September 25, 2007
Effect of a Probiotics Supplementation on Respiratory Infections and Immune and Hormonal Parameters During Intense Military Training
By Tiollier, Eve Chennaoui, Mounir; Gomez-Merino, Danielle; Drogou, Catherine; Et al
This study examined the effect of a probiotics supplementation on respiratory tract infection (RTI) and immune and hormonal changes during the French Commando training (3-week training followed by a 5- day combat course). Cadets (21 +- 0.4 years) received either a probiotics (n = 24) or a placebo (n = 23) supplementation over the training period. We found no difference in the RTI incidence between groups but a significantly greater proportion of rhinopharyngitis in the probiotic group (p
Military framing environment represents a situation which associates several stressors such as intense physical training, sleep deprivation, energy deficit, and psychological pressure. Singly or in combination these stressors are susceptible to exert detrimental effects on the immune system, and there is a prevalent assumption that this immunocompetence impairment might favors the development of infection and especially respiratory tract infection (RTl). This hypothesis is in accordance with data from studies on military framing related to infection.12
Many attempts have been made to find nutritional supplements that would prevent this framing-induced immune impairment and consequently incidence of infection.3 Probiotics have been shown to reduce respiratory infections and their severity among children4,5 and elderly subjects,6 but data are missing regarding the effectiveness of probiotics use in subjects undertaking intense training.7 To date, the only one study that has investigated the potential benefit of a probiotics supplementation during military framing has focused on gastrointestinal infections.8
Probiotics mechanisms of action remain unclear but one of the constant findings is the enhancement of the immunoglobulin A response.9,10
In recent studies, we noted mucosal immunosuppression and hormonal changes at the end of the French Commando fraining 11,12 - Tj16 purp0se 0f the present study was to investigate the effect of ingestion of fermented milk with the probiotic strain Lactobaculus casei on RTI, mucosal and cellular immune parameters, and hormonal parameters in subjects submitted to a multistressor environment such as the Commando training. It was hypothesized that the probiotics supplementation would lessen the negative impact of this multistressor environment on RTI and on immune and hormonal parameters.
A group of 47 male cadets from the French Military Officer School of Coetquidan in Brittany took part in the French Army Commando Training, including 3 weeks of commando framing followed by a 5-day combat course. The subjects were in good mental and physical condition. The study was approved by a French medical ethics committee (Faculty of Medicine, Paris V, France), and the participants had all given voluntary written consent.
Description of the French Army Commando Training
The 3-week training and the 5-day combat course (Fig. 1) took place at the National Center for Commando Training at MontLouis in the Pyrenees mountains in June. During the training, subjects spent most of their time engaged in realistic combat training in rough terrain, which included heavy physical activities: swimming, walking and running, and avoiding roads, lanes, and frails while carrying backpacks of 1 1 +- 1.2 kg. Several parts of the training involved mountain climbing.
The 5-day course took place from 6 a.m. on day 1 until 6 a.m. on day 6 and most of the physical activities were at night. Since the cadets slept outside at ambient temperatures during the 3 weeks of training, their sleep was disturbed, and since most of the physical activities were at night during the 5-day course, subjects were also sleep deprived.
During the 5-day combat course, subjects were estimated to have physical exercise activities corresponding to 35% of maximal oxygen uptake and a daily energy expenditure exceeding 5,000 kilocalories.13
During the training and the course, the subjects ate the commando ration which consisted of two main courses, bread in the form of biscuit, custard, energy bars, and candied fruit. The ration did not contain any fermented dairy products. In addition to this ration, they received one of two products described below. Water intake was not restricted.
Subjects received one of the two products: either a milk fermented by yogurt cultures and with L. casei strain DN- 1 14 00 1 (herein referred to as probiotics) or a nonfermented milk (herein referred to as placebo). The two drinks had the same taste and the same nutrient composition (2.5 g of protein, 1.5 g of fat, and 17.2 g of carbohydrates/100 mL). Products were manufactured at Danone Vitapole pilot laboratory following standard techniques and hygienic practices for fresh dairy products. Products were delivered in 100- mL portions with identical packaging and were distinguished by letter codes that were not revealed to the investigators. This protocol ensured that the study was performed under double-blind conditions.
The study design was randomized and paralleled probiotics versus placebo in double blind. Subjects were randomly assigned to the placebo (n = 23; 2 1 .3 +- 0.4 years) or the probiotics (n = 24; 21.3 +- 0.2 years) groups. During the 3 weeks preceding the beginning of the training, the subjects were allowed to eat no more than one fermented dairy product per day. Then, from the first day of the training to the last day of the combat course (1 month), subjects received daily 300 mL of product (3 pots of 100 mL). During the period of supplementation, they were asked to avoid other fermented dairy products, which was easy to control since all of the food was provided by the commandment. To assess the compliance, the subjects were asked to report in a logbook the quantity of tested product consumed each day.
