Eggshell Bacterial Load Research Articles

Effects of clove essential oil on eggshell bacterial load, antibacterial sensitivity, and hatchability

Background and objective Clove essential oil’s (CEO) physical and chemical properties allow for its potent antibacterial action. This study sought to determine the impact of various CEO disinfectant concentrations on the eggshell bacterial load, embryonic mortality, hatchability, and chick quality. Materials and methods A total of 1500 fertile chicken eggs were randomly divided into five treatment groups. Group one was sprayed with a commercial disinfectant (BioSentry 904), the second group was sprayed with ethyl alcohol 70%, and the last three groups were sprayed with 0.5, 1, or 2% of CEO. After spraying, eggs in each group were used for the determination of total bacterial load on the eggshell, hatchability, embryonic mortality, and chick quality. Results and conclusion Results showed that the bacterial load on the eggshell declined with the increase in the concentration of CEO. The use of CEO at 1, 2%, or BioSentry 904 resulted in the lowest bacterial load. However, hatchability of set and fertile eggs for the group treated with CEO at 0.5% was numerically greater than other groups, while chick quality grade (A) was numerically greater by CEO at 1% concentration. The chick yield of the egg group treated with CEO at 1% was significantly higher than in the control group. Embryonic mortalities for all groups were statistically similar. In conclusion, using CEO at different concentrations 1, 2%, or BioSentry 904 in disinfecting broiler breeder eggs can effectively reduce the bacterial load on eggshells. In addition, using CEO improved chick quality and chick yield without any adverse effect on hatchability. Consequently, it is considered a strong competitor to BioSentry 904 and a less hazardous disinfectant for hatching egg disinfection on a commercial scale.

Impact of Different Layer Housing Systems on Eggshell Cuticle Quality and Salmonella Adherence in Table Eggs.

The bacterial load on the eggshell surface is a key factor in predicting the bacterial penetration and contamination of the egg interior. The eggshell cuticle is the first line of defense against vertical penetration by microbial food-borne pathogens such as Salmonella Enteritidis. Egg producers are increasingly introducing alternative caging systems into their production chain as animal welfare concerns become of greater relevance to today’s consumer. Stress that is introduced by hen aggression and modified nesting behavior in furnished cages can alter the physiology of egg formation and affect the cuticle deposition/quality. The goal of this study was to determine the impact of caging systems (conventional, enriched, free-run, and free-range), on eggshell cuticle parameters and the eggshell bacterial load. The cuticle plug thickness and pore length were higher in the free-range eggs as compared to conventional eggs. The eggshells from alternative caging (enriched and free-range) had a higher total cuticle as compared to conventional cages. A reduction in bacterial cell counts was observed on eggshells that were obtained from free-range eggs as compared to the enriched systems. An inverse correlation between the contact angle and Salmonella adherence was observed. These results indicate that the housing systems of layer hens can modify the cuticle quality and thereby impact bacterial adherence and food safety.

Experimental old nest material predicts hoopoe Upupa epops eggshell and uropygial gland microbiota

Nest re‐use in birds has the potential cost of infection by parasites and pathogens but may also be a source of beneficial symbiotic bacteria transmitted horizontally. Eurasian hoopoes Upupa epops host antibiotic‐producing bacteria in their uropygial gland but only while breeding, which suggests that the nest‐hole may be a source of those symbionts. Interestingly, hoopoes do not build nests, thus might prefer for reproduction nest holes with soft materials from previous reproductions. Here, we tested experimentally this preference by installing in the field new nest boxes that were left empty or filled with either sawdust or a mixture of sawdust and hoopoe's nest material from the previous year. We explored the experimental effect on the composition of the uropygial secretion bacterial community, on eggshell bacterial loads, and on several proxies of reproductive success. Hoopoes bred significantly more often in nest boxes with nest material than in empty ones, but the type of nest material did not affect nest box occupancy. Eggs in nest boxes with old‐soft material harbored higher bacterial density on their shells, and the microbiota of the uropygial secretion of nestlings and females in these nest boxes differed from those in nest boxes without old‐soft material. Moreover, although the experiment did not affect breeding success or related proxies, several operational taxonomic units from female uropygial secretions were positively associated with hatching success. This is the first experimental evidence showing that re‐used nest material affects the bacterial community of the uropygial secretions of hoopoe females. This suggests that the nest material can be a source of strains for their incorporation to both the uropygial gland and eggshell communities, highlighting a possible advantage of nest re‐use previously unconsidered.

