Limited vertical transmission of gut bacteria in commercial chickens

doi:10.21203/rs.3.rs-2132073/v1

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Limited vertical transmission of gut bacteria in commercial chickens

https://doi.org/10.21203/rs.3.rs-2132073/v1

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Background: Efficient vertical transmission of commensal gut microbes is important for the host in order to promote gut microbiota functions such as protection from gut pathogens, and for gut microbes considering microbial competition over limited niche space. While the role of direct contact with parents in transmission is well established, conflicting reports exist regarding transmission in commercial settings where chicks are raised separated from adults, including the utilization of the chicken egg as a transmission mechanism. This question is also relevant to other vertebrates which leave their eggs after laying. Results: We compared the fecal microbiota of poultry chicks hatched and grown separately, with the microbiota of their mothers over three rounds of egg incubation and chick growth. We found that most bacterial strains identified in the hens did not appear at all in chicks up to two weeks of age. Furthermore, most of the strains common with the hens which did appear in chicks had a low incidence among the chicks. Thus, the gut microbiota of hens does not efficiently transmit to chicks when there is no contact with adults. That been said, a few bacterial strains common with the hens were good colonizers of chicks; these included members of Lactobacillales and Enterobacteriales. Finally, we performed two interventions in an attempt to disrupt transmission. In round two, we sprayed a disinfectant on half of the eggs. In round three, we treated half of the hens with an antibiotic cocktail which decimated their cecum and fecal microbiota. Both interventions resulted in a reduction in chick colonization. Interestingly, both interventions affected strains shared with the hens as well as strains not identified in hen samples, implying many ‘environmental’ opportunistic strains reach the chicks through the egg. Conclusions: To conclude, vertical transmission in commercial poultry grown separately from hens likely exists but is not efficient, possibly resulting in impairment of microbiota function as evidenced by sensitivity of chicks to gut pathogens. These results also imply that artificial exposure to adult hen bacterial strains might result in improved microbiota functioning.

Poultry

gut microbiota

vertical transmission

chick colonization

interventions

The gut microbiota provides important functions for the host, including protection from pathogens [1], breakdown of plant derived nutritional fibers, and signals that regulate the maturation and development of host systems, including the immune, neurological and intestinal systems [2]. Thus, efficient vertical transfer of gut bacteria from parents to offspring is important for the establishment of optimal microbiota functioning early on. Efficient vertical transfer is also important for gut microbes, especially when considering the advantages of early colonization of a niche [3].

In nature hens brood on their eggs and after hatch the chicks will stay for a while with the hen. Co-habitation, or direct contact between parents and offspring, is an important mechanism for vertical transmission including for poultry [4, 5]. Gut bacteria can also possibly utilize the egg for vertical transfer. Pathogens such as Salmonella are known to use the egg for vertical transmission [6, 7]. A vertical transmission mechanism utilizing the egg might be important in commercial settings where fertilized eggs are separated from the hens immediately after being laid as well as for other vertebrates in which the parents leave the fertilized eggs after laying them and never interact with their offspring. A third mechanism of vertical transmission might be the non-direct long-term survival of microbes in the environment as they wait to be picked up by a randomly passing newly hatched chick. It should be noted that in many commercial operations eggs and other surfaces are disinfected by a variety of chemical treatments aimed at inhibiting pathogen transmission [8]. Thus, it is not clear if vertical transfer of gut bacteria in the commercial chicken still happens or has been partially or fully severed.

On the one hand, a number of reports imply the existence of vertical transmission through the egg in poultry. First, as already mentioned, pathogens use the poultry egg for transmission [6, 7]. Furthermore, a reproductive tract microbiota was shown to exist [9], and a large overlap in the intestinal and reproductive tract microbiomes of chickens, as well as an association between the levels of relative abundance of bacteria in the gut and their chance of being present in the reproductive tract have been shown, implying gut contents are sampled into the reproductive tract and that gut bacteria are in the right place to possibly integrate into the forming egg [10]. Moreover, an embryo gut microbiota has been implied by 16S rRNA gene sequencing [9], and some similarity at the genera level between hens and their offspring has been shown [11]. Last, it has been shown that both the egg shell and the environment contribute to the intestinal microbiota of growing chicks [12].

