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University of Queensland (2011)

The bacterial community in the foregut of the dromedary camel

Anjas Samsudin

Titre : The bacterial community in the foregut of the dromedary camel

Auteur : Anjas Samsudin

Grade : Doctor of Philosophy (PhD) 2011

Université de soutenance : University of Queensland

The digestive systems of the Arabian camels are anatomically different from that of the true ruminants such as cattle and sheep. The forestomach of camelids consists of three chambers, whilst in the true ruminant, they have four. Despite having differences in anatomical structures, functionally they are the same. The molecular diversity of the foregut bacterial community in the dromedary camel (Camelus dromedarius) in Central Australia was investigated through comparative analyses of 16S rRNA gene sequences prepared from the foregut contents of 12 adult feral camels fed on native vegetation. A total of 267 near complete 16S rRNA clones were examined, with 151 operational taxonomic units (OTUs) identified at a 99% species-level identity cut-off criterion. The prediction of actual diversity in the foregut of the dromedary camel, using the Chaol’s approach, was 238 OTUs, while the richness and evenness of the diversity estimated, using the Shannon’s index, was 4.84. The majority of clone bacteria in the current study was affiliated with the bacterial phylum Firmicutes (67% of total clones) and was related to the classes Clostridia, Bacilli, and Mollicutes, and these were followed in number by the Bacteroidetes (25%) that were mostly represented by the family Prevotellaceae. The remaining phyla consisted of Actinobacteria, Chloroflexi, Cynophyta, Lentisphaerae, Planctomycetes, Proteobacteria, and Sphirochaetes. Moreover, 11 clones of cultivated bacteria were identified as Brevundimonas sp., Butyrivibrio fibrisolvens, Prevotella sp., and Ruminococcus flavefaciens. The novelty in this foregut environment is remarkable where 97% of the OTUs were distantly related to any known sequence in the public database. In the second study, numerous genera of cellulolytic bacteria, involved in fibre degradation in the rumen, and their functional role in the foregut of the dromedary camels (Camelus dromedarius) were investigated using 16S rRNA gene libraries. The foregut content from five adult feral camels, fed on native vegetation, was inoculated in three different fibre enrichment media, namely ; cotton thread (CT), filter paper (FP), neutral detergent fibre (NDF). A total of 283 near complete 16S rRNA clones were examined, with 33 OTUs identified at a 99% species-level identity cut-off criterion. The actual diversity was estimated using Chaol’s approach, with 42 OTUs predicted, while the richness of the diversity estimated using Shannon’s index was 2.82. LIBSHUFF analysis of the three 16S rRNA clone libraries revealed significant differences across all of them. Clostridium bifermentans were found in all three libraries, while Eubacterium sp, Pseudobutyrivibrio ruminis, Pyramidobacter piscolens and Schwartzia succinivorans were found in both CT and FP enrichment media only. The current results in the present study showed significant findings of fibre-degrading bacteria that inhabit the foregut of wild dromedary camels. In the third study, the total bacterial community, F. succinogenes and R. flavefaciens, in the foregut of the dromedary camel was measured by quantifying the cell number of each respective species using real-time PCR. The samples from this study were obtained from the same samples used in the second study. The standard curve gave a linear relationship (R2=​0.969) with the equation y=​ -2.975x+42.33. Using an absolute quantification method, the numbers of cells in one millilitre of each sample ranged from 4.07x106 to 2.73x109 for total bacteria, 1.34x103 to 2.17x105 for F. succinogenes and 5.78x101 to 3.53x104 for R. flavefaciens. The mean cell number for three set of primers used in the current study demonstrated that the number of general bacteria prevalent in the CT enrichment media was 242-fold higher than in rumen content. The mean cell number of F. succinogenes was highest in the FP enrichment media at approximately 107-fold, whereas for the R. flavefaciens targeted primer, the NDF enrichment media had the highest mean cell number with approximately 4-fold when compared to the rumen content. Despite having a low population density in the rumen samples compared to samples from domesticated ruminants, the data presented here would help farmers to develop feeding regimes for dromedary camels since they do not share an interest in the forage consumed by domesticated livestock, especially during a drought season. The final study of this thesis was to identify the unrealized fibre-degrading bacteria in this environment niche using a combination of culturation and molecular methods. Samples in this study were obtained from the same enrichment media (CT, FP and NDF) used in the second and third studies. In this study, only 66 partial-length (700bp) sequenced isolates were successfully recovered. The majority of the isolates in this study were represented by the phylum Firmicutes followed by Proteobacteria and Actinobacteria. The phylum Firmicutes was dominant in the CT and FP enrichment media, while a reasonable number of Proteobacteria was present in the NDF enrichment culture. The genus Clostridium was found in all enrichment media and was the most dominant genus with the highest frequency, 56.1% of isolates, followed by Escherichia coli (19.3%), Actinomyces ruminicola (15.8%) and Streptococcus lutetiensis (8.8%). The results of the culture dependent methods revealed the difficulties in culturing fibre degrading bacteria and more work is needed to address this. None of the isolates obtained from the fibre-enriched media was able to degrade cellulose independently, confirming the complexity of the microbial ecosystem and the importance of the interaction between bacterial groups to degrade fibre. Overall, results of this study provided an insight into a microbial system of a herbivore that has not been thoroughly investigated. Furthermore, the study of the fibre-degrading bacterial community in the foregut of the dromedary camel has provided data that, hopefully, will contribute to the sustainable farming of camels well into the future. These animals do not compete with domesticated ruminants such as cattle and sheep either in feeding behaviour or in the type of feed these animals consume, so the data could support plans for the commercial management of a potential new livestock industry in Australia.

Mots clés : S rRNA clone library, foregut, dromedary camel, bacterial community, fibre-degrading bacteria, cellulose type, real-time PCR (16)

Présentation National Library of Australia

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