Ectoparasite Activity During Incubation Increases Microbial Growth on Avian Eggs.

While direct detrimental effects of parasites on hosts are relatively well documented, other more subtle but potentially important effects of parasitism are yet unexplored. Biological activity of ectoparasites, apart from skin injuries and blood-feeding, often results in blood remains, or parasite faeces that accumulate and modify the host environment. In this way, ectoparasite activities and remains may increase nutrient availability that may favour colonization and growth of microorganisms including potential pathogens. Here, by the experimental addition of hematophagous flies (Carnus hemapterus, a common ectoparasite of birds) to nests of spotless starlings Sturnus unicolor during incubation, we explore this possible side effect of parasitism which has rarely, if ever, been investigated. Results show that faeces and blood remains from parasitic flies on spotless starling eggshells at the end of incubation were more abundant in experimental than in control nests. Moreover, eggshell bacterial loads of different groups of cultivable bacteria including potential pathogens, as well as species richness of bacteria in terms of Operational Taxonomic Units (OTUs), were also higher in experimental nests. Finally, we also found evidence of a link between eggshell bacterial loads and increased embryo mortality, which provides indirect support for a bacterial-mediated negative effect of ectoparasitism on host offspring. Trans-shell bacterial infection might be one of the main causes of embryo death and, consequently, this hitherto unnoticed indirect effect of ectoparasitism might be widespread in nature and could affect our understanding of ecology and evolution of host-parasite interactions.

Nest Material Shapes Eggs Bacterial Environment.

Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically environmental variation associated with risk of bacterial proliferation determining the strength of such effects. Because of costs associated to nest building, birds should adjust nest building effort to expected bacterial environments during incubation, a prediction that should be further explored.

Laying date, incubation and egg breakage as determinants of bacterial load on bird eggshells: experimental evidence.

Exploring factors guiding interactions of bacterial communities with animals has become of primary importance for ecologists and evolutionary biologists during the last years because of their likely central role in the evolution of animal life history traits. We explored the association between laying date and eggshell bacterial load (mesophilic bacteria, Enterobacteriaceae, Staphylococci, and Enterococci) in natural and artificial magpie (Pica pica) nests containing fresh commercial quail (Coturnix coturnix) eggs. We manipulated hygiene conditions by spilling egg contents on magpie and artificial nests and explored experimental effects during the breeding season. Egg breakage is a common outcome of brood parasitism by great spotted cuckoos (Clamator glandarius) on the nests of magpie, one of its main hosts. We found that the treatment increased eggshell bacterial load in artificial nests, but not in magpie nests with incubating females, which suggests that parental activity prevents the proliferation of bacteria on the eggshells in relation to egg breakage. Moreover, laying date was positively related to eggshell bacterial load in active magpie nests, but negatively in artificial nests. The results suggest that variation in parental characteristics of magpies rather than climatic variation during the breeding season explained the detected positive association. Because the eggshell bacterial load is a proxy of hatching success, the detected positive association between eggshell bacterial loads and laying date in natural, but not in artificial nests, suggests that the generalized negative association between laying date and avian breeding success can be, at least partially, explained by differential bacterial effects.

An experimental test in Mallards (Anas platyrhynchos) of the effect of incubation and maternal preen oil on eggshell microbial load

