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. 2025 Jan 8;15:1327. doi: 10.1038/s41598-025-85495-0

Characteristics of gut microbiota profiles in Asian elephants (Elephas maximus) with gastrointestinal disorders

Sarisa Klinhom 1, Chanon Kunasol 2,3, Sirawit Sriwichaiin 2,3,4, Sasiwan Kerdphoo 2,3, Nipon Chattipakorn 2,3,4, Siriporn C Chattipakorn 2,3,5,✉, Chatchote Thitaram 1,6,7,✉
PMCID: PMC11711614  PMID: 39779898

Abstract

Colic and diarrhea are common gastrointestinal (GI) disorders in captive Asian elephants, which can severely impact health and lead to mortality. Gut dysbiosis, indicated by alterations in gut microbiome composition, can be observed in individuals with GI disorders. However, changes in gut microbial profiles of elephants with GI disorders have never been investigated. Thus, this study aimed to elucidate the profiles of gut microbiota in captive elephants with different GI symptoms. Fecal samples were collected from eighteen elephants in Chiang Mai, Thailand, including seven healthy individuals, seven with impaction colic, and four with diarrhea. The samples were subjected to DNA extraction and amplification targeting the V3-V4 region of 16S rRNA gene for next-generation sequencing analysis. Elephants with GI symptoms exhibited a decreased microbial stability, as characterized by a significant reduction in microbiota diversity within individual guts and notable differences in microbial community composition when compared with healthy elephants. These changes included a decrease in the relative abundance of specific bacterial taxa, in elephants with GI symptoms such as a reduction in genera Rubrobacter, Rokubacteria, UBA1819, Nitrospira, and MND1. Conversely, an increase in genera Lysinibacillus, Bacteroidetes_BD2-2, and the family Marinifilaceae was observed when, compared with the healthy group. Variations in taxa of gut microbiota among elephants with GI disorders indicated diverse microbial characteristics associated with different GI symptoms. This study suggests that exploring gut microbiota dynamics in elephant health and GI disorders can lead to a better understanding of food and water management for maintaining a healthy gut and ensuring the longevity of the elephants.

Keywords: Gut microbiome, Elephants, Captive, Gastrointestinal disorder, Colic, Diarrhea

Subject terms: Microbiology, Molecular biology, Zoology, Health care

Introduction

The gut microbiota, which comprises of millions of microorganisms, plays a crucial and central role in various functions within the host. Gut microbiota maintain the integrity of intestinal-endothelial cell barrier, regulate host immune nutrient metabolism, modulate the immune system, and provide defense against pathogens1,2. An imbalance in microbiome homeostasis may arise from alterations in the relative abundance of microorganisms due to certain diseases, leading to changes in the microbiome composition, a condition referred to as dysbiosis3,4. Gastrointestinal (GI) disorders are the most common symptoms found in elephants, and these disorders can be significant and life threatening for animals. According to reports from the National Elephant Institute in Thailand, GI disorders showed a prevalence of 25%5, while those in the Myanmar Timber Enterprise in Myanmar reported an 18.9% incidence rate6. These disorders accounted for 9.8% of overall causes of death in Asian Elephants (Elephas maximus)7.

Colic and diarrhea are common GI symptoms in elephants and can be life-threatening for these animals. Both conditions can present as acute or subacute clinical signs and can be caused by either infection or non-infection factors. Suspected causes of GI discomfort include dietary changes (especially the source of hay), eating novel food items, ingestion of sand and/or rocks, and decreased exercise8. Colic is a broad clinical term that refers to abdominal pain with associated clinical signs such as restlessness, stretching, rolling, and abdominal distension9. This condition can be classified into two types: spasmodic colic and obstructive colic. Spasmodic colic describes a condition where bowel becomes overactive, leading to spasms. In horses, this condition has often been linked to intestinal tapeworm infestation10. Fortunately, spasmodic colic can usually be effectively managed with pain relief and antispasmodic medication11. On the other hand, obstructive colic results from intestinal tract obstruction, often induced by feed impaction, dental problems and/or inadequate chewing12, as well as and or foreign body ingestion13, which impedes normal passage through the intestinal tract. In their natural habitats, impaction colic may be attributed to heavy intestinal helminth infestations14,15. Diarrhea is characterized by loose, watery stools during bowel movements and is closely associated with an altered composition of gut microbiota. In humans and animals, diarrhea often occurs with disruptions in gut microbiota, and infections can also trigger diarrhea16,17. Infection-related diarrhea in elephants is caused by various factors, including bacteria such as Salmonella18, Clostridium19,20, and Escherichia21; protozoa such as Balantidium coli22; and viruses such as elephant endotheliotropic herpesvirus (EEHV)23. These infections can cause enteritis, resulting in inflammation of the intestines. This inflammation leads to severe symptoms such as diarrhea, dehydration, electrolyte disturbance, and metabolic acidosis, potentially resulting in death if prompt treatment was not performed24.

