Broiler Chickens and Early Life Programming: Microbiome transplant-induced cecal bacteriome dynamics and phenotypic effects

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first derive a stable source of inoculum. Subsequently, we compared microbiome development between chicks receiving this passaged cecal material, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa were identified across treatment types that may inform future studies aimed at identifying strains associated with beneficial phenotypes.


Abstract 31
The concept of successional trajectories describes how small differences in initial community 32 composition can magnify through time and lead to significant differences in mature 33 communities. For many animals, the types and sources of early-life exposures to microbes have 34 been shown to have significant and long-lasting effects on the community structure and/or 35 function of the microbiome. In modern commercial poultry production, chicks are reared as a 36 single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a 37 trajectory of microbial community development that is significantly different than non-38 industrial settings where chicks are exposed to a much broader range of environmental and 39 fecal inocula; however, the comparative effects of these two scenarios on microbiome 40 development and function remain largely unknown. In this work, we performed serial transfers 41 of cecal material through multiple generations of birds to first derive a stable source of 42 inoculum. Subsequently, we compared microbiome development between chicks receiving this 43 passaged cecal material, versus an environmental inoculum, to test the hypothesis that the first 44 exposure of newly hatched chicks to microbes determines early GI microbiome structure and 45 may have longer-lasting effects on bird health and development. Cecal microbiome dynamics 46 and bird weights were tracked for a two-week period, with half of the birds in each treatment 47 One promising approach to better understand how specific GI bacterial taxa may influence 89 growth performance and pathogen resistance in poultry is the use of microbiome transplants 90 receiving either CMT or EMT at day-of-hatch relative to PBS controls. The PBS gavage 159 (negative MT control) recipients lost approximately 20% of their average body weight between 160 12 and 14 days of age (5-7 days post-challenge) and at day 14 of age were significantly lighter 161 than MT (EMT and CMT) recipients. Also, at d14 of age, EMT recipients were significantly 162 heavier than CMT recipients. 163 164 4. Alpha-diversity 165 The number of observed taxa (genus-and 99% OTU-level) was lowest in 1-day old birds for 166 all treatment groups (Figure 3, A & B). However, significantly more taxa at the genus and 99% 167 OTU levels were observed at d1 for birds administered a CMT relative to the EMT treatment 168 or PBS controls ( Figure 3, A & B). From day 1 to day 7, significant increases in observed taxa 169 occurred for all treatment groups ( Figure 3). Subsequently, for birds that did not undergo a 170 pathogen challenge, there were no significant differences in genus-or OTU-level richness 171 between bird age 7 and 14 days ( Figure 3, A & B). For birds that were pathogen challenged at 172 7 days of age, a significant decrease in OTU-level richness at 14d relative to 7d was observed 173 in the group that received a day-of-hatch CMT ( Figure 3D). A day-of-hatch CMT 174 administration generally resulted in higher OTU richness at d7 versus d14 for both the non- A total of 9 OTU lineages, belonging to three genera within the phylum Bacteroidetes, 193 exhibited significant differences in abundance in cecal communities from unchallenged birds 194 that received EMTs compared to PBS controls ( Figure 5A). These OTUs were classified as 195 members of the Barnesiella, Parabacteroides, and Alistipes genera ( Figure 5A) were significantly differentially abundant in cecal communities from unchallenged birds that 200 received CMT relative to PBS controls ( Figure 5B). Specifically, 18 OTUs were significantly 201 more abundant in CMT versus PBS treatments ( Figure 4B). These OTUs were classified within 202 the following genera: Rikenella, Parabacteroides, Lactobacillus, Alistipes, and Barnesiella 203 ( Figure 5B). Five OTUs classified as Coprococcus, Barnesiella, Alistipes and Sporobacter 204 were significantly less abundant in CMT versus PBS treatments ( Figure 5B). Interestingly, 205 two genera, Alistipes and Barnesiella, had OTUs that were both significantly more and less 206 abundant in cecal communities of CMT recipients relative to PBS controls ( Figure 5B). 207 208

Pathogen Challenged Birds: EMT 209
A total of 54 OTU lineages, belonging to either the Firmicutes or Bacteroidetes, exhibited 210 significant differences in abundance in cecal communities from pathogen challenged birds in 211 the EMT group versus PBS controls ( Figure 5C). Specifically, thirty-six and nineteen OTU 212 lineages were significantly more and less abundant, respectively, in cecal communities from 213 EMT recipients relative to PBS controls. All OTUs classified as Lactobacillus, Butyricicoccus, 214 Bacillus, and Parabacteroides, were significantly enriched in EMT relative to PBS controls. 215 All OTUs classified as Faecalitalea, Barnesiella, Odoribacter, and Faecalibacterium were 216 significantly less abundant in cecal communities from birds that received an EMT relative to 217 PBS controls. Interestingly, three genera (Alistipes, Barnesiella, and Bacteroides) contained 218 some OTUs that were significantly enriched and some that were significantly less abundant in 219 cecal communities of EMT recipients relative to controls ( Figure 5C). 220 221