Microbial infection is one of the main factors reducing survival in the first stages of life in oviparous species, and recent studies have shown that the avian eggshell harbors an important variety of microorganisms that can rapidly multiply and penetrate the shell, leading to a decrease in hatchability. Here, we report the results of an experiment in which we examined how incubation and maternal preen oil affect the growth of avian eggshell microbes, using the Mallard (Anas platyrhynchos) as a model species. We compared the bacterial and fungal loads on the shell of non-incubated eggs and eggs incubated by females having free or blocked access to their preen gland. An increase of eggshell bacterial loads was observed in all conditions, but bacterial growth was higher on the shell of incubated eggs than on non-incubated eggs. We did not find any significant difference in eggshell bacterial growth for eggs incubated by females with free or blocked access to their preen gland. In addition, fungal growth during our experiment was not affected by incubation or the mother’s preen oil. Our findings are in contrast with those of previous studies which showed that incubation limited or had no effect on eggshell bacterial growth. Differences in environmental conditions and/or species ecology may explain the difference between the results of our experiment and those of previous studies. Our study provides the first data on the effect of maternal preen oil on eggshell microorganisms, showing that preen oil does not limit eggshell microbial growth.

Eggshell Bacterial Load Is Related to Antimicrobial Properties of Feathers Lining Barn Swallow Nests

The use of feathers to line bird's nests has traditionally been interpreted as having a thermoregulatory function. Feather-degrading bacteria growing on feathers lining nests may have antimicrobial properties, which may provide an additional benefit to lining nests with feathers. We test the hypothesis that the production of antimicrobial substances by feather bacteria affects the microbiological environment of the nest, and therefore the bacterial density on eggshells and, indirectly, hatching success. These effects would be expected to differ between nests lined with pigmented and white feathers, because bacteria grow differently on feathers of different colors. We experimentally manipulated the composition of pigmented and unpigmented feathers in nests of the barn swallow (Hirundo rustica) and studied the antimicrobial properties against the keratin-degrading bacterium Bacillus licheniformis of bacteria isolated from feathers of each color. Analyzed feathers were collected at the end of the incubation period, and antimicrobial activity was defined as the proportion of bacteria from the feathers that produce antibacterial substances effective against B. licheniformis. Our experimental manipulation affected antimicrobial activity, which was higher in nests with only white feathers at the beginning of incubation. Moreover, white feathers showed higher antimicrobial activity than black ones. Interestingly, antimicrobial activity in feathers of one of the colors correlated negatively with bacterial density on feather of the opposite color. Finally, antimicrobial activity of white feathers was negatively related to eggshell bacterial load. These results suggest that antimicrobial properties of feathers in general and of white feathers in particular affect the bacterial environment in nests. This environment in turn affects the bacterial load on eggshells, which may affect hatching success.

Efficacy of combination chemicals as sanitizers of Salmonella-inoculated broiler hatching eggshells

SUMMARY Sanitization of broiler hatching eggs provides an opportunity to reduce the eggshell bacterial load entering hatchery incubators, which can be subsequently carried with the chicks to the grow-out farm and ultimately into the processing plant. Two experiments evaluated combination chemicals containing ammonium chlorides (NH4Cl), biguanide (C2H7N5R; BG), bronopol (C3H6BrNO4; BP), quaternary ammonium (NR4), or hydrogen peroxide (H2O2) for efficacy in eliminating inoculated Salmonella from the shell of broiler hatching eggs. These chemicals were compared with no treatment, water sprayed (to account for spray rinsing of eggshells independent of sanitization), and to 1.4% H2O2, a demonstrated effective hatching egg sanitizer for Salmonella. All 4 combination chemicals tested (2NH4Cl+H2O2, 2NR4+BP+BG, 4NR4, and 4NR4+BG) significantly reduced the recovery of Salmonella (80% to 100%) in comparison to the water-sprayed controls (a 9% to 20% reduction following water spray). In the second experiment, the chemical 4NR4+BG (containing 4 quaternary ammoniums and a biguanide) at 200 mg/L was as effective as H2O2 at 1.4% in sanitizing hatching eggs for both Salmonella strains when the inoculum levels were 1 × 10 4 (0% positive) or 1 × 10 6 cfu/eggshell (3% positive). Following hatchability and chick quality trials for 4NR4+BG, this chemical should be considered for evaluation in commercial hatchery operations for the reduction of Salmonella on the shells of hatching eggs.