On the other hand, exposure of newly hatched chicks to a hen, even for just 24 hours, has been shown to greatly affect the gut microbiota of chicks [4]. Additionally, exposure of newly hatched chicks to adult gut contents has been shown to be protective against Salmonella infection [13, 14], further implying that microbiota functions were lacking in newly hatched chicks possibly because of lack of exposure. Thus, it is unclear if vertical transmission of gut bacteria occurs in chickens. Furthermore, vertical transmission might be different for different bacterial phylogenetic groups depending on their specific mechanism of vertical transmission.

Here we compared the fecal microbiota of hens and their specific progeny at the Amplicon Sequence Variant (ASV) level during three cycles of chick growth to understand if vertical transmission happens in commercial chickens growing apart from adults, and characterized the phylogeny of possibly vertically transmitted bacteria. We also rated different ASVs by their incidence in the chick population, indicating their ability to colonize the chick gut. Last, we disrupted possible vertical transmission by either disinfecting the eggshell, or by giving an antibiotic cocktail to the hens during the week of egg collection.

Experimental outline and ethics

All animal trials were conducted in accordance with the guidelines of the National Council for Animal Experimentation and were subjected to approval by the Hebrew University of Jerusalem’s Ethics committee, approval No. AG-18-15514-3.

In the first round of the experiment, eggs were collected for a week from ten broiler breeder hens of the Ross breed that were housed in the experimental farm in the Hebrew University of Jerusalem’s Faculty of Agriculture, and incubated until hatching (Supplementary Fig. 1). Individual fecal samples were collected from the hens during the egg-collection week. Chicks were raised for 14 days, and individual fecal samples were collected on days 2, 7 and 14. This was repeated twice more with the same hens, with a different intervention performed on half of the hens in each round. In the second round of the experiment, eggs laid by half of the hens were disinfected before incubation, while eggs from the other hens remained untreated. In the third round, half of the hens were treated with an antibiotic cocktail for two weeks, including egg-collection week. All hens were euthanized at the end of the experiment, and samples of internal organs (jejunum, cecum and magnum) were collected.

Hens’ growth conditions

Ten 37-weeks old female and ten male broiler breeders were brought to the experimental chicken house in the Hebrew University of Jerusalem’s Faculty of Agriculture from a commercial farm and were acclimatized for six weeks before the start of the experiment. Broiler breeders were housed in individual cages and were kept according to the Ross Parent Stock Management Handbook. Feed used in the experiment did not contain antibiotics. Every 5 days fresh semen was collected and pooled from the 10 males and used for artificial insemination of the females. Egg collection of rounds 1, 2 and 3 were performed when the hens were 43, 49 and 53 weeks old, respectively.

Egg incubation and chick growth conditions

During the egg collection weeks, eggs were collected with clean gloves, placed in clean plastic trays, and kept in 18°C for up to 7 days before incubation. Incubation was done in clean Maru 190 Delux incubators (Rcom, Korea) for 21 days at 37.8°C and 58% relative humidity, with automatic rotation every hour. Egg rotation was stopped at incubation day 18. Chicks were hatched in hatching trays, separated by clean individual metal cages. After a 24 h hatching period, chicks were moved to plastic containers (2,368 cm², up to 7 chicks per bin) with sawdust bedding, equipped with two feeders and two water bottles. Chicks had ad libitum access to feed and water. Chicks were raised according to the Ross Broiler Management Handbook. Chicks from interventions and control chicks were kept in separate containers. The first round included 41 chicks. The second round included 22 chicks from sprayed eggs and 25 control chicks. The third round included 20 chicks from antibiotic treated hens and 23 control chicks.