Metagenomics serves as a crucial tool for studying alterations in the gut microbiota in the context of diseases. Previous studies indicated a correlation between changes in specific bacterial groups, which were crucial for maintaining a healthy GI tract, and the occurrence of colic and diarrhea in equines25–27. However, there is limited information available about the gut microbiota of elephants, and even fewer studies have explored its composition and structure under different GI health conditions. Therefore, the goal of this study is to examine and compare the gut microbial composition in healthy elephants and those with GI symptoms, with the goal of filling the gap in understanding the elephant gut microbiota and providing insights into its role in GI health and disease.

Results

Analysis of microbial diversity in healthy elephants and elephants with GI symptoms

The differences in alpha diversity between gut microbiota of healthy elephants and those experiencing GI disorders (i.e. colic and diarrhea) were examined using of Pielou’s evenness, Observed features, Faith’s PD, and Shannon’s index to estimate species richness and evenness (Fig. 1). Alpha diversity, measured by Observed features and Shannon’s index, was significantly lower in elephants with colic and diarrhea than in healthy elephants (q < 0.05), with comparisons of colic and healthy yielding adjusted p-values of 0.006 and 0.01, respectively, and comparisons of diarrhea and healthy yielding adjusted p-values of 0.009 and 0.01, respectively (Fig. 1B,D, Supplementary Table 1). Meanwhile, alpha diversity analyses, including Pielou’s evenness (Fig. 1A) and Faith’s PD (Fig. 1C), showed no significant differences among the health groups. These findings suggested that GI disorders in elephants have an impact on the alpha diversity of their gut microbiota. In contrast, there was no difference in alpha diversity between elephants with colic and those with diarrhea. The compositional similarities of gut microbiota among distinct groups were assessed through beta diversity and represented via principal coordinate analysis (PCoA) plots, using of Bray–Curtis, Jaccard, Unweighted UniFrac, and Weighted UniFrac (Fig. 2). The Bray–Curtis (Fig. 2A) and Jaccard (Fig. 2B) analyses indicated no statistically significant differences in gut microbiota composition between healthy elephants and those with GI disorders. However, both Unweighted (Fig. 2C) and Weighted UniFrac (Fig. 2D) analyses revealed a statistically significant distinction between these groups, with a q-value < 0.05 in the pairwise PERMANOVA test. Specifically, comparisons of normal and colic yielding q-values of 0.003 and 0.003, respectively, and comparisons of normal and diarrhea yielding q-values of 0.006 and 0.003, respectively (Supplementary Table 2).

Fig. 1.

Fig. 1

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Alpha diversity of gut microbiota of captive infant Asian elephants categorized by GI status. Alpha diversity with (A) Pielou’s evenness, (B) Observed features, (C) Faith’s PD, (D) Shannon’s index, by Kruskal–Wallis test with significance threshold of * p < 0.05, ** p < 0.01.

Fig. 2.

Fig. 2

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Beta diversity of gut microbiota of infant captive elephants in Northern Thailand categorized GI status. Beta diversity including (A) Bray–Curtis, (B) Jaccard, (C) Unweighted UniFra, and (D) Weighted UniFrac.

Composition analysis of the gut microbial community in healthy elephants and elephants with GI symptoms

The taxonomy of gut microbiota was determined based on the data from the hypervariable region V3-V4 of the 16S rRNA gene. A total of forty-one phyla and 792 genera of gut microflora were identified within the complete set of elephant fecal samples, and specific details regarding the phyla, families and genera were described in Table 1. The relative abundance of gut microbiota at the phylum, family, and genus levels across all age groups is presented in Fig. 3.

Table 1.

The abundance of elephant gut microbiota at the phylum, family, and genus levels, categorized by health condition.

Group Phylum Family Genus
Normal 19 99 191
Colic 27 162 262
Diarrhea 34 186 302
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Fig. 3.

Fig. 3

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Comparison of the relative abundance of elephant gut microbiota at the (A) phylum, (B) family, and (C) genus levels. Elephants categorized by GI status included normal elephants, elephants with colic, and elephants with diarrhea.

Compositional analysis revealed that the gut microbiota of healthy elephants was predominantly composed of the phylum Firmicutes (61%), followed by Bacteroidota (22%), Actinobacteria (6%), Spirochaetota (4.7%), Proteobacteria (1%), and Euryarchaeota (0.7%), in descending order of abundance (Fig. 3A). Elephants with GI symptoms exhibited a gut microbiota composition predominantly dominated by Firmicutes (57% in colic cases and 54% in diarrhea cases) (Fig. 4A), accompanied by a significant reduction in the relative abundance of Bacteroidota, which was 14% in both colic and diarrhea groups (Fig. 4B). Accordingly, the Firmicutes/Bacteroidota (F/B) ratio in elephants afflicted with GI disorders increased to 4.04 ± 0.815 and 3.79 ± 0.922 for colic and diarrhea, respectively. Notably, the F/B ratio in elephants with colic was significantly higher than that in healthy elephants, which had a ratio of 2.79 ± 0.560 (p < 0.01) (Fig. 4C). Moreover, increases in the abundances of Proteobacteria and Acidobacteriota, and the emergence of Euryarchaeota were observed, alongside the presence of Myxococcota and Chloroflexi, in elephants afflicted with GI disorders (Fig. 3A). A comprehensive list of relative abundances is provided in Supplementary Table 3.

