Effect of Temperature and Humidity on Bacterial Growth and Infection of Eggs Pallavi Karunakaran

Effect of Temperature and Humidity on Bacterial Growth and Infection of Eggs Pallavi Karunakaran The University of Akron Biology Department Major: Natural Science Project Sponsor: Dr. Matthew Shawkey Number of Project Credits: 3

Microbial penetration of eggshells poses a threat to the development of avian embryos in environmental conditions that support bacterial proliferation. We hypothesized that high humid surroundings provide a suitable environment for bacterial growth on the eggshell surface and for microbial penetration into the egg contents. We tested this by exposing eggs right after oviposition to one of four treatment groups: high and low levels of humidity at two temperatures. After one week of exposure to the set environmental conditions, we compared relative microbial growth on the eggshell and bacterial abundance on the membrane and albumen. Enteric bacterial relative growth on egg surfaces was positively influenced by high temperature, as expected from other studies on microbial growth. When microbial penetration did occur, more heterotrophic bacterial abundance was present on the membrane and the albumen exclusively in the high temperature experimental groups. Ambient humidity does not seem to have a strong effect on microbial growth and on penetration into the egg contents. Adding to the key role that temperature plays in bacterial growth, humidity might affect susceptibility of eggs to microbial penetration but perhaps only when high humidity results in condensation and presence of liquid water on the egg surface. Introduction: Microbes are one of the main causes of death in egg embryos (Deeming 1995, Pinowski et al. 1994). Growth of bacteria on the surface of the eggshells and penetration of those microbes into the egg may be affected by ambient environmental conditions, in particular temperature and humidity (Walsberg and Schmidt 1992). Many bacteria grow maximally at temperatures around 37°C (Madigan et al. 2005). Moreover, infection of the egg may occur soon after oviposition and humidity may play a vital role. High relative humidity can allow for the proliferation of microbes (Bruce and Drysdale 1994). Past studies have shown that presence of

water on the eggshell aids in penetration of microbes as it is the mode of microbial transport into the egg (Board & Halls 1973). Humidity can cause the presence of liquid water by condensation, and this effect on bacterial growth on eggs needs to be studied. Previous studies on microbes on eggs have not isolated the effect of humidity on bacterial growth. Rather, studies where eggs have been placed in different natural conditions have shown that the probability of infection is highly dependent on the presence of fungal growth on the eggshells (Cook 2003). It has been demonstrated that incubation itself constitutes a mechanism that parent birds use to prevent bacterial growth (Cook et al. 2005, Shawkey et al. 2009). However, in the absence of parents in the nest, eggs are exposed to environmental conditions that contribute to increased microbial growth and potential microbial penetration into the egg contents. Infection of the eggs requires some humidity since water acts as vehicle by which microbes can enter the egg through the eggshell pores (Cook 2003). High humidity values are present in tropical regions of the world. A medial humidity value may be present in grasslands and forests that birds inhabit. Low humidity values are present in locations such as plains and plateaus with less moisture present in the air. All locations harbor nesting birds and may provide different risks of bacterial infection to the eggs. However, the relative severity of risks in these different habitats is largely unknown. Therefore, the purpose of this study is to test the effect of two different humidity and temperature values on bacterial growth in the course of incubation of eggs after oviposition. We investigated how bacterial growth and rate of microbial invasion varies between low and high humidity conditions as well as between low and high temperature. Hypothesis:

The question that will be explored is: Does humidity affect the growth and infection of bacteria on eggs? At a high humidity, the growth of bacteria is expected to be higher than at a lower humidity. In addition, temperature can affect humidity by altering the amount of liquid water present. This is expected since humid conditions can provide ideal environments for microbial growth. Furthermore, liquid water on the surface of the eggs can facilitate bacterial penetration into the egg contents via the eggshell pores. It is predicted that even at various temperature ranges, water is the key element that allows for increased bacterial growth. The main goal of the project is to learn how humidity affects the growth of bacteria on eggshells and penetration into the egg contents. Methods: Egg collection Fresh chicken eggs were collected in groups of 20 for each treatment group from Brunty Farms in Akron, OH. The eggs were collected directly from the chicken nests to minimize contact with pathogens and placed in a sterile casing during transport to the lab. The eggs were swabbed with sterile phosphate buffered saline (PBS) solution within an hour after collection to detect the initial bacterial count on each egg before treatment. The swabbing was used to collect pre-treatment microbial samples. Microbiology We swabbed one-fourth of the eggshell surface initially before the treatment to determine initial bacterial counts. The eggs were then put through the treatment for 1 week at different sets of temperatures and humidity (see experiment below). After 1 week treatment, a post-treatment swab was done of one-fourth of the eggshell. The shell surface was then sterilized with alcohol wipes. Using sterile materials, each egg shell was cracked and peeled away gently at the blunt

