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Essay Mom Knows Best: The Universality of Maternal Microbial Transmission Lisa J. Funkhouser1*, Seth R. Bordenstein1,2* 1Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America, 2Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee, United States of America Summary The sterile womb paradigm is an enduring premise in biology that human infants are born sterile. Recent studies suggest that infants incorporate an initial microbiome before birth and receive copious supplementation of maternal mi- crobes through birth and breastfeeding. Moreover, evidence for microbial mater- nal transmission is increasingly widespread across animals. This collective knowledge compels a paradigm shift—one in which maternal transmission of microbes ad- vances from a taxonomically specialized phenomenon to a universal one in ani- mals. It also engenders fresh views on the assembly of the microbiome, its role in animal evolution, and applications to human health and disease. Introduction While the human microbiota comprises only 1–3% of an individual’s total body mass, this small percentage represents over 100 trillion microbial cells, outnumbering human cells 10 to 1 and adding over 8 million genes to our set of 22,000 [1,2]. This complexity establishes a network of interactions between the host genome and microbiome spanning gut development , digestion [4,5], immune cell develop- ment [6–8], dental health [9,10], and resistance to pathogens [11,12]. Recent studies have also provided a greater understanding of how the composition of an individual’s microbiota changes throughout development, especially dur- ing the first year of life [3,13]. While the general dogma is that the placental barrier keeps infants sterile throughout pregnan- cy, increasing evidence suggests that an infant’s initial inoculum can be provided by its mother before birth [14–18] and is supplemented by maternal microbes through the birthing  and breastfeed- ing [20,21] processes. While maternal transmission of mi- crobes in humans has attracted consid- erable attention in the last few years, nearly a century’s worth of research is available for vertical transmission of symbionts in invertebrates . Similar to gut bacteria in humans that assist nutrient intake, many insect-associated bacteria function as nutritional symbi- onts that supplement the nutrient-poor diet of their host with essential vitamins or amino acids [23,24]. Since these indispensable symbionts cannot live out- side of host cells, they cannot be acquired from the environment and are faithfully transferred from mother to offspring [22,25]. Maternal transmission in inver- tebrates has been reviewed elsewhere [22,26,27], and Box 1 and Box 2 highlight examples of heritable symbio- ses across invertebrate phyla. By integrating previous studies in inverte- brates with recent evidence for maternal microbial transmission in humans and other vertebrates, we contend that maternal pro- visioning of microbes is a universal phenom- enon in the animal kingdom. As a result, a considerable new phase of study in heritable symbiont transmission is underway. Thus, this essay presents current evidence for maternal microbial transmission and pro- vides new insights into its impact on microbiome assembly and evolution, with applications to human health and disease. Internal Maternal Transmission At the turn of the twentieth century, French pediatrician Henry Tissier asserted that human infants develop within a sterile environment and acquire their initial bacterial inoculum while traveling through the maternal birth canal . More than a century later, the sterile womb hypothesis remains dogma, as any bacterial presence in the uterus is assumed to be dangerous for the infant. Indeed, studies of preterm deliveries have found a strong correlation between intrauterine infections and pre- term labor, especially when birth occurs less than 30 weeks into the pregnancy [29,30]. Since preterm birth is the leading cause of infant mortality worldwide , much attention has focused on identifying the bacterial culprits responsible for spon- taneous preterm labor. Surprisingly, most of the bacteria detected in intrauterine infections are commonly found in the female vaginal tract , and risk of preterm birth is markedly increased in women diagnosed with bacterial vaginosis during pregnancy . Interestingly, the vaginal microbial community varies signif- icantly among American women of differ- ent ethnicities (Caucasian, African-Ameri- can, Asian, or Hispanic), with African- American and Hispanic women more likely to have a