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Effects of liposomal-curcumin on five opportunistic bacterial strains found in the equine hindgut - preliminary study

Abstract

Background

The horse intestinal tract is sensitive and contains a highly complex microbial population. A shift in the microbial population can lead to various issues such as inflammation and colic. The use of nutraceuticals in the equine industry is on the rise and curcumin is thought to possess antimicrobial properties that may help to minimize the proliferation of opportunistic bacteria.

Methods

Four cecally-cannulated horses were utilized to determine the optimal dose of liposomal-curcumin (LIPC) on reducing Streptococcus bovis/equinus complex (SBEC), Escherichia coli K-12, Escherichia coli general, Clostridium difficile, and Clostridium perfringens in the equine hindgut without adversely affecting cecal characteristics. In the first study cecal fluid was collected from each horse and composited for an in vitro, 24 h batch culture to examine LIPC at four different dosages (15, 20, 25, and 30 g) in a completely randomized design. A subsequent in vivo 4 × 4 Latin square design study was conducted to evaluate no LIPC (control, CON) or LIPC dosed at 15, 25, and 35 g per day (dosages determined from in vitro results) for 9 days on the efficacy of LIPC on selected bacterial strains, pH, and volatile fatty acids. Each period was 14 days with 9 d for acclimation and 5 d withdrawal period.

Results

In the in vitro study dosage had no effect (P ≥ 0.42) on Clostridium strains, but as the dose increased SBEC concentrations increased (P = 0.001). Concentrations of the E. coli strain varied with dose. In vivo, LIPC’s antimicrobial properties, at 15 g, significantly decreased (P = 0.02) SBEC when compared to 25 and 35 g dosages. C. perfringens decreased linearly (P = 0.03) as LIPC dose increased. Butyrate decreased linearly (P = 0.01) as LIPC dose increased.

Conclusion

Further studies should be conducted with a longer dosing period to examine the antimicrobial properties of curcumin without adversely affecting cecal characteristics.

Background

Horses may suffer from inflammation in their gastrointestinal (GI) tract, such as colic, enterocolitis, diarrhea, and inflammatory bowel disease [1]. It has been reported that seasonal environmental changes, viruses, intestinal parasites, and infectious agents, such as C. difficile and C. perfringens, are a few causes of colitis, or inflammation of the large intestine, and diarrhea [2,3,4]. The intestinal tract contains a highly complex, yet sensitive microbial population, and a shift in this population can lead to serious gastrointestinal issues for horses [2]. C. perfringens, C. difficile, E. coli general and K-12, and Streptococcus bovis/equinus complex (SBEC) are common opportunistic bacteria found in the hindgut. In a study examining the hindgut of horses with starch-induced laminitis, it has been suggested that SBEC could precede the onset of laminitis due to the increased concentrations of Streptococcus spp. isolates [5].

Oral administration of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) to horses, with gastrointestinal disease, is a common practice in veterinary medicine [6]. However, studies examining the effects of NSAIDs suggest that these drugs are associated with adverse gastrointestinal effects [6]. Additionally, studies have shown that NSAIDs, in vitro, can affect hindgut mobility, which could lead to a change in the pH, volatile fatty acid concentration, and overall digestion [7]. Since nonselective NSAIDs are more preferable in equine medicine [6], the potential of additional adverse effects could lead to dysbiosis of the hindgut microbiota. The utilization of nutraceuticals, in an effort to prevent the aforementioned dysbiosis, offer a potential therapy to mitigate the adverse side effects associated with the traditional therapeutic use of NSAIDs.

Curcumin is the active ingredient in turmeric [Curcuma longa], and in human [8], chicken [9], and horse [10] studies it has demonstrated antimicrobial properties. While the targeted nature of the antimicrobial effects are yet unknown in horses; curcumin’s antimicrobial properties may help to minimize the proliferation of opportunistic bacteria in the equine hindgut. However, while curcumin could potentially be an alternative treatment for a wide variety of diseases, it has poor bioavailability [11]. Observed in humans [8], and mice [12], curcumin’s poor bioavailability is due to its hydrophobic properties and quick elimination from the body [12]. However, studies have speculated that encapsulating curcumin in liposomes could increase its bioavailability [12]. The objective of this research was to evaluate the antimicrobial properties of liposomal-curcumin and its effect on cecal characteristics.