Avian life history traits influence eggshell bacterial loads: a comparative analysis

Selection pressures due to parasitism play an important role in driving the evolution of life history traits of birds in general and of behaviour at the nest in particular. Eggshell bacterial load has been shown to predict hatching failure (i.e. the probability of embryo infection) but the relationships between the bacterial environment of the nest and life history characteristics of birds remain poorly investigated. We explored interspecific variation in eggshell bacterial load of mesophilic bacteria,Enterococcusspp.,Staphylococcusspp. andEnterobacteriaceaegroups across 24 bird species and assessed whether bacterial load is associated with breeding traits. Interspecific variation was much higher than intraspecific variation for all measures of bacterial load even after controlling for annual variation. Thus, we were able to assess the correlation between bacterial community characteristics and life history traits. After correcting for phylogenetic effects, we found that nest type, the use of feathers or plants as lining material, and incubation behaviour explained a significant proportion of the variance in bacterial communities on eggshells. The strength of these associations depended on study year, suggesting an important role of environmental conditions for eggshell bacterial load or community. Overall, these results suggest that bacteria on eggshells are associated with bird species traits, probably because birds are mediating the deleterious effect of eggshell microbes through behavioural traits that modify bacterial load.

The evolution of size of the uropygial gland: mutualistic feather mites and uropygial secretion reduce bacterial loads of eggshells and hatching failures of European birds

Potentially, pathogenic bacteria are one of the main infective agents against which a battery of chemical and physical barriers has evolved in animals. Among these are the secretions by the exocrine uropygial gland in birds. The antimicrobial properties of uropygial secretions may prevent colonization and growth of microorganisms on feathers, skin and eggshells. However, uropygial gland secretions also favour the proliferation of feather mites that feed on secretions and microorganisms living on feathers that would otherwise reach eggshells during incubation if not consumed by feather mites. Therefore, at the interspecific level, uropygial gland size (as an index of volume of uropygial secretion) should be positively related to eggshell bacterial load (i.e. the risk of egg infection), whereas eggshell bacterial loads may be negatively related to abundance of feather mites eating bacteria. Here, we explore these previously untested predictions in a comparative framework using information on eggshell bacterial loads, uropygial gland size, diversity and abundance of feather mites and hatching success of 22 species of birds. The size of the uropygial gland was positively related to eggshell bacterial loads (mesophilic bacteria and Enterobacteriaceae), and bird species with higher diversity and abundance of feather mites harboured lower bacterial density on their eggshells (Enterococcus and Staphylococcus), in accordance with the hypothesis. Importantly, eggshell bacterial loads of mesophilic bacteria, Enterococcus and Enterobacteriaceae were negatively associated with hatching success, allowing us to interpret these interspecific relationships in a functional scenario, where both uropygial glands and mutualistic feather mites independently reduce the negative effects of pathogenic bacteria on avian fitness.

Early Onset of Incubation and Eggshell Bacterial Loads in a Temperate-Zone Cavity-Nesting Passerine

It has been hypothesized that incubation begins before laying is completed because it controls bacterial growth on eggshells and infection of eggs. If so, early incubation should be associated with decreased abundance of bacteria on eggshells. There is no detailed information on incubation during daytime or nighttime during laying and its association with bird eggshells' bacterial loads. We used temperature sensors placed in contact with eggs and connected to data-loggers to measure early incubation by females of the Pied Flycatcher, Ficedula hypoleuca, a hole-nesting passerine, beginning with laying of the third egg (day 3). Incubation usually began with laying of the fourth egg (day 4), the modal clutch size in our population being six. We sampled heterotrophic bacteria on eggshells from the whole clutch soon after it was completed. We used colony-forming units counted on trypticase soy agar to estimate bacterial loads. Clutches incubated more intensively during daytime on day 4 and also during nighttime on days 3–5 had fewer bacteria on eggshells than clutches incubated less. The temperature of the air around the eggs seemed not the key explanatory variable for the eggshell's bacterial loads, suggesting that mechanisms other than thermal changes associated with incubation may be more relevant to control of bacterial proliferation. Ours is the first study showing a correlation between increased incubation on the day incubation begins during laying and a reduction in bacteria on the eggshells of wild birds. It supports the hypothesis that early incubation is an antimicrobial defense of the clutch.