microbiota traditionally associat- ed with bacterial vaginosis (predominance of anaerobic bacteria over Lactobacillus species)  and a higher rate of sponta- neous preterm deliveries (reviewed in ). While intrauterine infection and in- flammation is important in understanding the etiology of preterm birth, relatively few studies have examined the uterine microbiome of healthy, term pregnancies Essays articulate a specific perspective on a topic of broad interest to scientists. Citation: Funkhouser LJ, Bordenstein SR (2013) Mom Knows Best: The Universality of Maternal Microbial Transmission. PLoS Biol 11(8): e1001631. doi:10.1371/journal.pbio.1001631 Published August 20, 2013 Copyright: 2013 Funkhouser, Bordenstein. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported directly by an NSF Graduate Research Fellowship to LJF and NSF award DEB-1046419 and NIH award GM085163 to SRB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: FISH, fluorescent in situ hybridization; GI, gastrointestinal tract; TEM, transmission electron microscopy. * E-mail: firstname.lastname@example.org (LJF); email@example.com (SRB) PLOS Biology | www.plosbiology.org 1 August 2013 | Volume 11 | Issue 8 | e1001631 owing to the sterile womb paradigm. Investigations into the potential for bac- terial transmission through the placental barrier have detected bacteria in umbili- cal cord blood , amniotic fluid [14,35], and fetal membranes [35,36] from babies without any indication of inflammation (Figure 1). Furthermore, an infant’s first postpartum bowel movement of ingested amniotic fluid (meconium) is not sterile as previously assumed, but instead harbors a complex community of microbes, albeit less diverse than that of adults [18,37]. Interestingly, many of the bacterial genera found in the meconium, including Enterococcus and Escherichia, are common inhabitants of the gastrointesti- nal tract [18,37]. To test whether mater- nal gut bacteria can be provisioned to fetuses in utero, Jime ´nez et al.  fed pregnant mice milk inoculated with genetically-labeled Enterococcus faecium and then examined the meconium microbes of term offspring after sterile C-section. Remarkably, E. faecium with the genetic label was cultured from the meconium of pups from inoculated mothers, but not from pups of control mice fed noninocu- lated milk. Meconium from the treatment group also had a higher abundance of bacteria than that of the control group. Importantly, the study controlled for potential bacterial contamination from contact between skin and the meconium by sampling an internal portion of the meconium . Thus, this study provides foundational evidence for maternal mi- crobial transmission in mammals. Other than ascension of vaginal mi- crobes associated with preterm births, the mechanisms by which gut bacteria gain access to the uterine environment are not well understood. One possibility is that bacteria travel to the placenta via the bloodstream after translocation of the gut epithelium. While the intestinal epithelial barrier generally prevents microbial entry into the circulatory system, dendritic cells can actively penetrate the gut epithelium, take up bacteria from the intestinal lumen, and transport the live bacteria throughout the body as they migrate to lymphoid organs [38,39]. Interestingly, microbial translocation may even increase during pregnancy, as one study showed that pregnant mice were 60% more likely to harbor bacteria in their mesenteric lymph node (presumably brought there by den- dritic cells) than nonpregnant mice . Bacterial species normally found in the human oral cavity have also been isolated from amniotic fluid and likely enter the bloodstream during periodontal infections, facilitated by gingiva inflammation [14,16] (Figure 1). Overall, the study of internal maternal transmission of microbes in mammals is in its infancy due to the enduring influence of the sterile womb paradigm and to the ethical and technical difficulties of collect- ing samples from healthy pregnancies before birth. Thus, we still know very little about the number and identity of innocu- ous microbes that traverse the placenta, whether they persist in the infant, or whether their presence has long-term health consequences for the child. Similar- ly, we know almost nothing about non- pathogenic viruses or archaea that may be transferred from mother to child alongside their bacterial counterparts. Fortunately, the advent of culture-independent, high- throughput sequencing will serve as a tremendous resource for this field and will hopefully lead to a characterization of the ‘‘fetal microbiome’’ in