Fig. 4.

Fig. 4

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The number of relative abundance (%) of major gut microbiota phyla: (A) Firmicutes, (B) Bacteroidota, and (C) Firmicutes to Bacteroidota (F/B) ratio, compared among health status groups by Kruskal–Wallis test with significance threshold of * p < 0.05, ** p < 0.01.

In elephants with normal GI health, the gut microbiota was predominantly composed of families such as Lachnospiraceae, Oscillospiraceae, Rikenellaceae, Christensenellaceae, Anaerovoracaceae, Prevotellaceae, Spirochaetaceae, Clostridiaceae, and Streptococcaceae. However, in elephants experiencing GI disorders, the relative abundance of these families varied significantly across individuals, indicating fluctuations in microbial composition associated with GI disturbances (Fig. 3B). At the genus level, Christensenellaceae_R-7_group, an unidentified genus of the family Lachnospiraceae, Rikenellaceae_RC9_gut_group, NK4A214_group of family Oscillospiraceae, Treponema, p-251-o5 of order Bacteroidales, Sarcina, and Methanobrevibacter were predominant in the gut microbiota composition of healthy elephants (Fig. 3C). Meanwhile, elephants with GI problems showed a decrease in these genera proportion but exhibited increase in the abundance Methanobrevibacter (Fig. 3C). Interestingly, in one elephant afflicted with colic, there was a noticeable increase in the Streptococcus genus from the Streptococcaceae family (Fig. 3B,C).

Elephants with GI disorders exhibited a markedly distinct composition of gut microbiota in comparison to healthy elephants

Differential abundance analyses were carried out employing the Analysis of Composition of Microbiomes with Bias Correction (ANCOM-BC) method to ascertain phyla, families, and genera exhibiting notable differences in abundance across varying health statuses. The results detailing significantly disparate taxa at each level of fecal microbiota are illustrated in Fig. 5, whereas exhaustive information regarding all statistically significant taxa is available in Supplementary Table 4. Normal GI elephants were utilized as the reference group for comparison.

Fig. 5.

Fig. 5

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The differential abundance of gut microbiota of captive elephants categorized by their GI status: normal elephants, elephants experiencing colic, and elephants suffering from diarrhea. The data are presented through Log fold change, with healthy elephants serving as the reference group. Only log fold changes exceeding 0.5 are visualized in this figure. Full lists of significant differential abundances are provided in Supplementary Table 4.

In elephants afflicted with GI disorders, the abundances of the phyla Nitrospirota, Myxococcota, Methylomirabilota, Gemmatimonadota, Chloroflexi, and Acidobacteriota were lower than those observed in elephants with healthy GI tracts. Additionally, the abundances of the phyla NB1-j and Entotheonellaeota decreased only in elephants that experienced colic, whereas those of the phyla RCP2-54 and Crenarchaeota were notably lower only in elephants afflicted with diarrhea than in those with normal GI function (Fig. 5).

Taxa from the families including Vicinamibacteraceae, S0134_terrestrial_group, Rubrobacteriaceae, Rokubacteriales, Pyrinomonadaceae, Nitrospiraceae, Nitrosomonadaceae, Microscillaceae, MB-A2-108, Haliangiaceae, Gemmatimonadaceae, Dongiaceae, uncultured taxa of order Gaiellales, uncultured taxa of order Vicinamibacterales, Chitinophagaceae, Blastocatellaceae, bacteriap25, Anaerolineaceae, and 67–14 of order Solirubrobacterales presented lower abundances in elephants suffering from GI disorders. Conversely, the abundances of the families Planococcaceae and Bacteroidetes_BD2-2 in elephants with GI symptoms were greater than those in elephants with normal GI health (Fig. 5).

At the genus level, Vicinamibacteraceae, UBA1819, S0134_terrestrial_group, Rubrobacter, Rokubacteriales, RB41, Pedosphaeraceae, Nitrospira, MND1, MB-A2-108, Haliangium, Dongia, uncultured taxa of the families Gemmatimonadaceae, uncultured of the order Vicinamibacterales, bacteriap25, and 67-14 of the order Solirubrobacterales presented a decreased abundance in elephants with GI symptoms compared to those with healthy GI tracts. In contrast, taxa from the genera including Lysinibacillus of the family Planococcaceae, uncultured taxa of the family Marinifilaceae, and Bacteroidetes_BD2 - 2 of the family Bacteroidetes_BD2 - 2 in elephants afflicted with GI disorders were found to be more abundant in elephants afflicted with GI disorders than in their healthy counterparts. Moreover, the genus Agathobacter was more abundant solely in elephants suffering from colic, whereas the genera Solibacillus, MVP-15, Coriobacteriales, and Butyrivibrio were significantly more abundant only in elephants with diarrhea than in those with healthy GI tracts (Fig. 5).