Eggshell disinfection and sampling

During the second round, within 24 h of laying, eggs were thoroughly sprayed with 0.3% Virocid (CID LINES, Belgium) which was pre-heated to 45°C, and were then left to dry for 10 min. After drying, the eggs were sprayed again in the same manner and left to dry for another 10 min. A 2x2 cm area of each egg was sampled using a sterile flocked swab (Deltalab, Spain) slightly moistened with Difco D/E neutralizing buffer (BD, France). The swab was then directly suspended in 600 µl of the buffer. 200 µl of the suspension were plated on both aerobic and anaerobic YCFA plates [15]. Plates were incubated for 2 days at 37^oC and CFUs were counted.

Antibiotic cocktail treatment

Five hens were gavaged, twice daily for two weeks, with an antibiotic cocktail containing 150 mg Ampicillin, 100 mg Colistin, 400 mg Neomycin and 400 mg Metronidazole. These drugs represent different classes of antibiotics, directed at different targets in the bacterial cell, and covering a wide spectrum of bacteria [16–19]. Furthermore, published works show no residues of these antibiotics are found in laid eggs when given orally [20].

Sample collection

Chicks’ fecal samples were collected by individually placing each chick on a clean piece of paper; hens’ fecal samples were collected by placing a clean plastic sheet beneath each cage. For chicks, most fecal samples were collected within minutes while those of broiler breeders were collected within an hour. Fresh fecal samples were collected into 5 ml of sterile PBS and snap frozen in liquid nitrogen. At the end of the experiment broiler breeders were euthanized, and GI tract and oviduct samples were removed from the euthanized animal. Contents were squeezed out of the GI tract samples, and mucosa was scraped with a sterile glass slide from the oviduct samples. Samples were mixed with 5 ml sterile PBS and snap frozen in liquid nitrogen.

DNA extraction and 16S rRNA gene sequencing

16S rRNA gene library preparation and sequencing were performed according to the Earth Microbiome Project protocol [21] using V4 primers 515F (GTGYCAGCMGCCGCGGTAA) and 806R (GGACTACNVGGGTWTCTAAT). Thermocycling was performed under the following conditions: 94°C for 3 mins, followed by 35 cycles of 94°C for 45 s, 50°C for 60 s and 72°C for 90 s, and a final elongation step of 72°C for 10 min. 150 bp paired-end sequencing was performed on an Illumina Miseq platform using a V2 reagent kit by the sequencing unit of the Faculty of Medicine at the Hebrew University of Jerusalem. Sequences were processed and taxonomy assigned using QIIME2 [22]. ASVs were determined with Dada2 plugin version 2018.8.0 [23] using the denoise-paired method. ASVs with under 5 reads were discarded. All samples were normalized to 5,000 reads per sample. Taxonomy was assigned using a naive-bayes classifier [24] trained on the Greengenes database [25].

Colonization index

Colonization index was calculated for each ASV in each round as the percentage of chick samples containing the ASV out of the total number of chick samples in that round.

Statistical Analysis

All statistical analyses were done using GraphPad Prism 8.0.0 (GraphPad Software, San Diego California USA, www.graphpad.com), except ANOSIM tests which were performed using Past 4.05 [26]. Significance of the ratios between treated and untreated chicks (Fig. 5D) was inferred by shuffling the treated/untreated labels of the samples and calculating the ratios 1,000 times, and comparing the real value to the distribution of permuted data. Values were considered significant if they were lower than mean – 2 X SD. Only phylogenetic groups with at least 80 colonization events were analyzed.

