Influence of Precision Glycans on Layer Cecal Community

I. Introduction

Glycans are polysaccharides, essential biomolecules with well-established benefits for the host and abundantly used as prebiotics to support balanced microbiome function. Host produced glycans, such as mucins, are crucial for the defensive purpose of the intestinal epithelial barrier against pathogens (Coker et al., 2021). Glycans are often projecting on the cell surface and are frequently secreted molecules, thus presenting a perfect pathogen trap or barrier.

In addition to host produced glycans, many studies have investigated enormous benefits to intestinal health via supplementation of dietary glycans (Tannock, 2021). Dietary supplementations with specific glycans can modulate microbiota, improving enteric health and preventing dysbiosis by rebuilding diverse microbial communities (Tannock, 2021). This is presenting us with the opportunity to develop precision glycans, optimised to maximise and harness the best intestinal benefits and bring about the new generation of prebiotics (Tannock, 2021) to match or outperform next-generation probiotics.

Precision glycan inspired Precision Biotic (PB) is developed on a functional metagenomics platform to control pathogenic functions in the poultry gut and improve intestinal health. A natural coccidiosis challenge study reported that PB resulted in similar or better performance to the Avilamycin and consistently exhibited significantly enhanced performance compared to the phytogenic, essential oils-based treatments (Ramirez et al., 2021). In another study, two precision glycan metabolic modulators of different glycan sizes were investigated across 19 geographically distinct high powered trials conducted using 33,880 broiler chickens. One of the products displayed a reduction of cFCR of 0.06 g feed/ g BW gain compared to control, while the other precision glycan product reduced cFCR. While both products improved FCR, one of them increased and the other one reduced feed intake (Walsh et al., 2021). In this study, we investigated the effects of PB, previously shown beneficial in broiler enteric challenge (Ramirez et al., 2021), in a commercial layer trial comprised of 40,000 layers housed in experimental sheds, specifically designed for this type of experiment.

II. Method

The experiment was performed in a large commercial layer farm in a shed designed to take two groups of 20,000 birds each, with the two treatments on opposite sides of the shed, separated by a utility room in the middle and with a wire fence separating the birds in the range with an outdoor buffer zone between the two treatments. The 40,000 of 18 weeks old pullets from the same batch of Hyline chicks were randomly assigned to either control or PB supplemented group. The birds are given the same batch of feed with the supplement automatically premixed to the treatment side of the shed at the dose of 400ppm. The birds were provided with a standard company layer ration optimised for maximum health and performance under the current farm environmental conditions and layer breed. Ten weeks after the shed placement and treatment supplementation, when the birds were 28 weeks of age, caecal content was collected for microbiota analysis via 16S amplicon sequencing.

Microbiota analysis was done via amplification of the V3-V4 region of 16S rRNA. Sequencing was completed on the Illumina MiSeq platform using 2x300 bp paired-end sequencing. The data were analysed using Quantitative Insights Into Microbial Ecology 2 (QIIME 2). After demultiplexing with Cutadapt, Phred quality scores were set to a minimum of 30 using only top quality sequences. Dada2 was used to error correct and chimera clean the data and taxonomy was assigned using SILVA database. Data analysis and interpretation were done using Hellinger transformed ASV table at an ASV and genus level. DeSeq2 was used for univariate significance comparisons at the genus level.

III. Results

In total, 39 of 40 samples were successfully sequenced with the smallest sample size of 4,228 and the largest sample containing 12,056 sequences with no significant differences between the groups in a number of sequences per sample. The caecal microbial community was significantly different between control and PB supplement using Adonis multivariate analysis and Bray-Curtis distance at an ASV (P=0.0003) and a genus level (P=0.002). Supplementation marginally increased cecal diversity. Table 1 shows the genera affected. Genera increased in the supplemented group included Saccharimonadales, Mucispirillum, Odoribacter, Parasutterella and Mailhella, while reduced include Anaerofilum, Enterorhabdus, Blautia, Subdoligranulum, Ruminococcus, Romboutsia and Megamonas.

Table 1 – Significantly altered cecal genera

Linear discriminant analysis Effect Size (LEfSe), an algorithm for high-dimensional biomarker discovery, was used to determine main features related to the treatments (Figure 1) and it additionally identified clostridia and Corynebacterium as the biomarker for the control group and Eubacterium hallii group associated with PB.

Figure 1 - LEfSe analysis identified Clostridium sensu stricto and Corynebacterium as significantly associated with control, and Eubacterium hallii group associated with PB.

IV. Discussion

The inclusion of PB introduced distinct changes to the microbial community of layer caeca. One of the strongly inhibited microbial communities (no sequences detected in control birds) were members of the genus Saccharimonadales. As with other newly identified or reclassified genera, there is not much literature available to draw meaningful conclusions on its role in layer intestinal health. Recently, Padovan et al. (2021) thoroughly investigated the threat posed by pathogenic vibrio species in tropical Australia. They identified 42 Vibrio species and reported a significant negative correlation with Saccharimonadales. Mucispirillum (Robertson et al., 2005), another genus increased in the PB group, is better characterised, reported in insects, mice, poultry, dogs, pigs, goats and humans. The name is connected to the flagella and the ability to move through the mucus (Robertson et al., 2005). It is possible that novel glycans interacted with mucin to make the environment more favourable for Mucispirillum. Interestingly, Mucispirillum was also reported as a Salmonella antagonist protecting the mice against colitis (Herp et al., 2019).

Odoribacter are named by the foul smell they produce in the mouth. The effects are high strain-specific and members of this genus have been reported as significant members of oral and faecal microbiota, able to confer protective immunity against cancer (Foegeding and Byndloss, 2021) or, in contrast, as increasing the risk of multiple sclerosis relapse in children or being increased in lupus (Vieira et al., 2021) and obesity (Zeng et al., 2021). Based on our abundant data collected from poultry farms, this genus is a part of normal poultry microbiota, and its particular roles should be further investigated for both benefits and an opportunistic pathogen ability.

Another genus increased in the PB group, Parasutterella, should not be confused with Pasteurella genus responsible for fowl cholera. Parasutterella is a more novel and uncharacterised genus that represents one of the main prominent gut members (Ju et al., 2019). It has been reported as improving lipoprotein levels in human participants consuming resistant potato starch (Bush and Alfa, 2020) and as potentially promoting intestinal inflammation (Chen et al., 2018). Mailhella, a sulphate reducing genus, is also one of the more recently noticed genera detected in layers of the caecum (Huang et al., 2019). In addition to more neutral genera affected by PB, the benefits of reduction of true clostridia are well characterised.

A range of layer commensal bacterial genera was reduced in the PB group, including Blautia, Subdoligranulum and Ruminococcus, mostly considered as beneficial. Ruminococcus is one of the major mucin utilising genera (Crost et al., 2016) and is considered beneficial; however, as we stated above, the benefits and pathogenicity are often strain level specific. Ruminococcus was also reported as associated with prediabetes (Allin et al., 2018) and inflammatory bowel disease (Png et al., 2010) and there is growing evidence of the role of primary and secondary mucin degraders in intestinal inflammatory diseases (Png et al., 2010). More research is needed to further investigate possible PB and mucin intestinal interactions.

Presented at the 33th Annual Australian Poultry Science Symposium 2022. For information on the next edition, click here.