Noticeable variations in gut microbiota composition were noted between individuals presenting with colic and those presenting with diarrhea. Specifically, elephants that experienced diarrhea presented a decrease abundance of phylum Crenarchaeota, the families Pyrinomonadaceae and Nitrososphaeraceae, as well as the genera Rokubacteriales and Erysipelotrichaceae_UCG-009, compared with elephants that experienced colic (Fig. 5).

Prediction functions of elephant gut microbiota in healthy elephants and elephants with GI symptoms

Functional Annotation of Prokaryotic Taxa (FAPROTAX) was employed to investigate the metabolic functions of microbial communities based on their taxonomic composition. This analysis provided insights into how shifts in microbial communities are associated with GI disorders in elephants. The gut microbiota in elephants primarily engaged in anaerobic chemoheterotrophy and fermentation processes (Fig. 6A).

Fig. 6.

Fig. 6

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Barplot of predicted microbial functions derived from the FAPROTAX database. (A) Relative abundance (%) of the top 20 predicted functional categories. (B) Predicted functional categories presenting mean values and error bars representing standard error (SE). Statistically significant differences (p < 0.05) were determined using one-way ANOVA with Bonferroni correction.

Significant differences were observed in the predicted functional categories among the normal, colic, and diarrhea elephants. Elephants with GI disorders showed an increase in functions related to human gut function, human-associated functions, and mammalian gut functions compared to healthy elephants. This finding suggests a potential enrichment of pathogenic or dysbiotic taxa in these groups. Conversely, functions related to sulfate respiration and the metabolism of sulfur compounds were significantly reduced in the colic and diarrhea groups compared to the healthy elephants, indicating a disruption in sulfur metabolism under diseased conditions (Fig. 6B). This suggests that acute GI symptoms can significantly impact the functional profile of elephants’ gut microbiota.

Discussion

Recent advances in metagenomics have brought significant attention to the investigation of the gut microbiota in Asian elephants. Previous studies have highlighted a strong association between GI disorders and alterations in gut microbiota in both humans and horses28–30. Our study is the first to characterize and compare the gut microbiota composition in healthy Asian elephants and those afflicted with colic and diarrhea, utilizing high-throughput sequencing to analyze the fecal microbiome. We found that the bacterial community structures in elephants with GI disorders differed significantly from those in healthy elephants. Healthy elephants presented a relatively uniform bacterial composition, suggesting a balanced microbial community in the intestines. However, elephants with GI disorders presented changes in the abundance of certain bacterial species. Additionally, noticeable variations in gut microbiota were observed between elephants suffering from impaction colic and those suffering from diarrhea. Specifically, elephants with GI symptoms exhibited reduced gut bacterial diversity, with lower species richness and variable evenness compared to healthy elephants.

As expected, elephants with GI disorders presented a prompt decrease in alpha diversity in their microbiota compared with those with normal GI function. Additionally, significant differences were observed in phylogenetic relationships and the relative abundances of microbial species in the guts of elephants with GI symptoms compared with healthy elephants, as indicated by both the Unweighted and Weighted UniFrac measures. These findings suggest a less diverse and potentially less healthy gut microbiota in elephants with GI symptoms, similar to the findings from prior research on captive elephants with colic31. The reduction in diversity might result in dysbiosis, characterized by an imbalance in gut microbiome composition32,33. The factors that influenced these changes vary depending on the specific disease context. Similar reductions in gut microbiota diversity were observed in humans with GI disorders such as acute diarrheal disease34, and inflammatory bowel disease (IBD)35. Interestingly, a study by Keady, et al.36 revealed that captive African elephants with recent GI disorders exhibit lower bacterial richness compared to those without, a pattern not observed in Asian elephants. The shorter GI tract of African elephants may contribute to a higher sensitivity to dietary changes37,38 and a greater predisposition to IG issues, supporting observations that GI discomfort is more common in captive African elephants36. In equines, which share anatomical similarities in their GI tracts with elephants, a previous study demonstrated lower alpha diversity in diarrheic horses than in their healthy counterparts27. However, study by Costa, et al.39, suggested that equine colitis might not be consistently correlated with decreased diversity and richness. Changes in the abundance of specific bacterial groups within the gut microbiota of horses experiencing colic and diarrhea highlighted their critical role in maintaining GI health9,40–42.

The gut microbiota of elephants with normal GI function was primarily composed of phylum Firmicutes, followed by Bacteroidota, Actinobacteriota, and Spirochaetota, respectively. In this study, elephants that succumbed to colic and diarrhea exhibited reduced levels of the Bacteroidota phylum compared to healthy elephants, leading to an increase of F/B ratio in afflicted elephants. The phylum Bacteroidota is widely acknowledged as a predominant component of the healthy GI tract in humans43 and various mammalian species44,45. Therefore, a decrease in its relative abundance has been associated with chronic diarrhea in humans46. The F/B ratio is widely recognized as an indicator of intestinal equilibrium. An elevation or decrease in the F/B ratio indicates dysbiosis, commonly associated with obesity and IBD in humans47,48. Additionally, an increased F/B ratio has been reported in herbivorous animals, such as horses with hindgut fermentation, where those with intestinal diseases showed higher ratios compared to healthy horses49. However, relying solely on the F/B ratio for disease assessment is considered inadequate due to its inconsistent and limited systematic changes.

