Effect of incubation on bacterial communities of eggshells in a temperate bird, the Eurasian Magpie (Pica pica).
ABSTRACT: Inhibitory effect of incubation on microbial growth has extensively been studied in wild bird populations using culture-based methods and conflicting results exist on whether incubation selectively affects the growth of microbes on the egg surface. In this study, we employed culture-independent methods, quantitative PCR and 16S rRNA gene pyrosequencing, to elucidate the effect of incubation on the bacterial abundance and bacterial community composition on the eggshells of the Eurasian Magpie (Pica pica). We found that total bacterial abundance increased and diversity decreased on incubated eggs while there were no changes on non-incubated eggs. Interestingly, Gram-positive Bacillus, which include mostly harmless species, became dominant and genus Pseudomonas, which include opportunistic avian egg pathogens, were significantly reduced after incubation. These results suggest that avian incubation in temperate regions may promote the growth of harmless (or benevolent) bacteria and suppress the growth of pathogenic bacterial taxa and consequently reduce the diversity of microbes on the egg surface. We hypothesize that this may occur due to difference in sensitivity to dehydration on the egg surface among microbes, combined with the introduction of Bacillus from bird feathers and due to the presence of antibiotics that certain bacteria produce.
Project description:Urban environments present novel and challenging habitats to wildlife. In addition to well-known difference in abiotic factors between rural and urban environments, the biotic environment, including microbial fauna, may also differ significantly. In this study, we aimed to compare the change in microbial abundance on eggshells during incubation between urban and rural populations of a passerine bird, the Eurasian Magpie (Pica pica), and examine the consequences of any differences in microbial abundances in terms of hatching success and nestling survival. Using real-time PCR, we quantified the abundances of total bacteria, Escherichia coli/Shigella spp., surfactin-producing Bacillus spp. and Candida albicans on the eggshells of magpies. We found that urban magpie eggs harboured greater abundances of E. coli/Shigella spp. and C. albicans before incubation than rural magpie eggs. During incubation, there was an increase in the total bacterial load, but a decrease in C. albicans on urban eggs relative to rural eggs. Rural eggs showed a greater increase in E. coli/Shigella spp. relative to their urban counterpart. Hatching success of the brood was generally lower in urban than rural population. Nestling survival was differentially related with the eggshell microbial abundance between urban and rural populations, which was speculated to be the result of the difference in the strength of the interaction among the microbes. This is the first demonstration that avian clutches in urban and rural populations differ in eggshell microbial abundance, which can be further related to the difference in hatching success and nestling survival in these two types of environments. We suggest that future studies on the eggshell microbes should investigate the interaction among the microbes, because the incubation and/or environmental factors such as urbanization or climate condition can influence the dynamic interactions among the microbes on the eggshells which can further determine the breeding success of the parents.
Project description:Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components--pH, lysozyme and ovotransferrin--during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon's ?-diversity index increases during early incubation stages, and ?-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment.
Project description:The common cuckoo (Cuculus canorus) is an avian brood parasite, laying its eggs in the nests of other bird species, where these hosts incubate the parasitic eggs, feed and rear the nestlings. The appearance of a cuckoo egg in a host nest may change the bacterial community in the nest. This may have consequences on the hatchability of host eggs, even when hosts reject the parasitic egg, typically within six days after parasitism. The present study revealed the bacterial community of cuckoo eggshells and those of the great reed warbler (Acrocephalus arundinaceus), one of the main hosts of cuckoos. We compared host eggs from non-parasitized clutches, as well as host and cuckoo eggs from parasitized clutches. As incubation may change bacterial assemblages on eggshells, we compared these egg types in two stages: the egg-laying stage, when incubation has not been started, and the mid-incubation stage (ca. on days 5-7 in incubation), where heat from the incubating female dries eggshells. Our results obtained by the 16S rRNA gene sequencing technique showed that fresh host and cuckoo eggs had partially different bacterial communities, but they became more similar during incubation in parasitized nests. Cluster analysis revealed that fresh cuckoo eggs and incubated host eggs in unparasitized nests (where no cuckoo effect could have happened) were the most dissimilar from the other groups of eggs. Cuckoo eggs did not reduce the hatchability of great reed warbler eggs. Our results on the cuckoo-great reed warbler relationship supported the idea that brood parasites may change bacterial microbiota in the host nest. Further studies should reveal how bacterial communities of cuckoo eggshells may vary by host-specific races (gentes) of cuckoos.