end to expose the membrane from the airsac region. A 1cm2 sample of the membrane was cut away and placed in a sterile 1.5 ml microfuge tube with 1mL sterile PBS. The opening in the egg was widened slightly to pour the albumen into a petri dish. A 0.01 mL aliquot of albumen was taken from the contents using a calibrated 10μl loop and placed in a microfuge tube with 200μL PBS (Cook 2010). Bacterial plating was done by pipetting 0.1mL of the PBS solution in each microfuge tube into two types of growth media: Soy agar to grow heterotrophic bacteria and MacConkey agar to grow Gram-negative bacteria. Using two types of growth media allowed us to see the various types of bacterial that may occur on and in the egg. Plates were made with these growth media according to the directions provided by the manufacturer. After plating of the samples into both Soy and MacConkey dishes, they were incubated for 48 hours at 34 -36°. Control plates were set up using just PBS solution without egg components to ensure there was no contamination in the buffer solution that would cause false positives. After the incubation period, the bacterial colony forming unit (CFU) counts were found on each plate. These counts were used to determine bacterial growth on the eggshell and to compare amount of bacteria present among the treatment groups. When CFU’s were too abundant to count, we assigned a maximum number of 2,000 CFUs. Experiment To investigate the effect of humidity on bacterial growth, eggs were put in incubators that were set to a specific relative humidity (RH). Each treatment group had 10 eggs for each corresponding humidity and temperature. The eggs in each group were incubated for 1 week and after incubation, bacterial growth was determined by culture methods.

Two experimental treatments of high and low humidity were applied in the incubators at two constant temperatures. The varying humidity values and corresponding temperatures allow for comparison among different habitats and environmental conditions present in the real world. The experiment was replicated one month after the first trial using the same sample sizes and experimental groups. The experimental design was as follows: The eggs in treatment group 1 (low temperature and high humidity, LTHH) were incubated at 16°C and 80% RH. Treatment group 2 (low temperature and low humidity, LTLH) eggs were incubated at 16°C and 30% RH. Eggs in treatment group 3 (high temperature and high humidity, HTHH) were incubated at 36°C and 80% RH. Eggs in treatment group 4 (high temperature and low humidity, HTLH) were incubated at 36°C and 30% RH. An experiment replicate was done of all the treatment groups. These temperature and humidity values were used to represent various climates where avian incubation takes place. Statistical Analysis Bacterial counts did not show a normal distribution therefore data were rank-transformed before analyses. Relative microbial growth was found for each egg by the following equation: (CFU post treatment – CFU pre treatment)/CFU pre treatment. Two-way ANOVA test was used to compare the relative growth on eggshells among groups or the CFU counts (abundance) on membrane and albumen. We compared the initial bacterial counts on eggshell between the first and second trials using a non-parametric test (MannWhitney U test). All graphs show raw data while all statistical tests used ranked data. All tests done were two-tailed and p-values less than 0.05 were considered statistically significant. Results: Initial CFU counts

We found a significant difference in the initial heterotrophic bacterial CFU counts between the two trials run to conduct the experiment. A significant difference in the distribution (U = 1072, p = 0.009; Fig 1) and median (p = 0.044: Fig 1) of initial counts was found between the trials using a Mann-Whitney U test and a median test, respectively, with a greater initial CFU count in the first trial. We also found a significant difference in the initial gram-negative CFU counts between the two trials. A significant difference in the distribution (U=1109, p = 0.002; Fig 2) and median (p = 0.025: Fig 2) of initial counts was seen with the greater initial CFU counts present for the first trial. First Trial Egg surface: During the first trial of experiments there were no significant differences among groups in the relative growth of heterotrophic bacteria on the eggshell surfaces (F = 0.545, p = 0.655; Fig 3). Eggs in the LTHH group had slightly greater relative growth on the eggshell although no significance was found between the groups. Only two eggs, one in the LTLH group and the other in the HTLH group, showed enteric bacteria on the eggshell surface after treatment, therefore, analysis on bacterial growth were not performed. Similarly, the membrane and albumen of the eggs in the first trial did not show any presence of bacteria following the treatments. Second Trial Egg surface: During the second trial of experiments there were no significant differences among the different treatments in relative growth of heterotrophic bacteria on the eggshell surfaces (F =