Recent studies found that the gut microbiota of captive and wild Asian elephants predominantly harbors hemicellulose-degrading hydrolases rather than cellulose-degrading ones, indicating a potential source of novel, efficient lignocellulose-degrading enzymes50,51. However, elephants with colic and diarrhea experience disruptions in the delicate balance of commensal microorganisms in the GI tract. Specifically, Rubrobacter spp., associated with lignin degradation52, were less abundant in symptomatic elephants, further reflecting the impact of GI disorders on microbial composition. Rokubacteria spp., non-methanotrophic bacteria positively correlated with pH and total nitrogen content53, were less abundant in symptomatic elephants compared to those with healthy GI tracts. Elephants with GI symptoms also showed a reduction in UBA1819 spp., butyrate-producing bacteria from the family Ruminococcaceae54,55 which are important for gut motility by stimulating the release of gut hormones such as glucagon-like peptide-1 and peptide YY55. These hormones were essential for regulating GI transit time56,57. Consequently, a decrease in these beneficial bacteria could impair digestive efficiency and nutrient absorption, worsening GI symptoms and affecting overall health. In addition to the bacterial genera Nitrospira and MND1, was reduced in elephants with GI disorders. Previous pyrosequencing studies had detected Nitrospira bacteria in pediatric patients with irritable bowel syndrome58, highlighting their potential role as indicators of GI disorders.

Compared to healthy group, elephants with GI disorders showed an increased relative abundance of certain bacterial taxa, including the genus Lysinibacillus (family Planococcaceae), an uncultured genus belonging to the family Marinifilaceae, and the genus Bacteroidetes_BD2-2. The genus Lysinibacillus is known for its beneficial properties, such as inhibiting pathogenic bacteria and promoting beneficial bacteria, thereby enhancing microbial diversity59. In elephants with GI disorders, increased levels of Marinifilaceae, known for their nitrogen-fixing capabilities60,61, may represent an adaptive response aimed at stabilizing nitrogen balance and supporting the gut’s nitrogen cycle amid dysbiotic conditions. In contrast, human studies associate lower levels of Marinifilaceae with IBD62, where increased nitric oxide and reactive nitrogen species create a hostile gut environment that disrupts microbial diversity and function60,63. The contrasting abundance of the family Marinifilaceae between elephants with GI disorders and humans IBD highlights potential differences in gut microbiome adaptations across species. The observed differences suggest that in some elephants with food impaction, higher proportions of Marinifilaceae might offer temporary benefits by buffering the nitrogen cycle in response to nitrate-rich diets, as they may have ingested grass grown in areas close to fertilized crops, potentially leading to elevated nitrate levels in their feed. While these bacteria may contribute to nitrogen metabolism and resilience against dysbiosis, their elevated levels in a disrupted gut environment could also exacerbate microbial imbalances, thus playing a dual role in gut health.

The function prediction analysis revealed significant metabolic shifts in elephants with GI disorders. Both healthy and elephants with GI disorders showed bacterial functional groups primarily involved in energy generation and fermentation. However, elephants with GI disorders exhibited an increase in functions associated with human gut activity, human-associated processes, and mammalian gut functions compared to healthy elephants. These findings suggest that the altered gut environment in elephants with GI disorders promotes the growth of microbial species commonly found in human and mammalian guts. The enrichment of these functions may reflect a shift toward microbial communities typically associated with dysbiosis in elephants. Additionally, the increased presence of nitrate-reducing bacteria (NRB) in the gut of sick elephants might reflect a response to altered nitrate levels caused by dietary changes or a disrupted gut environment60. NRB convert nitrate to nitrite, producing nitric oxide, which can enhance blood flow, contribute to hypoxic signaling, and reduce inflammation60,64. This response might be compensatory, as elevated nitrate levels in GI disorders likely promoted NRB proliferation, potentially mitigating some adverse effects while contributing to dysbiosis.