utero. Maternal provisioning of microbes to developing offspring is widespread in animals, with evidence of internal micro- bial transmission in animal phyla as diverse as Porifera [41–45] (Box 1), Mollusca [46–49] (Box 1), Arthropoda [50–52] (Box 2, Figure 2), and Chordata [19,53,54] (Box 3, Figure 2). The presence of maternal transmission at the base of the Animalia kingdom and the surprising plasticity by which microbes gain access to germ cells or embryos in these systems signifies that maternal symbiont transmis- sion is an ancient and evolutionarily advantageous mechanism inherent in an- imals, including humans. Therefore, we can no longer ignore the fact that exposure to microbes in the womb is likely and may even be a universal part of human pregnancy, serving as the first inoculation of beneficial microbes before birth. External Maternal Transmission External maternal transmission encom- passes any transfer of maternal symbionts to offspring during or after birth. In invertebrates, it is often accomplished by ‘‘egg smearing,’’ in which females coat eggs with microbes as they are deposited , or through the provision of a microbe-rich maternal fecal pellet that is Box 1. Examples of Maternal Transmission in Marine Invertebrates Marine Sponges (Phylum Porifera) Sponges are ancient metazoans that evolved over 600 million years ago as one of the first multicellular animals . In marine sponges, a remarkably large consortium of extracellular microbial symbionts thrives within the sponge’s mesohyl, a gelatinous connective tissue located between the external and internal cell layers. Many of these bacterial residents are found in diverse species of sponges with nonoverlapping distributions but not in the surrounding seawater [84–86]. These ‘‘sponge- specific’’ microbes are hypothesized to have originated from ancient colonization events before the diversification of marine sponges and are maintained as symbionts through vertical transmission . Independent studies have estimated that up to 33 phylogenetically distinct microbial clusters spanning ten bacterial phyla and one archaeal phylum are vertically transmitted in sponges [41,84,86,88]. Both transmission electron microscopy (TEM) and fluorescent in situ hybridization (FISH) studies have confirmed the presence of microorganisms of different shapes and sizes in the oocytes of oviparous sponges  and in the embryos of viviparous sponges [43–45]. Vesicomyid Clams (Phylum Mollusca) Deep-sea hydrothermal vent communities rely upon chemosynthetic bacteria to harness chemical energy stored in reduced sulfur compounds extruding from the vents. Metazoans that live in this extreme environment harbor chemosynthetic endosymbionts in their tissues that provide most, if not all, of the host’s nutrition . Somewhat surprisingly, most invertebrates that live near hydrothermal events acquire their endosymbionts anew from the environment each generation [90,91], even though chemosynthetic bacteria are crucial for survival in such a harsh habitat. A major exception to this trend is found in the Vesicomyidae family of clams . Vesicomyid clams retain a rudimentary gut and rely primarily on sulfur-oxidizing bacteria sequestered intracellularly within specialized host cells called bacteriocytes in the clam’s large, fleshy gills . Vertical transmission via transovarial transmission appears to be the dominant mechanism for maintenance of these thioautotrophic bacterial symbionts given that follicle cells surrounding an oocyte and the oocyte itself are heavily infected with the chemosynthetic bacteria [46,94]. PLOS Biology | www.plosbiology.org 2 August 2013 | Volume 11 | Issue 8 | e1001631 Box 2. Examples of Maternal Transmission in Terrestrial Invertebrates Insects (Phylum Arthropoda) Insects that thrive on unbalanced diets such as plant sap, blood, or wood depend upon microbial symbionts for the provision of essential amino acids or vitamins lacking in their food source. In turn, hosts provide a wide range of metabolites to their symbionts as well as protection from environmental stressors. This codependence requires faithful transfer of symbionts to all offspring, usually through transovarial transmission [23,24]. Reproductive parasites, such as the obligate, intracellular bacteria Wolbachia, are also widespread in insects and hijack maternal transmission routes to ensure their spread within an insect population (reviewed in [95,96]). Pea Aphid (Acrythosiphon pisum) The pea aphid Acrythosiphon pisum (Figure 2A) and its nutritional endosymbiont Buchnera aphidicola are a preeminent example of obligate mutualism in insects. The ancestral