Methods

Four cecally-cannulated [13] horses, one gelding and three mares, weighing 522.95 ± 16.59 kg and having a body condition score (BCS) of 5.5 ± 0.5 on a scale of 1-9, with nine being obese, were used for the in vitro batch culture experiment and in the in vivo study. Southern Illinois University Animal Care and Use Committee (Protocol 14-048) approved care and handling of animals used in this study.

In vitro

The in vitro 24 h batch culture examined the effect of dose on bacteria concentrations when supplementing liposomal-curcumin (LIPC). Sixteen 125 mL Erhlenmeyer flasks were randomly assigned one of the following treatments in quadruplicate: 1) LIPC at the recommended dose of 15 g, (15); 2) 20 g of LIPC, (20); 3) 25 g of LIPC (25); or 4) 30 g of LIPC, (30). Based on the recommended dosage of 500 mg/g of turmeric at 15 g per 454.54 kg horse [10], the selected treatments were increased by 5 g, up to twice the recommended dose.

Composited cecal fluid mixed with McDougall’s buffer, at a 1:4 ratio [12], was poured (50 mL) into 16 separate 125 mL Erlenmeyer flasks, degassed with carbon dioxide (CO2), and placed in a water bath at 39 °C. The 125 mL Erlenmeyer flasks also contained 0.50 g [14] of ground alfalfa hay. Flasks were manually shaken every 2 h for 24 h.

Samples were collected at 0 and 24 h, pH measured (Oakton pH 110 Advanced Portable Meter (Vernon Hills, IL), stored in a 15 mL conical tube, and frozen at −80 °C for later analysis, including deoxyribonucleic acid (DNA) extraction (PowerSoil Mo Bio DNA Extraction Kits (Mo Bio Laboratories, Carlsbad, CA) and qPCR (Bio-Rad MyiQ Optical System Software 2.0). A Nano Drop ND-1000 Spectrophotometer (Wilmington, DW) assessed all DNA extractions for concentration and quality prior to PCR.

Performed in triplicates, all real-time PCR runs and each reaction mixture was prepared using Maxima SYBR Green/ROX qPCR (Thermo Scientific, Waltham, MA). The five opportunistic bacteria, E. coli general and K-12 [15] C. difficile [16, 17], C. perfringens [18], and SBEC [19] were amplified using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA), quantity analysis was performed by calculating the absolute value, using the cycle threshold. The primers sequences used are in Table 1.

Table 1 Forward and Reverse Primers used for real time PCR

In vivo

Four cecally-cannulated horses were utilized in a 4 × 4 Latin square to evaluate increasing doses of LIPC on the concentrations of the same opportunistic bacteria stated in the in vitro and to examine cecal characteristics, such as volatile fatty acids (VFA) and ammonia nitrate (NH3) concentrations. One of four treatments: 1) no LIPC, (0); 2) 15 g LIPC, recommended dose, (15); 3) 25 g LIPC (25); or 4) 35 g LIPC (35) for a total of 7500 mg, 12,500 mg, and 17,500 mg, respectively, of the active ingredient dosed daily, were randomly assigned to horses. Horses were fed 0.90-1.36 kg of Strategy® (Purina Mills, St. Louis, MO), once daily at 0600 and the treatments were top-dressed on the grain, for delivery and to maintain a BCS of 5-6. Post grain and treatment consumption, horses were then turned out to pasture (predominantly K31 Tall Fescue) and allowed to graze until 1600. This was the daily procedure with the exception for d 9 of each period, during which they were stalled all day and had ab libitum access to hay and water after complete consumption of Strategy® and treatment.

Each period was 14 days with a 9 d acclimation period and a 5 d withdrawal period [10, 20]. Cecal fluid was collected at 0 h on d 0 and 8, and again on d 9 at 0, 3, 6, 9, 12, 15, 18, and 21 h. Whole cecal contents (100 mL) were collected, pH recorded (Oakton pH 110 Advanced Portable Meter (Vernon Hills, IL), subsampled (15 mL), and immediately frozen for later analysis of opportunistic bacteria. On d nine, after pH was recorded, contents were filtered through eight layers of cheesecloth into a 15 mL collection tube and immediately frozen for later analysis of VFA and ammonia concentrations. Cecal NH3 concentrations were determined by the phenol-hypochlorite procedure [21]. VFA concentrations were determined [22] using a Shimadzu GC-2010 gas chromatograph (Shimadzu Scientific Instruments, Inc., Columbia, MD) and internal standards made with 2-ethyl butyrate [22].