The reduction of sulfate-reducing bacteria (SRB) in elephants with GI disorders likely results from disturbances in the gut environment, reflecting distinct dietary and physiological differences between humans and elephants. SRB typically thrived in neutral pH conditions, but GI disorders might have created more acidic environments65. In humans, sulfur is primarily sourced from animal-based proteins66, contributing to essential processes like protein synthesis and metabolism of bile acids67. In IBD sulfur metabolites, especially hydrogen sulfide (H₂S)68,69, are elevated and exacerbate inflammation and dysbiosis70. Elephants, however, consume a fiber-rich diet where sulfur comes mainly from plant-derived amino acids like cysteine and methionine71. In diseased states, elephants show a reduction in sulfate respiration and sulfur metabolism, indicating a suppression rather than an intensification of sulfur processing. Plants also defend themselves from herbivory using sulfur-containing compounds that become toxic upon tissue damage, which may impact herbivores’ gut environments71,72. Thus, while sulfur metabolism amplifies inflammation in human GI disorders, its disruption in elephants may reflect a distinct vulnerability tied to their high-fiber diet and reliance on microbial sulfur metabolism. In GI disorders, altered gut conditions may shift the microbial community structure, leading to changes in NRB and SRB activity and impacting overall gut health. Understanding these dynamics was crucial for developing therapeutic strategies that modulated microbial activity and gut health. Future metagenomic studies will be vital for distinguishing core microbial functions across species and improving our understanding of gut microbiota interactions with host health.

Comparative analysis of gut microbiota diversity between elephants with colic and those with diarrhea showed no significant differences in either alpha-diversity or beta-diversity. However, differences in specific bacterial taxa were observed between elephants afflicted with colic and those with diarrhea. Elephants with colic showed reduced abundance of the families Pyrinomonadaceae and Nitrospiraceae, and the genera RCP2-54 and Erysipelotrichaceae_UCG-009, compared to those with diarrhea. These findings suggest that colic and diarrhea are associated with distinct microbial signatures, likely reflecting differences in their underlying causes. Diarrhea might result from bacterial infections or dietary factors, whereas colic can stem from conditions such as impaction colic or constipation, each impacting the elephant gut microbiota differently. Nevertheless, notable fluctuations in microbial taxa were observed in one elephant afflicted with food impaction colic, characterized by a marked increase in the relative abundance of the Streptococcus genus. Streptococcus spp., commonly found in the normal enteric flora, was frequently associated with opportunistic infections and lactic acid production in horses73. An overabundance of the Streptococcus genus has been reported in horses with both large and small intestinal colic49,74, and strongly linked to high-concentrate forage diets73. This elephant was fed a dietary supplement, including feed pellets, alongside its usual roughage diet, which may have contributed to the overgrowth of Streptococcus spp. However, the observed discrepancies could be age-related, as the affected elephant was a juvenile while the others were adults. Age-related differences can significantly impact gut microbiota composition75,76, potentially complicating interpretation of the results. Future research is needed to comprehensively understand the extent of this influence.

In captivity, wild animals are exposed to environments and lifestyles that differ drastically from their natural habitats due to extensive human manipulation of their surroundings, diet, healthcare, and social interactions77. Previous studies have linked captivity to various health issues, including weight loss, immune system alterations, reduced reproductive capacity78,79, and induced microbiome alterations in captive Asian elephants80. Emerging research across multiple biological fields underscores the microbiome’s potential role in mediating these health conditions in captivity78. Diet and water management also play critical roles in GI health. Variations in diet, such as changes in forage type, hay sources, or novel foods, may contribute to dysbiosis, influencing the gut microbiota and GI symptoms in elephants8,81. High-fiber diets are recommended to prevent colic and support GI health82. Therefore, it is crucial to explore new methods to enhance animal welfare in captivity. Focusing microbiome research and interventions on a species-specific basis, while comprehensively consideration of immune, behavioral, reproductive, and metabolic health, can lead to more effective management and conservation strategies78.

This study faced several limitations, including the age and sex of the elephants, which are known to influence gut microbiota composition. Additionally, the variability in disease etiology among the subjects, including bacterial or viral infections, parasitic infestations, toxic ingestion, and other underlying health conditions, introduced significant heterogeneity into the study population. This variability makes it challenging to attribute observed changes in the microbiota solely to specific disease conditions. Future research should aim to control for these variables to better understand the relationship between the intestinal microbiota and GI disorders in elephants.

Conclusion

This study significantly enhances our understanding of the gut microbiota in Asian elephants, particularly those with GI disorders. Utilizing high-throughput sequencing, we observed distinct microbial profiles in healthy elephants compared with those succumbed from colic and diarrhea, indicating dysbiosis in afflicted animals. While healthy elephants exhibited a relatively uniform bacterial composition, those with GI disorders presented notable changes and unique patterns in the abundances of specific bacterial taxa. The observed increase in certain beneficial bacteria in ill elephants warrants further investigation to understand their potential role in the pathogenesis and their interactions with other gut microbes. Future research should focus on larger sample sizes and more homogenous study populations to address current limitations and provide deeper insights into the relationship between gut microbiota and GI health in elephants. Such research could pave the way for developing targeted interventions aimed at improving the well-being of these animals.

Methods

Animals and sample collection

This study was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand (FVM-ACUC; R3/2563) and all experiments were performed in accordance with relevant guidelines and regulations. All methods used in this study are reported in accordance with ARRIVE guidelines.