Buchnera gammaproteobacteria was acquired by aphids between 160 and 280 million years ago  and has since diverged in parallel with its aphid hosts through strict vertical transmission [26,97]. Buchnera are housed within the cytoplasm of bacteriocytes arranged into dual bacteriome structures located in the aphid body cavity adjacent to the ovaries , allowing efficient transfer of Buchnera symbionts to developing oocytes or embryos during the sexual and asexual phases of aphid reproduction, respectively. At the cellular level, symbiont transfer occurs when maternal bacteriocytes release Buchnera symbionts through exocytosis into the extracellular space between the bacteriocyte and oocyte or embryo, which then actively endocytoses the extracellular Buchnera symbionts . Cockroaches (Order Blattodea) Just as insects are morphologically diverse, the mechanisms by which insects transport symbionts to oocytes are highly varied. In cockroaches, Blattabacterium-filled bacteriocyte cells migrate from the abdominal fat body to the distantly located ovarioles where they adhere to the oocyte membrane [99,100]. Interestingly, the bacteriocytes remain associated with the oocyte for eight to nine days before finally expelling their symbionts through exocytosis. The Blattabacterium cells then squeeze between the follicle cells surrounding the oocyte and are engulfed into the oocyte cytoplasm via endocytosis just prior to ovulation . Whiteflies (Family Aleyrodidae) The whitefly circumvents exocytosis of its intracellular nutritional symbiont, Portiera aleyrodidarum, by depositing entire bacteriocytes into its eggs. These maternal bacteriocytes remain intact yet separate from the developing embryo until the embryonic bacteriomes form, at which point the maternal bacteriocytes deteriorate . Tsetse Flies, Bat Flies, and Louse Flies (Superfamily Hippoboscoidea) Members of the Hippoboscoidea superfamily (Order Diptera) are obligate blood feeders that have developed a unique reproductive strategy termed adenotrophic viviparity that offers a different solution to internal maternal transfer of symbionts. Females of this superfamily develop a single fertilized embryo at a time within their uterus (modified vaginal canal) until it is deposited as a mature third instar larva immediately preceding pupation. During their internal development, the larvae are nourished with milk produced by modified accessory glands that empty into the uterus . The milk primarily consists of protein and lipids , but it also serves as a reservoir for maternally transmitted microbial symbionts . For example, the obligate mutualistic symbiont of tsetse flies, Wigglesworthia glossinidia, is absent from the female germ line and surrounding reproductive tissues but is found extracellularly in the female milk glands and is first detected in tsetse offspring once milk consumption begins during the first larval stage . Stinkbugs (Superfamily Pentatomoidea) One of the most common mechanisms of external maternal transmission in insects is that of ‘‘egg smearing,’’ which occurs when a female contaminates the surface of her eggs with symbiont-laden feces during oviposition. Upon hatching, offspring probe or consume the discarded egg shells to acquire the maternal bacteria. This mode of transmission is commonly found in plant-sucking stinkbugs, including the Pentatomidae and Acanthosomatidae families . In the Cynidae family of stinkbugs, along with the Coreidae family of leaf-footed bugs, gut symbionts are transferred maternally via coprophagy, in which offspring consume maternal feces, sometimes directly from the mother’s anus [22,104]. Stinkbugs of the Plataspidae family, on the other hand, have developed a unique mode of transmission via a maternally provided ‘‘symbiont capsule’’ deposited on the underside of the egg mass . These capsules are comprised of bacterial cells dispersed throughout a resin-like matrix surrounded by a brown, cuticle-like envelope that protects the symbionts from environmental stressors such as UV irradiation or dissection . After hatching, plataspid nymphs immediately probe the capsules to ingest the symbionts [56,59]. European Beewolf (Philanthus triangulum) While nutritional symbionts appear to be the most common type of bacteria transmitted via external maternal transmission in insects, the European beewolf (Philanthus triangulum) instead cultivates a symbiotic bacteria that protects offspring against microbial infection during development. Beewolves are solitary digger wasps that deposit their offspring in moist, underground nests, making them susceptible to fungal and bacterial infections . To combat these pathogens, female beewolves cultivate Streptomyces philanthi bacteria in specialized glands in their antennae, which they copiously spread on the ceiling of the brood cell before oviposition [106–108]. After hatching, the larvae take up the bacterial cells and incorporate them into their cocoon that they build before pupation. When adult beewolves emerge from their cocoon in the summer, female beewolves acquire the maternally provided Streptomyces symbiont and house them in the female-specific gland reservoirs along each antenna [108,109]. PLOS Biology | www.plosbiology.org 3 August 2013 | Volume 11 | Issue 8 | e1001631 consumed by larval offspring upon hatch- ing [56–59] (see Box 2). Similarly, human infants are ‘‘smeared’’ with maternal vaginal and fecal microbes as they exit the birth canal [60–62] (Figure 1). Several studies have shown that the human neonatal microbiota across all body hab- itats (skin, oral, nasopharyngeal, and gut) is influenced by their mode of delivery [19,63–65], with infants born vaginally acquiring microbes common in the female vagina while C-section infants display a microbiota more similar to that of human skin . Furthermore, while the micro- biota of a vaginally delivered infant clusters with the vaginal bacteria of its mother, the microbiota of C-section babies is no more related to the skin flora of its mother than that of a stranger, indicating that most microbes are transmitted to the neonate from those handling the infant . Importantly, epidemiological data suggest that a Cesarean delivery can have long-term consequences on the health of a child, especially concerning immune-me- diated diseases. For example, children born via C-section are significantly more likely to develop allergic rhinitis , asthma , celiac disease , type 1 diabetes , and inflammatory bowel disease . These statistics are alarming given that 32.8% of all births in the United States in 2010 were delivered via C-section with similar rates on the rise in most developed countries . The higher rate of immune-mediated diseases in C-section children may indicate that maternally transferred vaginal or fecal microbes are unique in their ability to elicit immune maturation in the neonate. Development of the intestinal mucosa and secondary lymphoid tissues in the gut is contingent upon recognition of microbial components by pattern-recognition recep- tors on intestinal epithelial cells (reviewed in [71,72]). It is possible that these receptors cannot properly interact with the community of microbes acquired during Cesarean deliveries, leading to disrupted immune development and an increased risk for immune-mediated disor- ders in C-section children. Conversely, transmission for thousands of years of vaginal and fecal microbes at birth has likely produced specific human-microbe interactions important for neonatal gut development. In fact, a recent study found that the vaginal microbial community changes during pregnancy, becoming less diverse as the pregnancy progresses ; Figure 1. Sources of microbial transmission in humans from mother to child. Cut-away diagram highlighting the various internal and external sources of maternal microbial transmission as well as the species that are commonly associated with transfer from those regions. Regions discussed include the oral cavity [14,16], the mammary glands [40,78,79], the sebaceous skin surrounding the breast [78,80], the vaginal tract [19,33,73], and the intrauterine environment [14,15,17,18,29,35–37,40]. Illustration by Robert M. Brucker. doi:10.1371/journal.pbio.1001631.g001 PLOS Biology | www.plosbiology.org 4 August 2013 | Volume 11 | Issue 8 | e1001631 yet, in spite of the general decrease in richness, certain Lactobacillus bacterial spe- cies are enriched in the vaginal communi- ty during pregnancy and are hypothesized to be important for establishing the neonatal upper GI microbiota after vagi- nal delivery . Breastfeeding provides a secondary route of maternal microbial transmission as shown in humans (reviewed in , Figure 1) and nonhuman primates such as rhesus monkeys . In humans, maternal milk microbes are implicated in infant immune system development , resis- tance against infection , and protec- tion against the development of allergies and asthma later in childhood . High- throughput sequencing of breast milk from 16 healthy women identified 100–600 species of bacteria in each sample with nine genera present in every sample: Staphylococcus, Streptococcus, Serratia, Pseudo- monas, Corynebacterium, Ralstonia, Propionibac- terium, Sphingomonas, and Bradyrhizobiaceae . This ‘‘core’’ milk