Project description:CUMYL-PICA [1-pentyl-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide] and 5F-CUMYL-PICA [1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide] are recently identified recreationally used/abused synthetic cannabinoids, but have uncharacterized pharmacokinetic profiles and metabolic processes. This study characterized clearance and metabolism of these compounds by human and rat liver microsomes and hepatocytes, and then compared these parameters with in vivo rat plasma and urine sampling. It also evaluated hypothermia, a characteristic cannabimimetic effect. Incubation of CUMYL-PICA and 5F-CUMYL-PICA with rat and human liver microsomes suggested rapid metabolic clearance, but in vivo metabolism was prolonged, such that parent compounds remained detectable in rat plasma 24 h post-dosing. At 3 mg/kg (intraperitoneally), both compounds produced moderate hypothermic effects. Twenty-eight metabolites were tentatively identified for CUMYL-PICA and, coincidentally, 28 metabolites for 5F-CUMYL-PICA, primarily consisting of phase I oxidative transformations and phase II glucuronidation. The primary metabolic pathways for both compounds resulted in the formation of identical metabolites following terminal hydroxylation or dealkylation of the N-pentyl chain for CUMYL-PICA or of the 5-fluoropentyl chain for 5F-CUMYL-PICA. These data provide evidence that in vivo elimination of CUMYL-PICA, 5F-CUMYL-PICA and other synthetic cannabinoids is delayed compared to in vitro modeling, possibly due to sequestration into adipose tissue. Additionally, the present data underscore the need for careful selection of metabolites as analytical targets to distinguish between closely related synthetic cannabinoids in forensic settings.
Project description:Oviparous animals have evolved multiple defenses to prevent microbes from penetrating their eggs and causing embryo mortality. In birds, egg constituents such as lysozyme and antibodies defend against microbial infestation, but eggshell pigments might also impact survival of bacteria. If so, microbes could exert an important selective pressure on the evolution of eggshell coloration. In a previous lab experiment, eggshell protoporphyrin caused drastic mortality in cultures of Gram positive, but not Gram negative, bacteria when exposed to light. Here, we test this "photodynamic antimicrobial hypothesis" in a field experiment. In a paired experimental design, we placed sanitized brown, protoporphyrin-rich chicken eggs alongside white eggs that lack protoporphyrin. We deployed eggs for 48 hr without incubation, as can occur between laying and incubation, when microbial infection risk is highest. Eggs were placed on the open ground exposed to sunlight and in dark underground storm-petrel burrows. We predicted that the proportion of Gram-positive bacteria on brown eggs should be lower when exposed to sunlight than when kept in the dark, but we expected no such difference for white eggs. Although our data revealed variation in bacterial community composition, the proportion of Gram-positive bacteria on eggshells did not vary by egg color, and there was no interaction between egg color and location. Instead, Gram-positive bacteria were proportionally more common on eggs on the ground than eggs in burrows. Overall, our experiment did not support the photodynamic antimicrobial hypothesis. The diverse range of avian egg colors is generated by just two pigments, but over 10 hypotheses have been proposed for the evolution of eggshell color. If our results are generalizable, eggshell protoporphyrin might not play a substantial role in defending eggs against microbes, which narrows the field of candidate hypotheses for the evolution of avian eggshell coloration.