Pathogen Challenged Birds: CMT 222
A total of 90 OTU lineages, belonging to either the Firmicutes or Bacteroidetes, exhibited 223 significant differences in abundance in cecal communities from pathogen challenged birds that 224 received a CMT compared to PBS controls ( Figure 5D). 61 and 29 OTU lineages were 225 significantly more abundant or less abundant, respectively, in cecal communities from CMT 226 recipients relative to PBS controls. All OTUs classified as Butyricicoccus, Rikenella,227 Bacteroides, Parabacteroides, and Bacillus, were significantly enriched in CMT relative to 228 PBS controls. All OTUs classified as Odoribacter, Blautia, and Faecalibacterium, were 229 significantly less abundant in CMT relative to PBS controls. Four genera (Alistipes, 230 Barnesiella, Ruminiclostridium, and Eubacterium) contained some OTUs that were 231 significantly enriched and some that were significantly less abundant in cecal communities of 232 CMT recipients relative to PBS controls ( Figure 5D). 233 234

Taxa Differentially Abundant in Both Challenged and Unchallenged Groups 235
A total of 178 OTU lineages exhibited significant differences in relative abundance between 236 birds that received a MT (EMT or CMT) versus PBS controls ( Figure 6A). 125 and 13 of these 237 OTUs were observed exclusively in challenged and unchallenged groups, respectively. Twenty 238 differentially abundant OTUs, all classified as Bacteroidetes, were observed in both pathogen-239 challenged and unchallenged groups. Interestingly, these 20 OTUs exhibit similar trends in 240 magnitude and fold change direction as a function of MT administration in both pathogen-241 challenged and unchallenged groups even though these were independent experimental cohorts 242 ( Figure 6B). 243 244

Discussion 245
Applying the conceptual framework of successional trajectories (Fastie, 1995), similar to the 246 concept of "early life programming" (Rubio, 2019), we hypothesized that first exposure of 247 newly hatched chicks to environmental microbes determines early GI microbiome structure 248 and may have long-lasting effects on bird health and development. To test this hypothesis, we 249 tracked cecal microbiome dynamics and pathogen resistance of broiler chicks that received 250 complex microbiome transplants at day-of-hatch. To compare the effects of very different first 251 microbial exposure starting points, we compared a stable inoculum derived from serial 252 passages of cecal material to a complex environmental community derived from used poultry 253 litter and sterile PBS controls. To assess if early microbial exposure influences resistance to 254 pathogenic infection, we performed this study on two bird panels, one that was pathogen 255 challenged at 7d of age and one that was not pathogen challenged ( Figure 2A). 256 257

Microbiome dynamics through serial passages of cecal material 258
To obtain a transplant community inoculum selected by the cecal environment of broiler 259 chicks, we serially transplanted cecal material from 14-day-old birds to newly hatched chicks. 260 When the chicks reached 2 weeks of age, cecal contents were harvested and transplanted to a 261 new batch of chicks. This serial passaging was repeated for five generations of chicks. We 262 hypothesized that environmental filtering (Szekely and Langenheder, 2014) would result in an 263 overall reduction in community richness with each serial transfer of cecal material and 264 eventually lead to a stable microbial cohort consistently sorted by environmental and host-265 mediated factors. After just one passage, a relatively stable inoculum was derived ( Figure 1). 266 After the first serial passage, the starting inoculum had changed significantly in community 267 diversity and composition from Firmicutes to Bacteroidetes dominance and remained relatively 268 stable thereafter (Figure 1 C-E). These results suggest that a taxonomic subset of a community 269 is quickly selected in a deterministic fashion by the host. We speculate that, given its relative 270 stability, the selected community should be beneficial to the host. 271

