Figure 1), it was determined that all data from Cats 1 and 2 had optical densities beyond the range of the standard curve (Table 1). Therefore, the data from those two cats were unable to be analyzed and consequently omitted from further statistical analysis. A few data from Cat 3 were also beyond the range of the standard curve and omitted.

Chicken IgY

According to the IgY purification kit instructions, the IgY concentration from each egg should be 4 to 7 mg/ml and have a purity of 85% or greater. The yolk from chickens with outdoor cat exposure yielded a final purified IgY volume of 19.23 ml. The yolk from chickens with indoor cat exposure yielded a final purified IgY volume of 15.25 ml.

Treatment of Fel d 1 with IgY

Only data from Cat 3 was analyzed to determine if there was a significant difference between Fel d 1 levels following treatment with the IgY (Figure 2). The standard curve was used to determine Fel d 1 concentrations from the optical densities. The average Cat 3 Fel d 1 concentration for baseline was 366.06 ng/ml, for treatment with IgY from chickens with outdoor cat exposure was 234.59 ng/ml, and for treatment with IgY from chickens with indoor cat exposure was 449.43 ng/ml. From the Kruskal-Wallis test performed at a significance level of 0.05, a p-value of p = 0.1190 was determined, indicating that there is no significant difference between the baseline levels and either of the treatment groups. While not significantly different, a decrease of 131.47 ng/ml was observed between the averages of the baseline and after the treatment with the IgY from chickens with exposure to outdoor cats.

Discussion & Conclusion

Fel d 1 is the major cat allergen and many current cat allergy treatments aim to address Fel d 1 and IgY binding. One such treatment uses anti-Fel d 1 IgY produced by chickens inoculated with the allergen to create a blocking effect. The purpose of this study was to examine the effects of natural cat exposure on anti-Fel d 1 IgY production in chickens. Because the production of specific IgY is a natural phenomenon, it was hypothesized that if chickens were exposed to Fel d 1 or to cats through natural exposure, then anti-Fel d 1 IgY would be found in the chickens’ eggs because the chickens would produce specific antibodies in response to environmental Fel d 1 to provide immunity to their offspring.

Based on the experimental data and results, the initial hypothesis was not supported. The tests conducted do not provide evidence that levels of anti-Fel d 1 IgY significant enough to reduce active Fel d 1 can be produced by chickens naturally exposed to cats. Data from two of the three cats were completely omitted due to the data exceeding the standard curve. On average, the Fel d 1 levels for Cat 3 were lower than those of the other two cats, allowing analysis of Cat 3’s data. When comparing Fel d 1 concentrations following treatments with either baseline PBS, purified IgY from eggs with outdoor cat exposure, or purified IgY from eggs with indoor cat exposure, the p-value of p = 0.1190 for Cat 3 was higher than the established significance level (p > 0.05). While this p-value indicated that there was no statistically significant difference or decrease, a decrease in average Fel d 1 concentration between baseline and treatment with IgY from chickens with outdoor cat exposure was noted. This may suggest that smaller baseline Fel d 1 levels allow for a decrease in Fel d 1 levels when mixed with IgY. The average Fel d 1 concentration increased between the baseline and treatment with IgY from chickens with indoor cat exposure, though due to the small sample size that was able to be analyzed for this treatment, it was difficult to come to a definitive conclusion as to why this occurred. The Fel d 1 increase could have potentially been due to human error in testing or in the preparation of the materials.

A key component of this study was to examine the effects of the many uncontrolled environmental factors, in addition to cat exposure, on the effects of the production of anti-Fel d 1 IgY. The natural behaviors and habitats of the chickens were not altered in any way for this study. In addition, only two groups of chickens were used in this study. This may not have been representative of all or other chickens with exposure to cats. Due to the limited materials and budget (Appendix A), a Kruskal-Wallis test was used to account for the small sample size, nonparametric data, and lack of assumption of normality.

In the past, research utilizing anti-Fel d 1 IgY relied on the eggs of inoculated chickens. These studies found that this specific IgY could reduce active Fel d 1 levels, both in vitro and in vivo (Satyaraj et al., 2019a; Satyaraj et al., 2019b; Satyaraj et al., 2021). While past research has provided evidence treatment methods that reduce the need for treatments such as immunotherapy injections in humans or frequently bathing cats, the chickens used to produce the anti-Fel d 1 IgY must be injected or inoculated with Fel d 1. Also, the chickens are often kept in controlled environments, and it is recommended to use a cage unit (Schade et al., 2005). This study aimed to eliminate testing on live subjects or changing the behaviors of cats, chickens, or humans. It also aimed to address the comfort and needs of all species involved. While it failed to provide evidence of the production of anti-Fel d 1 IgY without inoculation in chickens naturally exposed to cats, the data collected can still be useful in future studies or expansions of this study.

Implications and Applications

The results found and data collected in this study can be applied to future studies or inspire future research in a similar direction. Further studies may adapt these methods to support hypotheses that also aim to create cat allergy solutions that do not interfere with the lives of any species involved. Moreover, future results may support the evidence displayed in this study to demonstrate that chickens cannot produce significant levels of anti-Fel d 1 IgY without inoculation in a controlled environment.

Future Research

It would be ideal for this study to be repeated more than once, but material limitations restricted the number of tests that could be run and the amount of samples that could be examined (Appendix A). A future extension would include introducing larger sample sizes and diluting the initial Fel d 1 samples by a greater amount in order to not exceed the standard curve values.

In the future, similar studies can be conducted using higher amounts of IgY to Fel d 1 on a larger sample size. In addition, Fel d 1 baseline levels can be determined prior to treating the samples with IgY in order to determine the ideal dilution ratios. Due to the equipment required, it may be unrealistic to produce and use specific IgY from chickens like the ones used in this study. Therefore, future studies examining the impact of cat exposure in a controlled environment may be pertinent to investigate. Using this method, inoculation could still potentially be avoided, but higher concentrations of anti-Fel d 1 may be produced. Finally, the effects of eating unpurified eggs containing anti-Fel d 1 IgY using chickens inoculated with Fel d 1 could also be examined. In a study aiming to research this, the cats or humans would eat the egg yolks of these inoculated chickens in an attempt to decrease active Fel d 1 levels or allergic responses.

Conclusions and Takeaways

While it did not have results in support of the original hypothesis, the study did aim to address the objective. Using a small sample of eggs from chicken groups exposed to cats and cat hair samples, IgY was purified and Fel d 1 was extracted. After using an ELISA to quantify active Fel d 1 levels with and without IgY treatment, the data was statistically analyzed. The data from two cats were omitted from analysis as their optical densities exceeded those that could be determined using the ELISA standard curve. The results determined that levels of anti-Fel d 1 IgY significant enough to reduce active Fel d 1 cannot be produced by chickens naturally exposed to cats, though future iterations of this test may provide more conclusive results. A need remains for a comprehensive and completely effective solution to the global problem of cat allergies that is ethical and safe for all species involved.

References

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