Project description:Incubation starts during egg laying for many bird species and causes developmental asynchrony within clutches. Faster development of late-laid eggs can help reduce developmental differences and synchronize hatching, which is important for precocial species whose young must leave the nest soon after hatching. In this study, we examined the effect of egg laying sequence on length of the incubation period in Wood Ducks (Aix sponsa). Because incubation temperature strongly influences embryonic development rates, we tested the interactive effects of laying sequence and incubation temperature on the ability of late-laid eggs to accelerate development and synchronize hatching. We also examined the potential cost of faster development on duckling body condition. Fresh eggs were collected and incubated at three biologically relevant temperatures (Low: 34.9°C, Medium: 35.8°C, and High: 37.6°C), and egg laying sequences from 1 to 12 were used. Length of the incubation period declined linearly as laying sequence advanced, but the relationship was strongest at medium temperatures followed by low temperatures and high temperatures. There was little support for including fresh egg mass in models of incubation period. Estimated differences in length of the incubation period between eggs 1 and 12 were 2.7 d, 1.2 d, and 0.7 d at medium, low and high temperatures, respectively. Only at intermediate incubation temperatures did development rates of late-laid eggs increase sufficiently to completely compensate for natural levels of developmental asynchrony that have been reported in Wood Duck clutches at the start of full incubation. Body condition of ducklings was strongly affected by fresh egg mass and incubation temperature but declined only slightly as laying sequence progressed. Our findings show that laying sequence and incubation temperature play important roles in helping to shape embryo development and hatching synchrony in a precocial bird.
Project description:The mosquito-borne West Nile virus (WNV) is a highly neurovirulent Flavivirus currently representing an emergent zoonotic concern. WNV cycles in nature between mosquito vectors and birds that act as amplifier hosts and play an essential role in virus ecology, being, thus, WNV a threat to many species. Availability of an efficient avian vaccine would benefit certain avian populations, both birds grown for hunting and restocking activities, as well as endangered species in captive breeding projects, wildlife reservations, and recreation installations, and would be useful to prevent and contain outbreaks. Avian vaccination would be also of interest to limit WNV spillover to humans or horses from susceptible bird species that live in urbanized landscapes, like magpies. Herein, we have addressed the efficacy of a single dose of a WNV recombinant subviral particle (RSP) vaccine in susceptible magpie (Pica pica). The protective capacity of the RSP-based vaccine was demonstrated upon challenge of magpies with 5 × 103 plaque forming units of a neurovirulent WNV strain. A significant improvement in survival rates of immunized birds was recorded when compared to vehicle-inoculated animals (71.4 vs. 22.2%, respectively). Viremia, which is directly related to the capacity of a host to be competent for virus transmission, was reduced in vaccinated animals, as was the presence of infectious virus in feather follicles. Bird-to-bird transmission was recorded in three of six unchallenged (contact) magpies housed with non-vaccinated WNV-infected birds, but not in contact animals housed with vaccinated WNV-infected magpies. These results demonstrate the protective efficacy of the RSP-based vaccine in susceptible birds against WNV infection and its value in controlling the spread of the virus.
Project description:Avian reoviruses (ARVs) cause a range of disease presentations in domestic, captive and free-living bird species. ARVs have been reported as a cause of significant disease and mortality in free-living corvid species in North America and continental Europe. Until this report, there have been no confirmed cases of ARV-associated disease in British wild birds.Sporadic individual magpie (Pica pica) mortality was detected at a single site in Buckinghamshire, England, April-September 2013. An adult female magpie was found moribund and subsequently died. Post-mortem examination identified hepatomegaly and splenomegaly as the most severe macroscopic abnormalities. Histopathological examination revealed extensive hepatic and splenic necrosis. Transmission electron microscopy (TEM) identified virions of a size (circa 78 nm diameter) and morphology consistent with ARV in both the liver and the small intestinal (SI) contents. Nucleic acid extracted from pooled liver and spleen was positive on both a pan-reovirus nested PCR targeting the RNA-dependent RNA polymerase gene and a PCR using primers specific to the ARV sigma C protein gene. Virus isolated from the liver and the SI contents was characterised by a syncytial-type cytopathic effect, a reovirus-like appearance on TEM and sequence identical to that from PCR of tissues. In situ hybridisation confirmed co-localisation of ARV with lesions in the liver and spleen, implicating ARV as the causative agent. Splenic lymphoid atrophy and necrotic stomatitis associated with Aspergillus fumigatus infection were consistent with generalised immunosuppression and resultant opportunistic infection.The pathology and comprehensive virus investigations in this case indicate ARV as the primary pathogen in this magpie, with concurrent secondary infection subsequent to immunosuppression, as has been observed with reoviral infections in other bird species. ARV should be considered as a differential diagnosis for magpie, and potentially other corvid, disease and mortality incidents. This is the first demonstration of ARV-associated mortality in a wild bird in Britain. The prevalence and significance of ARV infection in British wild birds, and its implications for poultry and captive bird health, are currently unknown.