The fecal microbiota of 14 day old chicks is different from that of adult hens

As a first step, we determined if the fecal microbiota of chicks up to the age of two weeks was different from that of adult hens. It should be noted that for broilers, 14 days constitute about a third or more of their lifespan and that for both broilers and layers the first week of life is considered important for overall growth as well as sensitivity to pathogens [27]. An analysis by Jaccard index showed that the chicks’ fecal microbiota changes from day 2 to 14, but that on day 14 it was still different than that of adult hens (Fig. 1A, Supplementary Table S1). Furthermore, an analysis of relative abundances showed the fecal microbiota of adult hens was dominated by genus Lactobacillus which accounted for 78.6% of the fecal community (Fig. 1B). While this genus was also an important genus in the chicks’ microbiota, it comprised on average just 9.6% on day 2, 38.9% on day 7 and 27.1% on day 14. Of note, a comparison of the chicks in the three rounds showed that the chick’s fecal microbiota was different between rounds for each of the three time points (Supplementary Table S2).Furthermore, an ASV identified as Clostridium perfringens was found in high abundance in many round 1 chicks (present in 64% of the chicks on day 2 with an average relative abundance of 17.4%) but was not found at all in round 2 chicks and only in low abundance in several round 3 chicks. Importantly, none of the animals included in this experiment displayed signs of illness. Thus, even though they originated from the same hens, the chicks of the three rounds had a different fecal microbiota. To conclude, after 14 days the chick’s fecal microbiota is still different from that of an adult, and a round specific effect on composition was identified.

After 14 days of life the chick’s fecal microbiota lacks most of the hen fecal ASVs

One possible reason for the difference in the microbiota of chicks and hens could be that the chicks lack bacterial strains found in the hens. To determine if this is the case, for each ASV in our database we determined if it was unique to hens or chicks or was shared by both. We compared only ASVs found in the hen fecal matter to ASVs found in chick samples, as only fecal samples were collected for the chicks. Furthermore, only a small number of ASVs that were not present in the hens’ feces were common between the hens’ GI tract and untreated chicks’ feces (37 ± 9). It was found that most of the hens’ fecal ASVs were not identified at all in chick samples (Fig. 2A). This implied that most hen ASVs were not reaching chicks in settings in which there is no interaction between hens and chicks. The percentage of hen ASVs found in chick fecal samples increased from 14.6% on day 2 to 30.7% on day 14 (Fig. 2B). Notably, the difference between day 7 and day 14 is smaller than that between day 2 and day 7. One hypothesis is that over time chicks are exposed to more ASVs shared with the hens, which then colonize the chicks. Another possibility is that most of the added shared ASVs colonized the chicks already at hatch but did not pass the 16S rRNA gene sequencing sensitivity threshold on day 2.

Most ASVs shared with the hen are weak colonizers of chicks

While the analysis of shared ASVs, described in the previous section, already showed vertical transmission to be inefficient, it is actually a permissive analysis because an ASV was considered shared even if it was found only once among all chick fecal samples. To better quantify the ability of specific ASVs to colonize chicks, a “colonization score” was calculated. The colonization score is the percentage of chick samples in which a certain ASV was identified. For this analysis fecal samples on day 2, 7 and 14 were counted together because we hypothesized that efficient vertically transmitted bacterial strains were likely to be identified already in day 2 fecal samples, while less efficient colonizers will colonize later on. An analysis of colonization score showed that most hen ASVs that were also identified in the chicks were poor colonizers of chicks found in less than 30% of the samples (Fig. 3A). Only 4.89 ± 1.34% of the hen fecal ASVs were found in 30–70% of the chick fecal samples, implying they had an intermediate ability to colonize chicks. In all 3 rounds just 3 ASVs, which account for 0.67% of all hen fecal ASVs, had a colonization score higher than 70%. To conclude, most ASVs found in the hens were not identified at all in the chicks, and of those who did, most were poor colonizers.

