The effect of long-term ethanol feeding on Brucella melitensis infection of rats

Zeki Yumuk*,, Sitki Ozdemirci1,, B. Faruk Erden1, and Volkan Dundar

Departments of Microbiology and
1 Pharmacology, Kocaeli University Faculty of Medicine, Kocaeli, Turkey

Received 1 November 2000; in revised form 2 February 2001; accepted 15 February 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The adverse effects of ethanol on Brucella melitensis have not been studied previously. In this study, a new model of B. melitensis infection was used in the setting of chronic ethanol administration in rats. It was found that the chronically ethanol-receiving rats exposed to B. melitensis infection had significantly greater numbers of B. melitensis in their spleen and liver than the rats in the control group.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alcoholic patients display an increased susceptibility to bacterial infections. Once such infections develop, they are usually more severe, and some are associated with a higher mortality than that found in the non-alcoholic population (reviewed in Sternbach, 1990). Ethanol exposure adversely affects the infections caused by bacteria such as Listeria monocytogenes (Saad et al., 1993Go), Streptococcus pneumonia (Davis et al., 1991Go; Lister et al., 1993Go), Legionella pneumophilia (Yamamoto et al., 1993Go), and Mycobacterium avium (Bermudez and Young, 1991Go). One of the main reasons for such adverse effects is that ethanol predominantly impairs the ability of mononuclear phagocytes to control the growth of the intracellular organisms (Jerrells and Sibely, 1995). Moreover, the infection with intracellular pathogens is prevalent because immunological abnormalities after both chronic and acute alcohol consumption appear to be consistent with a decreased Th1-type immune response based on reduced antigen-specific T cell proliferation and increased antibody and autoantibody levels (reviewed in Szabo, 1999).

On the other hand, Brucella species and the disease spectrum are partially explained by the ability of the organism to evade host defence mechanisms by virtue of its intracellular existence. The spectrum of disease depends on many factors, including the immune status of the host, the presence of other underlying diseases or conditions and the species of infecting organism (Koneman et al., 1997Go). Brucellosis is a disease of domestic and wild animals (zoonosis) and is transmittable to humans; the disease exists worldwide, especially in the Mediterranean basin, the Arabian Peninsula, the Indian subcontinent, parts of Mexico and Central and South America (Young, 1999Go). This infection is considered to be a problem, because Brucella abortus vaccines do not protect effectively against B. melitensis infection. Thus, bovine B. melitensis infection emerges as an increasingly serious public health problem in some countries (Corbel, 1997Go).

Since the clinical picture in human brucellosis can be misleading and ethanol abuse is a serious health problem, much attention should be paid to these two intersecting groups. Therefore, the aim of the present study was to investigate whether chronic ethanol consumption affects the course of B. melitensis infection in a rat model.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic ethanol administration procedures
Wistar rats weighing 210–290 g were obtained from a local supplier (Kocaeli, Turkey). The rats were housed individually. The rats were then matched by weight and paired so that one rat of the pair received the ethanol diet (n = 17) while the second rat was pair-fed (n = 12) isocaloric liquid diet containing sucrose as caloric substitute of ethanol. The nutritionally adequate liquid diet used in this study was formulated as previously described (Uzbay and Kayaalp, 1995Go). The maximum contribution of ethanol to total energy intake was 42.2% in the diet of ethanol-receiving rats. The liquid diets were prepared daily and given to rats every morning (10:00) throughout the study. For B. melitensis infection studies, rats were maintained on this dietary regimen for 16 days before challenge and 16 days after challenge. Five rats from the ethanol-receiving group were used to determine blood ethanol levels.

Determination of blood-ethanol levels
Blood ethanol levels were determined by headspace gas chromatography (Kumar and Gow, 1994Go). Blood samples were taken by intracardiac puncture from the rats under very light diethyl-ether anaesthesia.

