Fatigue and impaired performance in athletes is well recognised and has been loosely linked to “overtraining”. Reduced concentration of IgA in the saliva and increased shedding of Epstein Barr virus (EBV) have been associated with intense training in elite athletes.
To determine whether athletes presenting with fatigue and impaired performance had an immune defect relevant to defective containment of EBV infection, and whether a probiotic preparation (Lactobacillus acidophilus) shown to enhance mucosal immunity in animal models could reverse any detected abnormality.
The fatigued athletes had clinical characteristics consistent with re‐activation of EBV infection and significantly (p = 0.02) less secretion of interferon (IFN) γ from blood CD4 positive T cells. After one month of daily capsules containing 2 × 1010 colony forming units of L acidophilus, secretion of IFNγ from T cells had increased significantly (p = 0.01) to levels found in healthy control athletes. A significant (p = 0.03) increase in salivary IFNγ concentrations in healthy control athletes after the one month course of L acidophilus demonstrated in man the capacity for this probiotic to enhance the mucosal IFNγ concentration.
This is the first evidence of a T cell defect in fatigued athletes, and of its reversal following probiotic therapy.
Keywords: fatigue, overtraining, Epstein Barr virus re‐activation, interferon γ deficiency, Lactobacillus acidophilus
Subjects and methods
This study was designed to evaluate the effects of L acidophilus LAFTI®L10 (DSM Food Specialties, Moorebank, NSW, Australia) on immunity in healthy and “fatigued” athletes. All athletes were well trained recreational athletes. The fatigued athletes were self referred to a medical sports clinic complaining of fatigue, recurrent sore throats, and impaired performance. The control group included healthy, well trained recreational athletes who volunteered for this study. Table 11 summarises the characteristics of both groups. The study was conducted as a pre‐post intervention study.
EBV shedding in saliva was assessed using the real time polymerase chain reaction (PCR) technique7 in 24 saliva samples collected from the eight seropositive fatigued subjects (three samples from each subject) taken before and after the course of L acidophilus. The treatment protocol consisted of four weeks of L acidophilus, taken as a daily capsule containing 2 × 1010 colony forming units. Six of 24 saliva samples (25%) from five of the eight (63%) seropositive subjects with fatigue had detectable EBV DNA before the probiotic treatment, whereas afterwards only one of 24 saliva samples (4%) had detectable EBV DNA. The viability of the L acidophilus preparation was monitored and validated throughout the period of the study.
Unstimulated, non‐fasting, whole mixed saliva samples were collected into 5 ml plastic collection tubes and stored at −20°C until analysis. Salivary IgA was measured by enzyme linked immunosorbent assay (ELISA),10 and saliva osmolality by freezing point depression using an osmometer (The Advanced Micro Osmometer, model 3300; Advanced Instruments Inc, Norwood, Massachusetts, USA). Whole blood cultures were performed to measure cytokine secretion (interleukin (IL) 4, interferon (IFN) γ, IL12) as previously described.11 EBV DNA was measured by PCR.7
Statistical analyses were performed in Stata version 8. Data were checked for normality, and outliers and all continuous measures were found to have highly skewed distributions. Box‐Cox power and log transformations (bcskew0 and lnskew0 respectively) were applied to obtain normally distributed variables. The transformations for IFNγ, IL12, and IL4 did not provide distributions appropriate for parametric analysis. IgA and IgA/osmolality ratios were transformed to an approximately normally distributed variable. However, IgA and IgA/osmolality ratios were analysed using non‐parametric methods to be consistent with analyses for IFNγ, IL12, and IL4.
The pretreatment concentrations of salivary IgA and IFNγ and blood CD4 T cell secreted cytokines were compared between the healthy and fatigued groups of athletes using the Ranksum test for independent samples. Changes from pre to post treatment were compared separately for the healthy and fatigued groups, as well as for both groups combined using the Signrank test (for dependent or paired samples). Change scores were calculated by subtracting the pretreatment value from the post‐treatment value for each subject for all outcomes of interest. To determine whether the probiotic effect differed between healthy and fatigued athletes, the differences were compared between healthy and fatigued groups using the Ranksum test. Significance was accepted at p<0.05.
TablesTables 2 and 33 show the concentrations of salivary IgA and IFNγ and whole blood cultured secreted cytokines IFNγ, IL4, and IL12 before and after L acidophilus treatment for the healthy and fatigued groups respectively. There were no significant differences between the healthy and fatigued athletes with respect to salivary IgA (Z = −0.180, p = 0.86), the IgA/osmolality ratio (Z = −0.103, p = 0.92), salivary IFNγ (Z = −0.838, p = 0.40), or for the secretion from whole blood cultures of IL12 (Z = −1.132, p = 0.26) and IL4 (Z = 0.311, p = 0.76). However, the secretion of IFNγ from blood CD4 T cells measured in whole blood culture was significantly lower in fatigued athletes than in the healthy controls (Z = 2.291, p = 0.02).
After four weeks of treatment with L acidophilus, the whole blood culture secretion of IFNγ was significantly higher after probiotic treatment (Z = −2.384, p = 0.02). Similarly, the increase in salivary IFNγ concentration approached significance (Z = −0.878, p = 0.06). There were no significant changes in the combined group for salivary IgA, salivary IgA/osmolality ratio, or secretion of IL4 or IL12 in whole blood cultures after probiotic therapy. There were no other significant changes after the treatment. The changes did not differ between the healthy and fatigued athlete groups, for any variable, although the concentrations of salivary IFNγ after treatment were significantly higher in the healthy group (table 22)) and for whole blood culture secretion of IFNγ in the fatigued group (table 33).