1.759, p = 0.172; Fig 4). However, there was a significant difference in relative growth between just the low temperature low humidity group and the high temperature high humidity group (mean difference = 0.973, p = 0.028) as the former had a greater relative growth. After removing one outlier with extremely high relative growth (>3 times the interquartile range; Fig 5) in the low temperature low humidity group, no significant difference in relative heterotrophic bacterial growth was seen among the groups (F = 1.183, p = 0.330; Fig 5). We found significant differences in relative growth of gram-negative bacteria on the eggshell surface of the different treatment groups (F = 3.66, p = 0.021; Fig 6). Overall, relative bacterial growth was greater in the HTLH group compared to the high humidity groups: the relative difference in CFU counts was significantly higher in the HTLH group compared to the HTHH (mean difference = 1.066, p = 0.003) and was marginally non-significantly higher in the LTHH group (mean difference 0.625, p = 0.071). The relative difference in CFU counts was also significantly higher in the LTLH group, in which only 3 eggs showed growth, in comparison with the HTHH group (mean difference 0.745, p = 0.033). Both low humidity groups had higher bacterial growth than the high temperature and high humidity group. Egg membrane: We found significant differences in heterotrophic bacterial abundance on the membrane of eggs of the different treatment groups (F = 5.88, p = 0.002; Fig 7). Specifically, bacterial abundance was higher in the HTLH group in comparison with the LTHH group (mean difference = 0.94, p = 0.002) and the LTLH group (mean difference = 1.055, p = 0.001). In comparison with the low humidity groups, which did not show any presence of bacteria, the high temperature group had a greater bacterial abundance. Egg albumen:

In general, we did not find significant differences in heterotrophic bacterial abundance in egg albumen among the different treatment groups (F = 2.33, p = 0.091; Fig 8). However, bacterial abundance was significantly different between the LTLH and HTHH groups. The HTHH group had more bacterial abundance than the LTLH group (mean difference = 0.764, p = 0.014) since there was no bacterial presence in the LTLH group compared to the presence of bacteria in the HTHH group.

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Fig 1. Comparison of initial heterotrophic bacterial CFU counts on eggshell surface between trials. Sample sizes for each group is n= 40 eggs. The black asterisk denotes significant differences between groups. Raw data shown. The red asterisk and dot indicate initial CFU counts which deviate from the interquartile range.

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Fig 2. Comparison of initial gram-negative bacterial CFU counts on eggshell surface between trials. Sample sizes for each group is n= 40 eggs. The black asterisk denotes significant differences between groups. Raw data shown. The smaller asterisks and dot indicate initial CFU counts which deviate from the interquartile range.

Fig 3. Comparison of relative difference in heterotrophic bacterial pre and post treatment counts on the eggshell among treatment groups in trial 1. Sample sizes for each group is n= 10 eggs. Raw data shown. The smaller asterisks and dot indicate values which deviate from the interquartile range. No significant differences between groups were found.

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Fig 4. Comparison of relative growth of heterotrophic bacteria on the eggshell among treatment groups in trial 2. Sample sizes for each group is n= 10 eggs. The black asterisk denotes significant differences between groups. Raw data shown. The smaller asterisks and dot indicate values which deviate from the interquartile range.

Fig 5. Comparison of relative growth of heterotrophic bacteria on the eggshell among treatment groups in trial 2. One extreme value (>3 times the interquartile range) was removed from the lowT-lowRH group. Sample sizes for each group is n= 10 eggs, except the lowT-lowRH (n=9) group. Raw data shown. The smaller asterisks and dot indicate values which deviate from the interquartile range. No significant differences between groups were found.

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Fig 6. Comparison of relative growth in gram-negative bacteria on the eggshell among treatment groups in trial 2. Sample sizes for each group is n= 10 eggs. The black asterisk denotes significant differences between groups. Raw data shown. The smaller asterisks and dot indicate values which deviate from the interquartile range.

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Fig 7. Comparison of heterotrophic bacterial CFU counts on the egg membrane among treatment groups in trial 2. Sample sizes for each group is n= 10 eggs. The black asterisk denotes significant differences between groups. Raw data shown. The smaller asterisks and dot indicate values which deviate from the interquartile range.

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Fig 8. Comparison of heterotrophic bacterial CFU counts in the egg albumen among treatment groups in trial 2. Sample sizes for each group is n= 10 eggs. The black asterisk denotes significant differences between groups. Raw data shown. The smaller asterisks and dot indicate values which deviate from the interquartile range.