Analysis of efficient chick colonizers

While most hen ASVs were poor chick colonizers, there were a few exceptions. Three ASVs were consistently excellent colonizers of chicks in all three rounds of the experiment (Fig. 4A). These were identified as unclassified Enterobactericeae, Enterococcus, and Lactobacillus. It should be noted that most high colonizers, including the 3 excellent colonizers as well as the intermediate colonizers, were consistent in the three rounds (Fig. 4A). This implied that colonization of chicks by these ASVs is not stochastic but rather the result of the biology of these ASVs and their interactions with chickens, and perhaps the environment. Interestingly, a comparison of colonization score with relative abundance in the chick fecal samples showed a good correlation (Supplementary Figure S2). Thus, the ability to spread in the flock was connected to the ability to compete and reach high numbers in the microbial community. In fact, 75.3% of the chick samples were dominated by a single ASV which accounted for 30% or more of the relative abundance, and in 70.3% of them this was one of the three excellent colonizers (Fig. 4C). To conclude, there were a few good consistent colonizers of chicks that were common with the hen. These ASVs are either transmitted through the egg or are good at surviving in the environment, colonizing the newly hatched chick’s intestinal tract and spreading through the flock.

Lack of correlation between chick colonization and hen relative abundance

The excellent colonizer unclassified Lactobacillus was found in high abundance in chick feces as well as in the hen’s fecal and jejunal samples, implying it might be a symbiont vertically transmitting to progeny. In contrast, both the unclassified Enterobactericeae and the unclassified Enterococcus were found in low abundance in the hens’ fecal and internal samples, implying an adapted opportunist label might better describe these two ASVs (Fig. 4D). Further analysis of the relative abundance in the hens of ASVs with different colonization abilities of the chicks showed that most of the relative abundance in the hens represented ASVs that either did not colonize chicks or were poor colonizers (Fig. 4D). This implied that relative abundance in the hen was not correlated with chick colonization ability. Indeed, a direct comparison between the colonization score with relative abundance in hen fecal samples showed no correlation between the two (Supplementary Fig. 3A-C). Because it was possible that bacteria that reached the reproductive tract of the hen were integrated into the forming egg and vertically transmitted, colonization score was also compared with magnum relative abundance. However, no correlation was identified (Fig. 4D and Supplementary Fig. 3D). To conclude, while ASVs differed in their ability to colonize chicks, this could not be correlated with relative abundance in the hens. However, it is also important to note that most of the relative abundance in hens belongs to ASVs that are shared with the chicks (Fig. 4D). This implies that even though vertical transmission is not efficient on the single chick level, together with horizontal transmission among the chicks over time, it might be enough to ensure the transmission of the prominent hen gut microbes to the next generation.

Analysis of chick ASVs which were not identified in hens

The above results show transmission of hen bacteria to chicks is inefficient, implying that environmental opportunists might be able to fill the gap. Indeed, a very large number of ASVs found in chick fecal samples but not hen fecal samples were observed (Fig. 2A). A small number of these ASVs were observed in internal hen samples, implying these were hen ASVs that were below detection in hen fecal samples (Fig. 2A). However, most were not observed in the hens at all. It was also found that the number of chick ASVs not shared with the hens went up over time (Fig. 2C). This implies that as time goes by, the chicks are exposed to additional environmental strains that colonize them. However, it is important to note that a large group of non-shared strains was identified already on day 2. Of note, an analysis of the colonization index distribution showed the vast majority, 98%, of these non-shared chick colonizers are poor colonizers with a colonization score under 30% (Fig. 3B). The rest are intermediate colonizers with colonization scores of 30–70%. None of the non-shared ASVs were excellent colonizers. An analysis of the best non-shared colonizers (Fig. 4B) showed a phylogenetic distribution of bacteria which was similar to that of shared bacteria. Finally, an analysis of the dominant ASVs in fecal samples showed just 15.2% of the samples had a non-shared dominant ASV (Fig. 4C). To conclude, many non-shared ASVs colonized the chicks, but these were mostly poor colonizers.