Blood was also collected via jugular venipuncture on d 0 and 8 into a vacutainer serum separator tube and a 7.5% Ethylenediaminetetraacetic acid (EDTA) tube (Coviden, Mansfield, MA) for chemistry panel analysis, and complete blood count analysis, respectively.

Statistical analysis

The bacterial concentrations from in vitro experiment were analyzed as a completely randomized design using the MIXED procedure of SAS (SAS 9.4 Inst., Inc., Cary, NC).

For the in vivo experiment, bacterial concentrations, chemistry panel data, and complete blood count data were analyzed using the MIXED procedure of SAS (SAS 9.4 Inst., Inc., Cary, NC) using the model for a Latin square design with a Tukey post-hoc adjustment. Cecal fermentation data (NH3, pH, and VFA) were analyzed using the MIXED procedure of SAS for repeated measures. An autoregressive covariance structure (AR1 of the MIXED procedure of SAS) was determined to be most appropriate based on Akaike’s Information Criterion. Comparisons of main effects were determined using least square means and Fisher’s protected LSD. Calculation of coefficients for linear orthogonal polynomials with unequal spacing was done using IML of SAS [23]. Significance was set at (P ≤ 0.05) and tendency was set at (P ≤ 0.10).

Results

In vitro

The bacteria concentrations for the in vitro study are summarized in Table 2. Every flask had a pH within the normal equine cecum pH range of 6.5-7.1 [24]. Concentrations of SBEC were significantly lower (P < 0.0001) at the recommended dose (15) when compared to the 20, 25, and 30 dose treatments. E. coli substrain K-12 concentrations increased (P = 0.01) in the 25 and 30 treatments compared to 15 and 20 treatments. Concentrations of E. coli general were significantly less (P = 0.03) for 15, 20, and 30 compared to the 25 treatment.

Table 2 The effects of liposomal-curcumin on bacteria (ng/μL) found in equine cecal fluid, in vitro (24 h)a

In vivo

Based on the results of the batch culture, the authors decided to investigate 15 g, 25 g, and 35 g of 95% LIPC. SBEC bacterial concentrations increased linearly (P = 0.008) as LIPC dose increased (Table 3). However, as the dose of LIPC increased, the concentration of C. perfringens decreased linearly (P = 0.03).

Table 3 Effects of liposomal-curcumin on opportunistic bacteria (ng/uL) found in equine cecal fluid and on cecal fluid characteristics (9 d)a

Cecal fluid pH and ammonia concentration were not significant among treatments (P = 0.82) and (P = 0.21), respectively (Table 3). However, ammonia concentrations decreased numerically in a linear fashion as LIPC dose increased. Valerate was significantly different (P = 0.02) among treatments with 0 having the greatest concentration compared to all other treatments. Moreover, valerate decreased linearly (P = 0.005) as LIPC dose increased. As LIPC dose increased, butyrate and iso-valerate decreased linearly (P ≤ 0.03). However, acetate tended to increase linearly (P < 0.10), as the dose of LIPC increased. Lastly, increasing doses of LIPC tended (P = 0.10) to linearly increase total VFA concentrations when compared to 0.

Discussion

In vitro

Previous work, with human subjects, showed E. coli substrain K-12 possesses curcumin-converting activity, allowing this substrain to utilize curcumin as a substrate for growth [8]. It is possible that in the current study, increasing the dosage of LIPC increased the concentration of curcumin that E. coli general and K-12 could utilize as a substrate thus, allowing for an increase in these bacterial concentrations.

In vivo

Although increasing the dose of LIPC decreased C. perfringens, the observation that increasing the dose also increases SBEC and C. difficile would suggest that there may be no additional benefit of dosing LIPC above the recommended rate and could lead to potential problems, such as an dysbiosis of the hindgut microbiota leading to colic, diarrhea, and enterocolitis. In addition, this would also suggest that the nutraceutical is not compromised in the stomach or small intestine during the digestion process before reaching the cecum.

The tendencies to increase acetate and total VFAs would suggest that a longer dosing period of LIPC might increase fiber digestibility [25]; however, a decrease in butyrate may decrease intestinal lining repair. In addition, when dosing higher than the recommended dose (15 g) for long periods, caretakers should take caution. The numerical decrease in ammonia and isobuytrate along with a decrease in isovalerate suggests that rate of protein degradation may decrease when LIPC is administered above the recommended dose for longer periods.