microbiome repre- sented approximately 50% of all bacteria in each sample, with the other half representing individual variation in micro- bial composition . A similar study found that the bacterial composition in breast milk changes over time: milk produced immediately after labor har- bored more lactic acid bacteria along with Staphylococcus, Streptococcus, and Lactococcus, while breast milk after six months of lactation had a significant increase in typical inhabitants of the oral cavity, such as Veillonella, Leptotrichia, and Prevotella , perhaps to prime the infant for the switch to solid food. However, as with any DNA- based, culture-independent study that does not discriminate between live and dead bacteria, the number and identity of bacteria detected in these studies should be interpreted with some caution. Given that milk is only produced temporarily in a woman’s life, the origin of milk microbes is still somewhat of a mystery. Breast milk was traditionally thought to be sterile; however, colostrum (the first milk produced after delivery) collected aseptically already harbors hun- dreds of bacterial species . Breast milk does share many taxa with the microbiota found on sebaceous skin tissue around the nipple [78,80], and high levels of Strepto- coccus in breast milk may be a result of retrograde flow from an infant’s oral cavity back to the milk ducts during suckling  since Streptococcus is the dominant phylo- type in infant saliva . However, the presence of anaerobic gut bacteria in human milk suggests that an entero- mammary route of transfer also exists that may utilize phagocytic dendritic cells to traffic gut microbes to the mammary glands, similar to microbial transfer to amniotic fluid as discussed earlier. To support this hypothesis, Perez et al.  found identical strains of bacteria in milk cells, blood cells, and fecal samples from lactating women, but more work is needed Figure 2. Examples of animals that exhibit microbial maternal transmission. (A) Pea aphid (Acyrthosiphon pisum), photo credit: Whitney Cranshaw, Colorado State University/Bugwood.org/CC-BY-3.0-US; (B) Domesticated chicken hen (Gallus gallus domesticus), photo credit: Ben Scicluna; (C) Sockeye salmon (Oncorhynchus nerka), photo credit: Cacophony; (D) South American river turtle (Podocnemis expansa), photo credit: Wilfredor. All photos were obtained from Wikimedia Commons (www.commons.wikimedia.org). doi:10.1371/journal.pbio.1001631.g002 PLOS Biology | www.plosbiology.org 5 August 2013 | Volume 11 | Issue 8 | e1001631 to directly connect bacterial translocation in the gut to incorporation in breast milk. Overall, maternal transmission of ben- eficial microbes in humans has widespread relevance for human health. Evolution with these microbes has resulted in our dependence on them for the proper maturation and development of the immune system and gastrointestinal tract. Somewhat paradoxically, modern medicine designed to prevent infant mor- tality (such as emergency Cesarean sec- tions and formula feeding) has likely contributed to the rise in immune- mediated diseases in developed countries due to the inherent lack of exposure to maternal microbes associated with these practices. Fortunately, biomedicine is also making strides in finding effective probio- tic supplements to promote immune development and ameliorate some of the risks that C-section or formula-fed infants face as children and adults. Hopefully, as we gain understanding of the diversity and function of maternally transmitted mi- crobes in humans, more complete and effective probiotic blends will recapitulate the microbial communities found in vag- inally delivered, breast-fed infants and restore the microbe-host interactions that humans depend upon for proper develop- ment. Conclusions Since the early twentieth century, the study of maternal microbial transmission has focused heavily on animal systems in which maternal transmission maintains sophisticated partnerships with one or two microbial species. However, with the development of high-throughput se- quencing technologies, it is now possible to identify entire microbiomes that are transferred from mother to offspring in systems not traditionally considered to exhibit maternal transmission, such as humans. By expanding the definition of maternal transmission to include all internal and external microbial transfers from mother to offspring, we contend that maternal transmission is universal in the animal kingdom and is used to provision offspring with important mi- crobes at birth, rather than leave their acquisition to chance. Finally, with microbes