The effect of eggshell disinfection on chicks’ colonization

To try and sever any vertical transmission that may occur, two interventions were performed. In round 2 half of the eggs were sprayed with a disinfectant, Virocid, before incubation. To determine the effectiveness of the disinfection, a 4 cm² area of eggshell was swabbed and then plated for CFU counts. A 38.5- and 40.4-fold reduction in CFU counts after disinfection was found in aerobic and anaerobic conditions, respectively (Supplementary Figure S4). An analysis of the treated chicks’ fecal microbiota showed a drop in richness in day 2 samples but not on days 7 and 14 (Fig. 5A). A comparison by Jaccard index showed that the fecal microbiota of chicks hatched from treated eggs was different from that of chicks hatched from untreated eggs in the same round for all three time points, however the difference was small, as indicated by the R values (Fig. 5B). Furthermore, there were less non-shared ASVs in treated chicks in days 2 and 7 compared to untreated chicks (Fig. 2C, one-sample t-test p < 0.05). An analysis of colonization scores showed a marked reduction in the number of non-shared ASVs with a score of 10% or lower (Fig. 3B, one-sample t-test p = 0.0079). To understand how specific phylogenetic groups were affected by the disinfection, colonization events of ASVs from each phylogenetic group in treated and untreated chicks were counted. This analysis showed several phylogenetic groups were affected by the disinfectant, and mostly groups of non-shared ASVs (Fig. 5D). Interestingly, Clostridia and Erysipelotrichia were unaffected. As these two groups contain many spore formers, it can be hypothesized that this is the reason they were not affected. Finally, an analysis of the high colonizing ASVs showed that while a few ASVs seem to have been affected by the disinfection, including Clustridium celatum, Curvibacter, Geobavillus and Thermus, most were not affected (Fig. 4A). To conclude, at least some percentage of the ASVs which colonize chicks, including both shared and non-shared ASVs, utilize the eggshell to reach the chicks.

The effect of a disruption of the hen’s microbiota on the chick’s colonization

In round 3, half of the hens were treated with an antibiotic cocktail for two weeks, including the week of egg collection. The antibiotic cocktail significantly reduced the richness of treated hens’ cecal and fecal samples (Fig. 5C) and modulated the composition of all hen samples (Supplementary Figure S5). Of note, cecum richness dropped from an average of 221 ASVs to just 6. Interestingly, the relative abundance of two of the excellent colonizers, the unclassified Enterococcus and unclassified Enterobactericeae, in treated samples was increased (Supplementary Figure S6). This implied that these two bacterial strains are very adapted to the antibiotic intensive commercial environment. The third excellent colonizer, the unclassified Lactobacillus, had a reduced level in hen fecal samples but not in cecal or jejunal samples. Thus, the antibiotic cocktail had a profound effect on the treated hens’ microbiota but not on the top three suspects for vertical transmission.

An analysis of the treated chicks’ fecal microbiota showed a marked drop in richness at all three time points (Fig. 5A). Furthermore, a comparison by Jaccard index showed chicks from treated hens had a different fecal microbiota than chicks from untreated hens at all time points, again with low R values (Fig. 5B). Next, an analysis of the shared and non-shared ASVs showed a reduction in non-shared bacteria at all time points and a lesser effect on shared bacteria which were only statistically significantly reduced on day 14 (Fig. 2B and C). Additionally, the number of shared and non-shared ASVs in treated chicks with colonization score < 10 were reduced, and more hen ASVs were never identified (colonization score = 0) (Fig. 3A and B). An analysis of the colonization ability of specific phylogenetic groups showed a marked reduction in several groups, mainly Firmicutes (Fig. 5E). In this case, both shared and non-shared ASVs were affected. Last, while a few of the intermediate colonizers seem to have been affected by the treatment, such as Lactobacillus salivarius, most excellent and intermediate colonizers were unaffected by this disruption (Fig. 4A). This implies that most of the effect was on poor colonizers. It should be noted that while the antibiotics used in the cocktail are not thought to reach the egg, and are not expected to reach the chick at effective concentrations [20], this option cannot be excluded. To conclude, treatment with an antibiotic cocktail disrupted the colonization ability of many ASVs, but mainly of weak colonizers.