Recording of Respiratory Infections
As part of the commando training, cadets were under medical surveillance throughout the study. Every cadet had a medical file in which all medical data were recorded. Moreover, from the begirming of the commando training to the end of the recovery, cadets were requested to report daily, via a logbook, their signs and symptoms of infection, giving detailed information on RTI. Also, as part of the study, medical examinations were carried out on four occasions: before and at the end of the 3-week framing, at the end of the 5- day course and after 1 week of recovery (Fig. 1). Based on the collected data and on the examinations, the medical officers filled in a standardized log sheet giving details on the symptoms (rhinopharyngitis [defined as inflammation of the mucous membranes of the nasal and the pharyngeal cavities], tonsillitis, sinusitis, otitis, bronchitis, pneumonia, and asthma), the severity of the symptoms (mild, moderate, or severe), and the duration of the symptoms so far. More than one symptom could be diagnosed on the same day for any one subject. RTI was recorded when subjects reported infectious symptoms on at least 2 consecutive days. The number of RTI episodes, the number of days with symptoms, the number of symptoms, and the number of occurrences of each symptom for both groups were calculated.
Saliva samples were collected before and after the 3-week training, at the end of the 5-day course, and after 1 week of recovery. The saliva samples were obtained between 7 a.m. and 8 a.m. The subjects were told not to force salivation, and it was not (artificially) stimulated. Immediately after, the saliva sample was placed on ice and then stored at -8O0C until subsequent determinations.
Quantification of Salivary Immunoglobulin A
Salivary immunoglobulin A concentrations were assayed in duplicate with a modified nephelometric method (IGALC, immunoglobulin A low concentration; Immunochemisfry IMMAGE system; Beckman Coulter, Paris, France) generally used for assay of human immunoglobulin A in serum and cerebrospinal fluid. The limit of sensitivity for immunoglobulin A in cerebrospinal fluid was: 0.25 mg - L"1. The infra- and extra-assay coefficients of variation were 4% and 6%, respectively. All samples were determined within the same assay.
Blood samples were collected before training and after the combat course, between 7 a.m. and 8 a.m. Twenty milliliters of venous blood were sampled from an antecubital vein after a 10-minute rest supine. A sampling of 5 mL was transported in a sealed container from the location of collection to the immunology laboratory (Grenoble, Isere, France). The remaining 15 mL were centrifuged at the location of collection, and serum or plasma was collected and stored at - 18O0C in carbonic ice from collection to our laboratory. Serum and plasma were stored at -8O0C in our laboratory and used for subsequent determinations. Quantification of Leukocytes, Lymphocytes, and Their Subsets
Complete blood cell counts were measured from ethylenediaminetetraacetic acid-treated blood using an automated hematology analyzer (Penfra 120; ABX, France). The cell concenfra- tion of each leukocyte subpopulation was calculated using the same apparatus: the lymphocyte concentration allowed us to calculate the respective absolute lymphocyte subset count using the percentage of lymphocyte subsets that was determined by flow cytometry analysis as described below.
Lymphocyte subset markers for CD3+, CD4+, CD8CD19+ (B cells), and CD16+/56+ (NK cells) were analyzed by a Multitest IMK Kit (BD Biosciences, San Jose, CA) with a FACSCalibur flow cytometer (BD Biosciences) using the whole-blood lysis method and dual-color immunofluorescence assay.
Quantification of Hormones
Cortisol, dehydroepiandrosterone sulfate (DHEAS), and prolactin concentrations were assayed in duplicate by radioimmunoassay using commercial kits (DiaSorin; Beckman Coulter, Lineo Research, France). The normal fasting range for lean men was 193 to 690 nmol - L"1 for morning Cortisol, 1,330 to 4,410 ng - mL"1 for DHEAS, and 2.6 to 7.2 ng - mL'1 for prolactin. The limits of sensitivity were: 5.79 nmol - L-1 for Cortisol, 60 ng - mL"1 for DHEAS, and 0.5 ng - mL"1 for prolactin. The intraand extra-assay coefficients of variation were: 7.7% and 9.8% for Cortisol, 4.2% and 7.2% for DHEAS, and 1.58% and 8.03% for prolactin.