A total of unrelated 18 elephants were included in this study, comprising of seven healthy captive elephants, seven that were diagnosed with food impaction colic, and four exhibiting diarrhea. Among the four diarrheic elephants, one was found to have intestinal parasites, another was affected by geophagia, and the etiology of the other two cases was undetermined. Comprehensive information regarding the cause of disease for each elephant is detailed in Table 2. The elephants in this study were housed in elephant tourist camps in Chiang Mai, Thailand, where they regularly interacted with their mahouts and tourists. They were managed under consistent conditions, including diet and housing. The primary diet of most elephants consisted of Napier grass (Pennisetum purpureum), with only one individual primarily fed corn stalks (Zea mays L.) supplemented with elephant feed pellets. Seasonal supplements, including banana, sugarcane, mango, tamarind, watermelon, and corn, were also provided. Comprehensive details of dietary management for each elephant are presented in Table 3. Clinical health was assessed by veterinarians specializing in elephants through comprehensive historical and clinical examinations. Healthy elephants had no reported GI symptoms and had not been exposed to antibiotics or other drugs for at least six months prior to the study. Elephants with colic exhibited symptoms such as rapid abdominal bloating, abdominal discomfort, absence of defecation for 12 h, and decreased alertness. Those with diarrhea exhibited a decrease in appetite, sudden watery stools, and lethargy, without fever. Importantly, these elephants had been feeding normally on roughage until the evening before the onset of symptoms. Samples from elephants with GI symptoms were collected before treatment began, preferably before the administration of antibiotics.

Table 2.

Elephant characteristics, GI problem etiology, and camp locations.

Elephant ID Age (years) Sex GI status Causes of GI problems Elephant camp
BCG401 15 Female Normal – Mae Taeng district, Chiang Mai, Thailand
BCG402 20 Female Normal – Mae Taeng district, Chiang Mai, Thailand
BCG403 20 Female Normal – Mae Taeng district, Chiang Mai, Thailand
BCG405 20 Male Normal – Mae Taeng district, Chiang Mai, Thailand
BCG406 40 Female Normal – Mae Taeng district, Chiang Mai, Thailand
BCG407 40 Female Normal – Mae Taeng district, Chiang Mai, Thailand
CL01 45 Female Colic Food impaction Mae Taeng district, Chiang Mai, Thailand
CL02 22 Female Colic Food impaction Mae Sa district, Chiang Mai, Thailand
CL03 19 Female Colic Food impaction Mae Taeng district, Chiang Mai, Thailand
CL04 14 Female Colic Food impaction Mae Taeng district, Chiang Mai, Thailand
CL05 17 Female Colic Food impaction Mae Taeng district, Chiang Mai, Thailand
CL06 23 Male Colic Food impaction Mae Taeng district, Chiang Mai, Thailand
CL07 5 Male Colic Food impaction Mae Wang district, Chiang Mai, Thailand
DI01 3 Male Diarrhea Unknown Mae Taeng district, Chiang Mai, Thailand
DI02 42 Male Diarrhea Intestinal parasite Mae Sa district, Chiang Mai, Thailand
DL03 4 Female Diarrhea Geophagy Mae Taeng district, Chiang Mai, Thailand
DL04 1 Female Diarrhea Unknown Mae Taeng district, Chiang Mai, Thailand
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Table 3.

Diet management and feed types for elephants in this study.

Elephant ID Diet volume (kg/day) Feeding time** (meal per Day)
Roughage type Supplementary
Napier grass Corn stalk Banana Sugarcane Mango Tamarind Water melon Corn Feed pellets*
BCG401 160 0 10 8 2 0.5 0 5 0 3
BCG402 200 0 10 8 2 0.5 0 5 0 4
BCG403 200 0 10 8 2 0.5 0 5 0 4
BCG405 200 0 10 8 2 0.5 0 5 0 4
BCG406 200 0 10 8 2 0.5 0 5 0 4
BCG407 200 0 10 8 2 0.5 0 5 0 4
CL01 200 0 5 10 0 0 0 0 0 4
CL02 210 0 10 5 0 0 0 0 0 4
CL03 180 0 10 8 2 0.5 0 5 0 4
CL04 140 0 10 8 2 0.5 0 5 0 4
CL05 180 0 10 8 2 0.5 0 5 0 4
CL06 200 0 10 8 2 0.5 0 5 0 4
CL07 80 0 5 5 0 0 0 0 0 4
DI01 60 0 10 0 0 0 5 0 0 3
DI02 240 0 5 5 0 0 0 0 0 Ad libitum
DL03 0 80 5 5 0 0 0 2 0 3
DL04 0 40 3 2 0 0.5 1 1 1 3
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*Elephant feed pellet (Erawan, Charoen Pokphand Foods, Thailand) (contained more than 8% protein, 2% fat, and not more than 20% roughage).

**Feeding time was defined as follows: 3 meals per day at 08:00, 12:00, and 16:00; 4 meals per day at 08:00, 12:00, 16:00, and 20:00.