Drying eggs to inhibit bacteria: Incubation during laying in a cavity nesting passerine

Early incubation has been suggested as a defensive adaptation against potentially pathogenic bacteria colonizing avian eggshells in the wild. The inhibitory mechanisms underlying this adaptation are poorly understood and only recent experimental evidence demonstrates that keeping eggs dry is a proximate mechanism for the antimicrobial effects of avian incubation. We estimated partial incubation (the bouts of incubation that some birds perform during the egg-laying period, days of lay 3-5 in our population) intensity of female pied flycatchers breeding in nest-boxes using data loggers that allowed a precise measurement of temperature just between the eggs in the nest-cup. We also measured relative humidity within the nest-boxes and related it to incubation intensity, showing that more intense incubation during laying contributes to drying the air near the eggs. We analyzed separately the effects of incubation and of relative humidity on loads of three types of culturable bacteria known to be present on eggshells, heterotrophic bacteria, Gram-negative enterics and pseudomonads. Our results show an association of early incubation with an inhibition of bacterial proliferation through a drying effect on eggshells, as we found that incubation intensity was negatively and relative humidity positively associated with eggshell bacterial loads for heterotrophic bacteria, Gram-negative bacteria and pseudomonads, although the significance of these associations varied between bacterial groups. These results point to microclimatically driven effects of incubation on bacterial proliferation on eggshells during laying in a temperate cavity nesting passerine.

Innate humoural immunity is related to eggshell bacterial load of European birds: a comparative analysis

Fitness benefits associated with the development of a costly immune system would include not only self-protection against pathogenic microorganisms but also protection of host offspring if it reduces the probability and the rate of vertical transmission of microorganisms. This possibility predicts a negative relationship between probabilities of vertical transmission of symbionts and level of immune response that we here explore inter-specifically. We estimated eggshell bacterial loads by culturing heterotrophic bacteria, Enterococcus, Staphylococcus and Enterobacteriaceae on the eggshells of 29 species of birds as a proxy of vertical transmission of bacteria from mother to offspring. For this pool of species, we also estimated innate immune response (natural antibody and complement (lysis)) of adults, which constitute the main defence against bacterial infection. Multivariate general linear models revealed the predicted negative association between natural antibodies and density of bacteria on the eggshell of 19 species of birds for which we sampled the eggs in more than one nest. Univariate analyses revealed significant associations for heterotrophic bacteria and for Enterobacteriaceae, a group of bacteria that includes important pathogens of avian embryos. Therefore, these results suggest a possible trans-generational benefit of developing a strong immune system by reducing vertical transmission of pathogens.

Number and colour composition of nest lining feathers predict eggshell bacterial community in barn swallow nests: an experimental study

Summary 1. The use of feathers as nest lining material has traditionally been explained by the thermoregulatory properties of feathers. Feather nest lining could additionally affect nest detectability by predators, or play a role in a sexually selected context. Furthermore, feather nest lining harbours microorganisms that may influence environmental conditions where eggs and nestlings develop. 2. Microorganisms growing on nest lining feathers could affect the bacterial load of eggshells because they occupy space and/or produce antimicrobial substances against other bacteria, including egg pathogens. Feathers of different colours are known to differ in their bacterial community (i.e. feather degrading bacteria) and, thus, colour composition of nest lining feather could also affect the bacterial environment of avian nests. 3. Here we tested this hypothesis in the barn shallow (Hirundo rustica) by exploring the relationship between eggshell bacterial loads and number of feathers, and the effect of experimentally modified colour composition of nest lining feathers on eggshell bacterial load. 4. In agreement with the hypothesis we found that, before treatment, the number of nest lining feathers (mainly that of unpigmented‐white colour) predicted eggshell bacterial load, and that, at the end of the incubation period, eggshells of experimental nests with white feathers had a lower bacterial density than those in experimental nests with black feathers. 5. We failed to detect a relationship between bacterial load and hatching success. However, since evidence of that relationship exists for other species, these results would explain the previously detected experimental effect of colour composition of nest lining feathers on hatching success of swallows. 6. Nest design in general, and the use of nest‐lining white feathers in particular, may therefore have important consequences for reproductive success of birds. The reduced eggshell bacterial loads of experimental white nests would explain preferences by barn swallows for feathers of white colour.