Distributions of variables were checked for normality and homogeneity of variances. Results are expressed as median (range) for variables that did not follow a normal distribution and as mean +- SEM otherwise.
The difference in proportion of subjects who presented an infection during the trial between the two groups was assessed by a x^sup 2^ test. Comparisons of the incidence of infection, the number of symptoms, and the number of days with infection between the two groups were carried out using a Wilcoxon test. Analysis of the daily mean number of symptoms per group over the study was assessed by comparing the area under curve with a Wilcoxon test (Fig. 2). Comparisons of proportions of each symptom between groups were analyzed by a x^sup 2^ test.
Changes in salivary immunoglobulin A concentrations during the study were analyzed by a Friedman test (nonparametric test for analysis of variances). If a significant effect was found (p
The effects of supplementation on changes in cellular immune parameters and hormones in response to commando training were assessed using Student's ? test for paired data or a Wilcoxon test (if the variables did not follow a normal distribution).
For all statistical analyses, tests were performed at two-tailed level of significance with a 0.05. All analyses have been performed on the intention-to-treat population.
The compliance for the tested product was very good in both groups and not different between groups (93.5 +- 0.9% vs. 90.9 +- 2.4%, ? = 0.84 for placebo and probiotics, respectively).
Incidence of RTI
In the placebo group, 13 (57%) subjects presented at least one infection during the study, whereas only 1 1 (46%) in the probiotics group did, although this difference is not significant (p = 0.46). The incidence of RTI was not significantly different in the two groups (0.6 +-0.1 vs. 0.8 +- 0.2 episodes for placebo and probiotics, respectively, ? = 0.98). The mean number of days with symptoms was 6.1 +- 1.7 and 5.5 +- 1.6 days for the placebo and probiotics groups, respectively (p = 0.67). The mean number of symptoms was almost double in the placebo group compared with the probiotics group, but this difference was not significant (1.3 +- 0.3 vs. 0.7 +- 0.2 for placebo and probiotics, respectively, ? = 0.23). The daily mean number of symptoms was not significantly different between groups (Fig. 2). During the study, 30 vs. 17 RTI symptoms were recorded in the placebo and in the probiotics group, respectively. The breakdown of these RTI symptoms as a function of the type of symptom for both groups is presented in Figure 3. Results showed a greater proportion of rhinopharyngitis in the probiotics group compared with the placebo group (p
Salivary Immunoglobulin A Concentration
The mean individual percentage of change of the salivary immunoglobulin A concentrations over time for both groups is illustrated in Figure 4. There was no significant difference between the two groups at any sampling time. However, there was a significant decline in salivary immunoglobulin A concentra- tions after the combat course compared with both before training and after training values in the placebo group (p
Total and Differential Leukocyte and Lymphocyte Subsets
Cell number (leukocytes, leukocytes subset, and lymphocytes subset) changes in blood did not differ between the two groups (Table I). However, there was a trend for a greater increase in blood concentration of leukocytes (p = 0.06), neutrophils (p = 0.07), and CD19^sup +^ (p = 0.07) in the placebo group at the end of the combat course compared with the probiotics group, although the p did not reach significance.
The result showed a significant greater increase in mean DHEAS plasma concentrations in the probiotics group compared with the placebo group (p
The purpose of the present study was to investigate the effect of probiotics intake on RTI, mucosal and cellular immune parameters, and hormonal parameters in subjects submitted to a multistressor environment such as the commando training. The results did not show any difference between the two groups on incidence of RTI, although several elements suggest that probiotics might lessen the negative impact of the multistressor environment on host defense.
Analysis of the clinical symptomatology of our subjects during the trial showed that the two groups exhibited different profiles with a significantly greater proportion of rhinopharyngitis in the probiotics group compared with the placebo group. Indeed, the profile of the probiotics group was characterized by a very large majority (70%) of rhinopharyngitis, whereas in the placebo group the diagnosed symptoms were more evenly distributed. Moreover, no otitis was reported in the probiotics group, meanwhile three subjects of the placebo group presented this symptom, although the difference was not significant. This is in keeping with another study which found that probiotics consumption diminished the number of children suffering from respiratory infections with complications, particularly a reduction of 21% in the occurrence of acute otitis media.4 Also, Rio et al.5 demonstrated a decrease in bronchitis and a suppression of pneumonia in children supplemented with Lactobacillus compared to their counterparts drinking only milk. Despite no difference in RTI incidence, our finding, showing that infections were mainly confined to the nasopharyngeal area in the probiotics group, suggests that probiotics consumption had prevented the infection from spreading throughout the respiratory tract.