Conclusion

The utilization of the nutraceutical, liposomal-curcumin, in an effort to prevent microbial dysbiosis, was thought to offer a potential therapy to mitigate the adverse side effects associated with the traditional therapeutic use of NSAIDs. However, the results of this study would suggest that liposomal-curcumin at doses above the recommended rate have the potential to increase the concentration of opportunistic bacteria, which would contribute to microbial dysbiosis rather than mitigate it. These preliminary data provide some insight of the effects of liposomal-curcumin on selected opportunistic bacteria. A more comprehensive and thorough examination of the cecal microbiota is needed understand the antimicrobial effects of the active ingredient in liposomal-curcumin on the equine microbiota. Additionally, further research is needed to assess long-term effects of the active ingredient in liposomal-curcumin on digestion because of the decrease in butyrate production.

Abbreviations

BCS:

Body condition score

CO2 :

Carbon dioxide

CON:

Control

DNA:

Deoxyribonucleic acid

EDTA:

Ethylenediaminetetraacetic acid

GI:

Gastrointestinal

LIPC:

Liposomal-curcumin

NH3 :

Ammonia nitrate

NSAIDs:

Nonselective nonsteroidal anti-inflammatory drugs

SBEC:

Streptococcus bovis/equinus complex

VFA:

Volatile fatty acids

References

  1. Gustafson A. Antibiotic associated diarrhea in horses with special reference to Clostridium difficile. Diss. Swedish Univ Agr Sci. 2004;166.http://pub.epsilon.slu.se/440/1/AGfin_kappa.pdf. (Accessed 20 Jan 2016).

  2. Costa MC, Arroyo LG, Allen-Vercoe E, Stampflii HR, Kim PT, Sturgeon A, et al. Comparison of fecal microbiota of healthy horses and horses with colitis by high throughput sequencing of the V3-V5 region of the 16s rrna gene. PLoS One. 2012;7:e41484.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Julliand V, Grimm P. The microbiome of the horse hindgut: history and current knowledge. J Anim Sci. 2016;64 doi:10.2527/jas2015-0198.

  4. Mackie RI, Wilkins CA. Enumeration of anaerobic bacterial microflora of the equine gastrointestinal tract. Appl Environ Microbiol. 1988;54:2155–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Milinovich GJ, Klieve AV, Pollitt CC, Trott DJ. Microbial events in the hindgut during carbohydrate-induced equine laminitis. Vet Clin Eq. 2010;26:79–94.

    Article  Google Scholar 

  6. Marshell JF, Blikslager AT. The effects of nonsteroidal anti-inflammatory drugs on the equine intestine. Eq Vet J. 2011;43:140–4. doi:10.1111/j.2042-3306.2011.00398.x.

    Article  Google Scholar 

  7. Van Hoogmoed LM, Synder JR, Nieto J, Harmon FA. In vitro evaluation of a customized solution for use in attenuating effects of ischemia and reperfusion in the small intestine of horses. Am J Vet Res. 2002;63:1389–94.

    Article  PubMed  Google Scholar 

  8. Hassaninasab A, Hashimoto Y, Tomita-Yokotani K, Kobayashi M. Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism. Proc Natl Acad Sci. 2011; doi:10.1073/pnas.1016217108.

  9. Lawhavinit QA, Kongkathip N, Kongkathip B. Antimicrobial activity of curcuminoids from Curcuma longa L. on pathogenic bacteria of shrimp and chicken. Kasetsart J Nat Sci. 2010;44:364–71.

    CAS  Google Scholar 

  10. Farinacci M, Gaspardo B, Colitti M, Stefanon B. Dietary administration of curcumin modifies transcriptional profile of genes involved in inflammatory cascade in horse leukocytes. Ital J Anim Sci. 2009;8:84–6. doi:10.4081/ijas.2009.s2.84.

    Google Scholar 

  11. Ukil AL, Maity S, Karmakar S, Datta N, Vedasiromoni JR, Das PK. Curcumin, the major component of food flavour turmeric, reduces mucosal injury in trinitrobenzene sulphonic acid-induced colitis. Brit J Pharmacol. 2003;139:209–18. doi:10.1038/sj.bjp.0705241.