The main result of this study is that under rearing conditions in which there is no interaction with adults and in which the environment is kept relatively clean, the representation of adult bacterial strains in chicks is very low, implying that vertical transmission is minimal. These conditions closely represent the growth practices of many commercial operations that do so for reasons of biosecurity. Moreover, in many commercial operations eggs are treated with disinfectants which reduce vertical transmission even further. This data agrees with studies in which exposing chicks to the gut contents of adults or to live adults enables the transmission of many bacterial strains [4, 13, 14]. Thus, it seems that the quest to inhibit the vertical transfer of pathogens also disrupts the vertical transfer of commensal gut microbes.

As the gut microbiota is thought to provide a number of functions for the host and since, as this report shows, vertical transmission is minimal, it can be hypothesized that many microbiota functions are impaired. One function which might be impaired because of minimal vertical transmission is the ability of the gut microbiota to digest nutritional fibers, which the host cannot do on its own. Because chicken feed contains some nutritional fibers it is possible that feed utilization in commercial operations can be improved by artificial exposure to adult gut bacterial strains. Furthermore, there is an increased appreciation for the functions of microbiota derived signals on the maturation and development of host systems, such as the intestinal, immune and neural systems [2]. Our results imply that these signals might be impaired in commercial chickens, with unknown implications.

Another function of the gut microbiota for the host is protection against gut pathogens. Indeed, young chicks are sensitive to gut pathogens, such as the zoonotic pathogen Salmonella, with financial implications in commercial production [27]. It has been shown that exposure to live adults or to the gut content of adults renders chicks resistant to Salmonella infection [13, 14]. It has also been shown that as they age, chicks become resistant to Salmonella, likely because they acquire commensal gut bacteria which protects them [13]. The results presented in the current study, showing minimal vertical transmission in commercial growth conditions, complete this picture, and show that modern growth practices are the cause of sensitivity to at least Salmonella infection.

While this work shows most hen bacterial strains did not efficiently reach and colonize chicks, we also show that vertical transmission is also likely to occur in these conditions, where there is no interaction with adults. We found that 3 bacterial strains present in hens did reach the chicks and were excellent colonizers of the chick gut, about 20 more were intermediate colonizers and many more were poor colonizers. Furthermore, both interventions resulted in a decrease in the colonization of strains shared with hens. This implies that vertical transmission via the egg is a viable mechanism for vertical transmission even if not efficient. These results are in agreement with previous studies which showed that gut material is sampled into the female reproductive tract, implying gut bacteria were in the right place to integrate into forming eggs [10], as well as studies identifying bacterial DNA in embryos [9, 11, 28] and studies showing some resemblance between the microbiota of hens and chicks [9, 11]. Thus, vertical transmission through the egg is likely to exist, but is inefficient.

Interestingly, all medium and excellent shared bacterial strains were consistently also medium and excellent colonizers, respectively, in all three rounds. Thus, it seems a consistent colonization pattern of chicks by a specific set of bacterial strains is obtained at least when the same hens are involved. It should be noted that, by definition, medium colonizers reach only some of the chicks within the 14-day time frame.

These results raise two interesting questions. The first is how did the hens receive their gut microbiota? It can be hypothesized that while vertical transmission through the egg or the external environment is inefficient, it is sufficient, given the longer life spans of hens. In the present experiment many of the shared bacterial strains did make it to a few of the progeny. It can be hypothesized that as the flock ages, bacterial strains would spread through the flock, giving each bird a full and complete gut microbiota population as it matures. The second question is, did the commercial chicken lose commensal gut bacterial strains in the last decades due to biosecurity practices? While our results cannot answer this question directly, a likely answer is yes. The bacterial strains represented in this study are likely those which are still able to be vertically transmitted in current commercial practices. As this study shows many of these strains are barely vertically transmitted, it implies that others might have been lost. To conclude, in commercial operations in which there is no contact with adult hens, the efficient mechanism of vertical transmission is lacking. However, inefficient vertical transmission through the egg or the environment are the main mechanisms of vertical transmission.