Either CMTs or EMTs enhance resistance to pathogen infection 273
We observed two significant effects of day-of-hatch MT on bird weight. First, in unchallenged 274 birds, day-of-hatch EMT administration had no effect on weight while CMT administration led 275 to significantly lower bird weight relative to controls ( Figure 2C). In pathogen challenged 276 birds, administration of either MT type resulted in higher bird weight relative to controls; 277 however, birds administered the EMT gavage were significantly heavier than CMT recipients 278 ( Figure 2C). These observations lend credence to the notion that MT-elicited modulations of 279 the GI flora, are both a consequence of host genetics and health status (Schokker et al., 2015), 280 and also a cause of changes in host phenotype. Because EMT rather than CMT administration 281 resulted in increased weight gain, independent of pathogen challenge status, we concluded that 282 gavage composition drives phenotypic outcomes and that EMT inoculation alone may be 283 sufficient to produce desirable phenotypes. The EMT gavage was largely comprised of 284 Firmicutes lineages assigned as Lactobacillus spp. while the CMT was primarily comprised of 285 Bacteroidetes lineages within the Alistipes, Bacteroides, and Barnesiella genera. Notably, 286 despite being sourced from used commercial poultry litter, the EMT composition 287 (predominantly Firmicutes, Figure 2B to CMT. However, we note that the CMT gavage, comprised primarily of Bacteroidetes 295 lineages, also resulted in increased weight gain relative to controls in pathogen challenged 296 birds. This suggests that Firmicutes dominance (Lactobacillus spp., specifically) is not the sole 297 determinant of the phenotypic effects elicited by both MT types in pathogen challenged birds. 298 Overall, enhanced resistance to pathogen infection, inferred from weight gain, during early 299 development (< 2 weeks of age) appears to be a global benefit conferred by administration of 14d, the microbial community clustering at 7d, where both CMT and EMT communities are 320 similar to each other and dissimilar to controls, is particularly intriguing. Both MT types altered 321 the cecal microbiome relative to controls prior to the observed phenotypic differences. These 322 short-lived patterns in cecal bacteriome structure completely dissipate by 14d ( Figure 4E To better understand the potential mechanisms of action of MTs, we identified taxa that were 332 significantly differentially abundant between MTs and control communities at 7d ( Figure 5). 333 In non-pathogen challenged birds, significantly higher abundances of 9 lineages belonging to 334 the Barnesiella, Parabacteroides, and Alistipes genera were observed in the EMT treatments 335 relative to controls at 7d ( Figure 5A). The differential abundance of these taxa at 7d did not 336 result in significant differences in bird weight at 14d ( Figure 2C). Conversely, day-of-hatch 337 CMT administration did result in lower bird weights at 14d relative to controls ( Figure 2C were both positively and negatively associated with experimental treatment and phenotype, 385 reinforcing existing dogma that 'strains matter', i.e. specific bacterial strains can elicit 386 significantly different phenotypes (). We note that in pathogen challenged birds, day-of-hatch 387 MT administration yielded significantly higher bird weights relative to controls, however, the 388 highest weight gains were observed in EMT recipients ( Figure 1C). Two OTU lineages of 389 Lactobacillus spp. were significantly more abundant in the EMT recipients at 7d relative to 390 controls. Butyrate producers are known to cross-feed with lactic acid produced by 391 Lactobacillus and, for example, Butyricicoccus spp. in the 7d cecal community of EMT 393 recipients relative to controls, not observed in CMT recipients, suggests that the observed 394 benefits of MT administration may result from enhanced cecal SCFA production. 395

Conclusions 397
To advance our knowledge of microbiome-induced modulation of host health outcomes, 398 microbiome transplants can provide predictive and testable guidance by identifying specific 399 taxa that are differentially represented between treatments. Here we used MTs to better 400 understand microbiome establishment from diverse inocula and to identify specific strains 401 associated with pathogen resistance. Our results show that i) a relatively stable community was 402 derived after a single passage of transplanted cecal material, ii) this cecal inoculum 403 significantly but ephemerally altered community structure relative to the environmental 404 inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch 405 appeared to have some protective effects against pathogen challenge relative to uninoculated 406 controls. We identify lineages that significantly differ in abundance in cecal contents from 407 birds treated with MTs at day-of-hatch relative to controls that may drive observed phenotypic 408 effects. These results suggest that environmental exposure to used poultry litter may provide 409 an effective inoculum that could protect against pathogens and identifies specific taxa that may 410 be responsible for this effect. 411 412

Microbiome Transplant Source Materials 414
The CMT source material was developed as follows: Frozen cecal material from 6 week-old 415 broiler chickens was reconstituted by diluting 3:1 (w:v) in PBS and 0.2 mL administered via 416 oral gavage to ten day-of-hatch chicks. When these chicks reached 2 weeks of age, their cecal 417 contents were similarly prepared and administered to the next set of ten chicks. This serial 418 passaging was repeated for five sets of chicks, with 10 chicks belonging to each group for a 419 total of 50 birds. Chicks in each cohort were housed together. Cecal contents from each bird 420 were sequenced as described below. The cecal contents from the final 10 birds were suspended