Fresh fecal samples (approximately 50 g) were collected directly from the rectum or immediately post-defecation and promptly stored in a cooler box at 4℃ and transported to the laboratory within 3 h. Once in the laboratory, the samples were immediately transferred to a freezer at − 20℃ until DNA extraction, which employed a commercial genomic DNA isolation kit, specifically the QIAamp PowerFecal Pro DNA Kit by QIAGEN (Germany). To analyse changes in the gut bacterial community, bacterial genomic DNA was amplified, targeting the hypervariable region V3-V4 of the 16S rRNA gene using specific primers (F 5′-CCTAYGGGRBGCASCAG-3′, R 5′-GGACTACNNGGGTATCTAAT-3′). This was followed by sequencing using paired-end reads method on an Illumina MiSeq platform. The processes of DNA amplification, data quality control, and sequencing were meticulously carried out by Novogene Inc (Singapore City, Singapore). Employing a double-blind study design with systematically categorized samples was instrumental in mitigating potential biases.

Bioinformatics and data analysis

The initial data from Illumina MiSeq sequencing were performed a quality assessment to obtain effective data. DNA sequence data from NGS were processed using the Quantitative Insights Into Microbial Ecology 2 (QIIME 2 version 2023.2.0) open-source software83. The raw datasets containing pair-ended reads and quality scores were merged, denoised, trimmed according to quality scores, and assigned to amplicon sequence variants (ASVs) by using q2‐dada2 plugin version 2023.2.084. A feature table including the number of each ASV per sample was also generated85. The ASVs were aligned with MAFFT and used to generate a phylogenetic tree for further analysis86,87. The diversity analyses were conducted with rarefication at the sequencing depth of 23,000 as this is the minimum reads to retain all the samples in this study88. The alpha diversity measures, including Observed features, Pielou’s evenness89, Faith’s PD90, and Shannon’s index91 were calculated and analysed using Kruskal–Wallis test in QIIME2. The beta diversity including Bray Curtis, Jaccard, unweighted UniFrac92, and weighted UniFrac93 distance matrices were analyzed and were illustrated as (PCoA). The taxonomy was assigned to ASVs by using q2‐feature‐classifier classify‐sklearn naïve Bayes taxonomy classifier94 by using the data reference from SILVA database version 13895. For visualization of population structure and relative abundance, taxonomical bar plots indicating the relative abundance at the phylum, class, and genus levels of each sample from the different age groups were generated. The differences in taxon abundance between categories and the associations between taxa were estimated with a statistical framework: analysis of the composition of microbiomes with Bias Correction (ANCOM-BC)96. The data visualization was conducted through R version 4.4 by using ‘qiime2R’ package. Functional capabilities of the microbial community were predicted using FAPROTAX based on 16S rRNA gene data and nd KEGG functional databases. A one-way analysis of variance (ANOVA) with Bonferroni correction (p < 0.05) was performed using R software.

Supplementary Information

Supplementary Table 1. (17.4KB, docx)
Supplementary Table 2. (15.7KB, docx)
Supplementary Table 3. (208.9KB, xlsx)
Supplementary Table 4. (233.8KB, xlsx)

Acknowledgements

We would like to express our gratitude to the Center of Excellence in Cardiac Electrophysiology Research, Faculty of Medicine; the Center of Elephant and Wildlife Health Animal Hospital, Faculty of Veterinary Medicine; and the Erawan HPC Project, Information Technology Service Center (ITSC), Chiang Mai University, Chiang Mai, Thailand for their support. We also appreciate the elephant camp owners, their managers, and the mahouts for providing samples and data.

Author contributions

S.Kl., N.C., S.C.C., and C.T. conceptualized the idea and designed the research. S.Kl. and S.Ke. handled the preparation of materials and conducted the measurements. S.Kl., C.K., and S.S. analyzed and investigated the data. S.Kl. wrote the original draft manuscript. S.Kl., C.K., N.C., S.C.C., and C.T. reviewed and edited the manuscript. N.C., S.C.C., and C.T. secured the funding. N.C., S.C.C., and C.T. supervised the research.

Funding

This study was supported by the Thailand Research Fund (C.T.), the CMU Presidential Scholarship and Chiang Mai University (grant number 59/2565 to S.Kl.), the Distinguished Research Professor Grant from the National Research Council of Thailand (N42A660301 to S.C.C.); the Research Chair Grant from the National Research Council of Thailand (N42A670594 to N.C.); and a Chiang Mai University Center of Excellence Award (N.C., C.T.)

Data availability

The raw 16S rRNA gene sequence data and metadata files in this study are available in the NCBI sequence read archive under the accession number PRJNA1144818.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Siriporn C. Chattipakorn, Email: siriporn.c@cmu.ac.th

Chatchote Thitaram, Email: chatchote.thitaram@cmu.ac.th.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-85495-0.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1. (17.4KB, docx)
Supplementary Table 2. (15.7KB, docx)
Supplementary Table 3. (208.9KB, xlsx)
Supplementary Table 4. (233.8KB, xlsx)

Data Availability Statement

The raw 16S rRNA gene sequence data and metadata files in this study are available in the NCBI sequence read archive under the accession number PRJNA1144818.

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