Athletes complaining of fatigue, particularly associated with recurrent infections and declining performance, had significantly less secretion of IFNγ from blood CD4 positive T cells in cultures than healthy control athletes. After treatment with L acidophilus there was a significant increase in secretion of whole blood IFNγ to levels similar to those found in the control athlete group. As IFNγ is a cytokine intricately linked to mechanisms of control of both virus shedding and disease re‐activation, these observations are relevant to putative preventive intervention for relapsing EBV infection.
Review of the illness records of the fatigued athletes showed features consistent with a syndrome of re‐activated EBV infection. Ninety per cent of the fatigued athletes were seropositive for previous EBV infection and 63% of this group were PCR positive for EBV DNA in saliva on one or more occasions before the probiotic treatment. Compared with the healthy control athletes, the fatigued athletes had more frequent and protracted episodes of upper respiratory tract symptoms, usually linked to periods of intense training. In a prospective study of elite swimmers over a 30 day period of intense training, EBV shedding was detected in saliva in two thirds of the subjects studied with physician verified symptoms of viral illness, including sore throat at times of EBV re‐activation.6 Clinical observation of the illness pattern in the presence of EBV shedding, together with negative evidence for alternative aetiologies, suggest that re‐activation of EBV infection is a significant cause of recurrent sore throats and fatigue seen in some athletes after intense training.6 Non‐primary EBV infection has also been suggested as a cause of oropharyngeal symptoms in otherwise healthy young adults.12 Re‐activation of EBV, with the detection of EBV DNA in saliva, has been reported in other groups exposed to physical, psychological, and environmental stressors, including Antarctic winter expeditioners13 and astronauts preparing for space flight.14 The clustering of throat symptoms in athletes seropositive for EBV further supports the linkage.6
The fluctuating nature of virus shedding, the reasonable health of most affected individuals, the association of shedding with recognised stressors, and the known biology of EBV containment, all suggest that a subtle T cell defect in control mechanisms contributes to EBV re‐activation and virus shedding. It is likely that models of virus shedding in serious disease or after cytotoxic therapy with profound suppression of host T cell function have limited value in understanding the pathogenesis of EBV re‐activation in otherwise normal but stressed patients. In the latter, traditional assessments of T cell function are usually normal.
Changes in immune variables in athletes have been studied over the past two decades.1,2 High intensity exercise is associated with transient changes in many measures of immune function, including a fall in salivary IgA,1 and protracted training regimens can cause more persistent reductions in salivary IgA concentrations.2,3 Both the incidence and timing of “sore throats” and EBV shedding show an inverse correlation with salivary IgA.6 It has been proposed that salivary IgA monitoring can identify those at risk of developing recurrent upper respiratory “sore throat” symptoms.1 The demonstration of a low level of secretion of IFNγ from CD4 positive T cells in fatigued athletes suffering recurrent and prolonged episodes of sore throats is likely to be relevant to the pathophysiology of re‐activation EBV disease. The whole blood culture assay has been used to detect subtle but relevant defects in cytokine secretion.11 The presence of reduced IFNγ secretion, with normal IL4 secretion, may reflect a skewed T cell response to dendritic cell signals.15 The “antigen processing cell–CD4 positive T cell” unit, reliant as it is on finely tuned and complex cell to cell communications, would be an appropriate target for modulation and dysregulation by mediators influenced by stressors such as intense exercise. Salivary IgA concentrations are likely surrogate markers of protection, and the suppression of salivary IgA after intense exercise is itself a probable consequence of altered T cell function.
The L acidophilus isolate used in this study enhanced mucosal immunity in a murine model of Candida albicans stomatitis.16 In this model the DBA/2 strain clears infection slowly compared with the BALB/c strain because of less efficient generation of cytokines and subsequent cytokine dependent effector mechanisms.16 Defective cytokine secretion and cytokine dependent effector mechanisms were reversed by treatment with the L acidophilus, as a result of activation of the Peyer's patch dependent common mucosal system.16 The stimulation of IFNγ secretion in the fatigued athletes after L acidophilus administration resembles the situation in the DBA/2 model. A possible mechanism to explain the observed changes is the activation of antigen presenting cells to modulate a defective signal to the T cell receptor. Certain L acidophilus isolates can engage toll‐like receptors on the surface of antigen presenting cells, which in turn affects the balance of signal to the T cell and the subsequent cytokine secretion pattern.17 An increase in IFNγ secretion following polyclonal stimulation of blood lymphocytes in under‐nourished children18 and children with cow's milk allergy19 given different probiotic preparations is consistent with the cytokine secretion patterns in this study of athletes. This study, however, is the first to identify an increase in IFNγ secretion from whole blood cells and an increase in IFNγ concentration in saliva in man, which suggest a possible link between probiotic use in man and the observed reduction of mucosal infection in animal models, where saliva concentrations of IFNγ have increased after ingestion of L acidophilus.16 However, there is a need to document a significant reduction in symptoms in a larger and extended study, a conclusion also suggested by review of data presented in a study of an antiviral agent, Valtrex.7
This study shows for the first time reduced IFNγ secretion in fatigued athletes, a defect that was reversed after treatment with L acidophilus. The relation of these findings to other groups with chronic fatigue illnesses requires further study to clarify mechanisms and define therapeutic guidelines. It is important to extend the present studies to include clinical outcome measures to assist practitioners in developing management strategies for athletes presenting with fatigue, recurrent sore throats, and impaired athletic performance.