Concerning immune parameters, no significant difference was observed between groups. However, several interesting trends emerged. For instance, the reduced magnitude of changes of the leukocytes (p = 0.06), neutrophils [p = 0.06), and CD19^sup +^ cells (p = 0.07) at the end of the combat course in the probiotics group was indicative of a less severe impact of the commando training in this group.14 Moreover, probiotics supplementation abolished the combat course-induced decline in salivary immunoglobulin A concentrations observed in the placebo group. The latter observation is suggestive of a stimulating effect of probiotics on immunoglobulin A production that is consistent with previous studies on human9 and animal models.10 Finally, our findings, although not statistically significant, represented a body of elements suggesting that probiotics supplementation had lessen the negative impact of the training on immune parameters.
The mechanisms of action of probiotics remain unclear. Among the possible mechanisms of probiotics are the promotion of the nonimmunologic defense barrier in the gut, the stimulation of the nonspecific immunity, and also an improvement of the intestine's mucosal immune response and particularly intestinal immunoglobulin A response.15 We found that in the probiotics group, infections were mainly confined to the nasopharyngeal area, suggesting that probiotics consumption had prevented the infection from spreading throughout the respiratory tract. Taking into account the fact that, as part of a common mucosal system, an immune response initiated in the gut can provide effective protection at distal mucosal sites,16 it can be hypothesized that probiotics ingestion had resulted in enhanced regional immunity of the respiratory tract. This hypothesis is supported by a recent finding in an animal model which demonstrated that oral administration of lactic acid bacteria, among which L. casei, enhanced immunoglobulin A+ cells on the mucosa surface of the bronchus.10 The fact that probiotics supplementation abolished the combat course-induced decline in salivary immunoglobulin A concentrations observed in the placebo group lends support to this hypothesis. Finally, although attractive, the hypothesis of an enhanced regional mucosal immunity of the respiratory tract by probiotics supplementation remains speculative but provides tracks for further studies investigating the mechanisms of probiotics' protection against infection. Our results revealed a significantly greater increase of DHEAS in the probiotics group compared with the Placebo group at the end of the combat course. To date, to our knowledge, the relationship between probiotics and DHEAS has not been documented. Nevertheless, this finding is in favor of a beneficial effect of probiotics on immunity since DHEAS is known to be an immunostimulatory hormone.17
Moreover, it is interesting to note that probiotics and DHEAS shared some similar effects on immunity such as stimulation of immunoglobulin,9,18 reduction in pro-inflammatory tumor necrosis factor gamma release15,17, and protection against infection.4,19 Although the meaning of this result is not clear, it is of interest and warrants further investigations, in particular, to examine, first, the possible mechanisms underlying the probiotics effects on DHEAS and, second, whether beneficial effects of probiotics could be, in part, mediated by DHEAS.
Finally, the beneficial effect of probiotics intake on RTI during heavy military training seems to rely mainly on its capacity to prevent the infection from spreading throughout the respiratory tract. This could be achieved by an enhancement of regional immunity of the respiratory tract, although this hypothesis remains speculative at this stage. The potential capacity of probiotics to suppress salivary immunoglobulin A decline and to promote DHEAS during strenuous training deserve to be thoroughly examined. It is too early to recommend probiotics as a prophylaxis against RTI in individuals undertaking intense framing with associated stressors; however, our data are promising and warrant further investigations, especially using largescale studies.
We thank Mr. Sautivet for his technical assistance, Mr. Burnat and Dr. Mathieu for analysis of immune parameters, and Dr. Bourrilhon, Dr. Duforez, and Dr. Jouanin for their help with the medical examinations. We acknowledge Mrs. Rolf-Pedersen, Mrs. Tondu, and Mr. Guyonnet for their involvement in this project research.
This research was supported by grants from the General Delegation of Armament (PEA 980816) and from Danone Vitapole.
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Guarantor: Eve Tiollier, PhD
Contributors: Eve Tiollier, PhD*; CDT Mounir Chennaoui*; Danielle Gomez-Merino, PhD*; TLCN Catherine Drogou*; Edith Filaire, PhD[dagger]; GEN Charles Yannick Guezennec*
*IMASSA, Departement Physiologie, BP 73, 91223 Bretigny-sur- Orge, France.
[dagger]Laboratoire LAPSEP, 2 Allee du Chateau, Campus de la source UFR STAPS, 45062 Orleans, France.
This manuscript was received for review in July 2006. The revised manuscript was accepted for publication in April 2007.
Reprint & Copyright (c) by Association of Military Surgeons of U.S., 2007.
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