A surprising finding in this study is that interventions designed to disrupt vertical transmission had a large effect on bacterial strains that were found in the chicks but not in the hens. Such strains are viewed as environmental opportunists, bacterial strains which are not specifically adapted to the gut of chickens but colonize this niche because it is relatively empty. Environmental opportunists are thought to come from feed, cage surfaces or even carried on dust particles. In this study, each of the two interventions reduced non-shared colonization significantly. This implies that a considerable part of environmental opportunists utilizes the egg to colonize chicks. This portion is likely larger than that observed in this study, as both interventions performed were not optimal – Virocid spraying reduced the numbers of bacteria on the eggshell but did not sterilize it, and the antibiotic cocktail application had little effect on many excellent and intermediate colonizers including non-shared strains, implying many have inherent antibiotic resistance. One possibility is that these non-shared strains do come from the hen’s body, perhaps from the skin or cloaca or are even found at very low levels, below the detection level, in the gut and feces, and that a more appropriate label would be adapted opportunists. To conclude, it is likely that a large portion of the non-shared bacterial strains colonizing the chicks are not environmental opportunists per se but are rather chicken adapted opportunists which are not able to forge a niche for themselves in the adult chicken gut, but take advantage of the newly hatched chick’s relatively empty gut, and then are able to survive on or in the hen at low numbers and vertically transmit through the egg to the next generation.

We show here that vertical transmission in commercial conditions in which there is no contact between chicks and adults is likely to occur, but is very inefficient. On the whole flock level, while coupled with horizontal transmission through the flock over time it is likely sufficient for the transfer of the current modern chicken microbiota. However, on the single chick level, this results in a long delay in the acquisition of an adult gut microbiota. This might explain why chicks are sensitive to gut pathogens and why they become resistant after exposure to an adult’s cecum content. These results imply other gut functions might likewise be suboptimal, and that artificial exposure to adult bacteria might correct these problems.

Availability of data and materials

The raw sequencing reads of this study have been deposited in SRA under the accession number PRJNA885292.

Funding

This work was supported by the Israel Ministry of Agriculture (grant No. 12-04-0022 and 12-04-0025) and by the Israeli Science Foundation (grant No. 1272/20). Erez Mills is chair of the Vigevani Senior Lectureship in Animal Sciences.

Author information

Authors and affiliations

Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel

All authors.

Contributions

NS, ZU and EM designed the study. All authors participated in different parts of the experimental work. NS and EM analyzed the data and wrote the paper.

Corresponding author

Correspondence to Erez Mills

Ethics approval

Competing interests

This work was partially funded by a commercial company, Phibro Israel, through the above mentioned Ministry of Agriculture grant (No. 12-04-0022).

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Competing interest reported. This work was partially funded by a commercial company, Phibro Israel, through the above mentioned Ministry of Agriculture grant (No. 12-04-0022).

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Limited vertical transmission of gut bacteria in commercial chickens

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Abstract

Figures

Background

Methods

Experimental outline and ethics

Hens’ growth conditions

Egg incubation and chick growth conditions

Eggshell disinfection and sampling

Antibiotic cocktail treatment

Sample collection

DNA extraction and 16S rRNA gene sequencing

Colonization index

Statistical Analysis

Results

The fecal microbiota of 14 day old chicks is different from that of adult hens

After 14 days of life the chick’s fecal microbiota lacks most of the hen fecal ASVs

Most ASVs shared with the hen are weak colonizers of chicks

Analysis of efficient chick colonizers

Lack of correlation between chick colonization and hen relative abundance

Analysis of chick ASVs which were not identified in hens

The effect of eggshell disinfection on chicks’ colonization

The effect of a disruption of the hen’s microbiota on the chick’s colonization

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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