    Article  CAS  Google Scholar 

  12. Prasad S, Tyagi AK, Aggarwal BB. Recent developments in delivery, bioavailability, absorption, and metabolism of curcumin: the golden pigment from golden spice. Cancer Res Treat. 2014;46:2–18. doi:10.4143/crt.2014.46.1.2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Beard WL, Slough TL, Gunkel CD. Technical note: a 2-stage cecal cannulation technique in standing horses. J Anim Sci. 2011;89:2425–9.

    Article  CAS  PubMed  Google Scholar 

  14. Bailey SR, Baillon ML, Rycroft AN, Harris PA, Elliott J. Identification of equine cecal bacteria producing amines in an in vitro model of carbohydrate overload. Appl Environ Microbiol. 2003;69:2087–93. doi:10.1128/AEM.69.4.20872093.2003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lee C, Lee S, Shin S, Hwang S. Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli. Appl Microbiol Biotech. 2008;78:371–6. doi:10.1007/s00253-007-1300-6.

    Article  CAS  Google Scholar 

  16. Avbersek J, Cotman M, Ocepek M. Detection of Clostridium difficile in animals: comparison of real-time PCR assays with the culture method. J Med Microbiol. 2009;60:1119–25. doi:10.1099/jmm.0.030304-0.

    Article  Google Scholar 

  17. Karpowicz E, Novinscak A, Bärlocher F, Filion M. qPCR quantification and genetic characterization of Clostridium perfringens populations in biosolids composted for 2 years. J Appl Microbiol. 2009;108:571–81. doi:10.1111/j.1365-2672.2009.04441.x.

    Article  PubMed  Google Scholar 

  18. Hastie PM, Mitchell K, Murray JMD. Semi-quantitative analysis of Ruminococcus flavefaciens, Fibrobacter succinogenes and Streptococcus bovis in the equine large intestine using real-time polymerase chain reaction. Brit J Nutr. 2008;100:561–8. doi:10.1017/s0007114508968227.

    Article  CAS  PubMed  Google Scholar 

  19. Magdesian KG, Leutenegger CM. Real-time PCR and typing of Clostridium difficile isolates colonizing mare-foal. Vet J. 2011;190:119–23.

    Article  CAS  PubMed  Google Scholar 

  20. Weese JS, Anderson MEC, Lowe A, Monteith GJ. Preliminary investigation of the probiotic potential of lactobacillus rhammosus strain GG in horses: fecal recovery following oral administration and safety. Can Vet J. 2003;44(4):299–302.

    PubMed  PubMed Central  Google Scholar 

  21. Broderick GA, Kang JH. Automated simultaneous determinations of ammonia and total amino acids in ruminal fluid and in vitro media. J Dairy Sci. 1980;63:64–75.

    Article  CAS  PubMed  Google Scholar 

  22. Goetsch AL, Galyean ML. Influence of feeding frequency on passage of fluid and particulate markers in steers fed a concentrate diet. Can J Anim Sci. 1983;63:727–30. doi:10.4141/cjas83-084.

    Article  Google Scholar 

  23. Robson DS. A simple method for constructing orthogonal polynomials when the independent variable is unequally spaced. Int Bio Soc. 1959;15:187–91. doi:10.2307/2527668.

    Google Scholar 

  24. Willard JG, Willard JC, Wolfram SA, Baker JP. Effects of diet on cecal pH and feeding behavior of horses. J Anim Sci. 1997;45:87–93. doi:10.2134/jas1977.45187x.

    Article  Google Scholar 

  25. Bergman EN. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev. 1990;70:567–90.

    CAS  PubMed  Google Scholar 

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Authors’ contributions

SB – Made substantial contributions to conception and design, acquisition of data and analysis, and interpretation of data. Involved in drafting the manuscript, revised it critically for important intellectual content, and gave approval of the version to be published. EV – Made contributions to concept and design and revised the manuscript critically for important intellectual content, and given approval of the version to be published. JM – Acquisition of data and analysis. BA – Made substantial contributions to conception and design, acquisition of data and analysis, and interpretation of data. Involved in drafting the manuscript, revised it critically for important intellectual content, and gave approval of the version to be published. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Ethics approval and consent to participate

Southern Illinois University Animal Care and Use Committee (Protocol 14-048) approved care and handling of animals used in this study.

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Bland, S.D., Venable, E.B., McPherson, J.L. et al. Effects of liposomal-curcumin on five opportunistic bacterial strains found in the equine hindgut - preliminary study. J Anim Sci Technol 59, 15 (2017). https://doi.org/10.1186/s40781-017-0138-4

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