contributing 99% of all unique genetic information present in the human body, maternal microbial transmission should be viewed as an additional and important mecha- nism of genetic and functional change in human evolution. Similar to deleterious mutations in our genetic code, disruption of maternal microbial acquisition during infancy could ‘‘mutate’’ the composition of the microbial community, leading to improper and detrimental host-microbe interactions during development. Mater- nal transmission is also a key factor in shaping the structure of the microbiome in animal species over evolutionary time, since microbes that promote host fitness, especially in females, will simultaneously increase their odds of being transferred to the next generation. Thus, whether inter- nal or external, the universality and implications of maternal microbial trans- mission are nothing short of a paradigm shift for the basic and biomedical life sciences. Box 3. Examples of Maternal Transmission in Vertebrates Aside from studies in human and mouse models, very little is known about maternal transmission of microbial communities in vertebrates, especially outside Class Mammalia. Furthermore, research on vertical transmission in nonmamma- lians has largely focused on maternally transmitted pathogens, especially in animals of agricultural importance like chickens and fish. Domesticated Chickens ( G a l l u s g a l l u s d o m e s t i c u s ) Zoonotic Salmonella infections acquired from contaminated chicken eggs is estimated to cause more than 100,000 illnesses each year in the United States . In addition to horizontal transmission of Salmonella on eggs through surface contamination, direct transovarial transmission also occurs when Salmonella colonizes the reproductive tissues of hens (Figure 2B). Depending on the infection location within the female reproductive tract, the bacteria are deposited into the yolk, albumen, eggshell membrane, and/or eggshell of the developing egg before oviposition (reviewed in ). Other poultry pathogens, such as Mycoplasma synoviae in chickens  and M. gallisepticum, M. cloacale, and M. anatis in ducks , have also been cultured from the yolk of embryonated eggs, though whether commensal flora are incorporated into the egg is not known. Ray-Finned Fish (Class Actinopterygii) Several bacterial pathogens of economically important fish are transmitted transovarially in the egg yolk including Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids (Figure 2C), and Flavobacterium psychrophilum, which causes bacterial cold water disease in salmonids and rainbow trout fry disease in trout (reviewed in ). F. psychrophilum has also been found in ovarian fluid and on the surface of eggs of steelhead trout . Additionally, an obligate, intracellular eukaryotic parasite, Pseudoloma neurophilia, is a common pathogen found in zebrafish (Danio rerio) facilities and has been observed in spores of the ovarian stroma and within developing follicle cells of spawning females, suggesting that it can be vertically transmitted, though it is primarily spread from fish to fish in contaminated water (reviewed in ). Turtles (Order Chelonii) The formation of egg components in the uterine tube and uterus of turtles takes approximately two weeks, providing ample opportunity for maternal transmission of intestinal or reproductive microbes to the egg . One study of unhatched (dead) eggs from loggerhead sea turtle (Caretta caretta) nests found several potential pathogens, including Pseudomonas aeruginosa and Serratia marcesans, in fluid from the interior of the eggs, though environmental contamination of the eggs cannot be ruled out . A similar study of eggs from two species of South American river turtles, Podocnemis expansa (Figure 2D) and P. unifilis, identified several Enterobacteriaceae species, including Escherichia coli, Shigella flexneri, and Salmonella cholerasuis, in the eggs but not in the environmental samples taken from the turtle nests , suggesting that they may have a maternal origin. In support of this hypothesis, a separate study in green turtles (Chelonia mydas) that collected eggs directly from the maternal cloacal opening during egg laying isolated Pseudomonas, Salmonella, Enterobacter, and Citrobacter from the eggshell, albumen, and yolk. In fact, the yolk was the egg component most heavily infected with bacteria . Altogether, many potentially pathogenic species have been isolated from turtle eggs, but whether these bacteria actually cause disease in turtles or are part of their natural flora remains to be determined. 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