Discussion: In this study, we investigated the effects of different humidity and temperature conditions on the growth and penetration of microbes in eggs. Four experimental groups were set up to see if humidity under two set temperatures affected bacterial presence and growth. Previous studies have shown high temperature and humidity causing condensation of water on the eggshell favors microbial growth on the surface and aids penetration into the egg (Cook et al. 2005, Board & Halls 1973). We expected to see greater bacterial growth in the groups with high humidity under high temperature since those conditions seem optimal for microbial proliferation by the presence of liquid water on the egg surface. Overall and against our predictions, we showed that high temperature and low humidity were most favorable to growth of enteric bacteria on the egg’s surface, and on growth of heterotrophic bacteria in the egg’s contents. Surprisingly, with one exception, we did not see heterotrophic bacterial growth on eggs in the first trial. This may be explained by the period of one month that passed from when the first trial was conducted till the second trial was conducted. The natural variation of microbial communities with season could have caused different levels of contamination on the eggshell (Fierer and Jackson 2006). Figures 1 and 2 exhibit that more initial bacterial counts were found in the first trial. This indicates that initially higher bacterial loads could decrease subsequent growth, as the nutrients on the eggshell could have been used up by the presence on bacteria before the experiment was run. Seasonal variation can alter microbial processes, which makes experiments conducted at different times in the year difficult to compare. We found that when enteric bacteria were present on the eggshell surface, bacterial growth was significantly greater in high temperature and low humidity conditions. This was against our prediction that high humidity would increase bacterial growth. Also, we found that

microbial penetration to the membrane occurred only in eggs under high temperature conditions. No bacteria were found in eggs of the two groups with low temperature. The effects of humidity on bacterial penetration into the egg contents were mixed. For example, we found that invasion of egg membrane was higher at high temperature low humidity than at high temperature high humidity. The opposite was true for the bacterial presence in the egg albumen; eggs in the high temperature high humidity showed higher abundance compared to the high temperature low humidity groups. There was more heterotrophic bacterial abundance on the membrane in the high temperature high humidity groups compared to the low temperature groups, and bacterial presence was greater in the albumen at high temperature with high humidity, supporting the idea that these conditions are suitable for microbial processes. Perhaps our results indicate that high temperature is needed for penetration by microbes through the eggshell to occur, but the addition of high humidity is needed for penetration into the albumen to occur. In the second trial, no significant differences in heterotrophic bacterial relative growth on the eggshell surface were seen when one outlier was removed. The egg with the outlier could have had some remains of materials from the nest on the eggshell surface, which provided nutrients for bacterial growth and would have not been present on the other eggshells. Since relative enteric bacterial growth was greater in the high temperature low humidity group compared to the high humidity groups, high temperature is seen as supporting enteric bacterial proliferation on the surface of eggshells. Also, increased bacterial abundance for high temperature low humidity group compared to the low temperature groups may indicates that high temperature is enough to facilitate the penetration of eggshells by gram-negative bacteria, which are known to enter shell components (Godard et al. 2007).

Our results for bacterial abundance in the egg albumen in the second trial matched our expectations of having increased bacterial abundance at high temperature and high humidity. The microbial counts were significantly different than those in the low temperature groups. A variety of microbes including fungal hyphae can penetrate the eggshell barrier and enter the egg contents when the conditions of temperature and humidity allow for such microbial trans-shell infection (Board et al. 1964, 1994). Although we did not observe any condensation on the surface of eggs in trial 2, condensation of water vapor may have aided microbial penetration of the eggshell. This research provides evidence that eggs in natural populations are susceptible to penetration of the eggshell by bacteria when conditions of temperature, and to a lesser extent of humidity, are optimal for microbial growth. Our data also suggest that ambient humidity does not have a strong effect on microbial growth and on penetration into the egg contents. Indeed, it may only be significant under conditions where it condenses into liquid water on the surface. Parental care provided by incubation plays a role in the risk of infection by microbes. Incubation works in various ways; one way is by keeping the surface of the egg dry, it decreases chance of infection (Board & Tranter 1995; D’Alba et al. 2010). Environments where water vapor is able to condense on the eggshell, such as locations with heavy rain or large temperature fluctuation, would be good places to continue further research on microbial penetration of eggs. Wild birds may have specific antimicrobial behaviors to prevent infection through incubation in those environmental conditions. Studying these techniques used by birds in the wild will allow for increased understanding of the risk of microbial infection on bird eggshells and embryos.

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