Bacteria and culture conditions
A standard smooth strain of B. melitensis (16M) used in this study was provided by Pendik Institute of Veterinarians (Turkey). Bacteria were cultured on Brucella agar (Difco, Detroit, MN, USA) at 37°C to the logarithmic phase, which was assumed to have occurred after 48-h incubation (stationary phase entered after a 4-day incubation) and stored at 4°C until use. Identification of B. melitensis isolated from experimental animals was based on typical colonial morphology, growth pattern and gram stain appearance.

Experimentally induced B. melitensis infection
Brucella melitensis strain 16M cells were suspended in saline (cells in the logarithmic phase were used) and were adjusted (based on live counts) to yield 2 x 104 to 4 x 104 colony-forming units (CFU). Inoculation was performed by injecting one dose of 0.5 ml saline containing 2 x 105 to 4 x 105 intraperitoneally. Sixteen days after challenge to B. melitensis, rats were weighed and assessed for the weight of the spleen, liver and the number of B. melitensis isolated from those organs. Spleens and livers were aseptically removed, weighed and a small piece of each organ was homogenized in 1.0 ml of sterile saline. Aliquots of 0.1 ml of the homogenates were diluted 10-fold in saline and plated on to Brucella agar plates to obtain a viable count. Plates were incubated at 37°C for 72–96 h and a colony count was performed. Each procedure was repeated in triplicate. The mean count was calculated and expressed as log10. If no bacterial growth was apparent after 4 days of incubation, the plates were incubated for 3 additional days before being considered sterile (modified from Shasa et al., 1994).

The experiments reported in this study were carried out in accordance with the declaration of Helsinki. Ethical approval was granted by Kocaeli University Ethics Committee (Kocaeli, Turkey).

Statistical analysis
The ratio of spleen and liver weight to rat weight and the mean count of bacteria isolated from the ethanol-receiving and pair-fed groups were calculated. The mean count of bacteria and the spleen/body weight and liver/body weight isolated from ethanol-receiving rats were compared to that of pair-fed rats by using Student's t-test for independent groups. P > 0.01 was considered to be insignificant. The relationship of daily ethanol consumption and the number of B. melitensis in spleen and liver were analysed by using Pearson correlation test with a level of significance of P = 0.05.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The weights of the rats were recorded on a daily basis and daily ethanol intake was also measured and expressed as g per kg body weight per day.

Daily ethanol consumption of the ethanol-receiving rats was on average 14.0 ± 0.4 (mean ± SEM) (range 12.5–14.9) g/kg. The blood-ethanol level was 203 ± 16 mg/dl (mean ± SEM) on day 16 of the ethanol exposure. Ethanol consumption decreased body weight in the ethanol-fed group compared to pair-fed rats. Thus, the body weight gains (in g) minus initial experimental body weights were 32.0 ± 11.7 and 52.5 ± 16.1 (mean ± SEM) for alcohol-fed and pair-fed rats, respectively.

The number of B. melitensis isolated (log10 CFU) from spleen (Table 1Go) and liver (Table 2Go) in ethanol-receiving rats was significantly (P < 0.01) greater than in control rats. However, although there was a moderate correlation between level of ethanol consumption and the number of B. melitensis in spleen (r = –0.062), it was not significant (P > 0.05) (Fig. 1Go). There were no physical signs of infection observed in rats after they were challenged with B. melitensis. In order to show any possible enlargement of spleen and liver, the organs/body weights ratio were calculated. There were no significant differences found in the spleen (P = 0.204) and liver (P = 0.977) body wt ratios between the ethanol and control groups.


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Table 1. The number of B. melitensis isolated from spleen
 

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Table 2. The number of B. melitensis isolated from liver
 


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Figure 1. Relationship of long-term ethanol feeding and B. melitensis infection. The number of B. melitensis in spleen (r = –0.48, P > 0.05) and liver (r = –0.062, P > 0.05) varies independently from daily ethanol consumption. CFU, colony forming units.

 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, a new model of B. melitensis infection was used in the setting of chronic ethanol administration in rats. It was found that the chronically ethanol-receiving rats exposed to B. melitensis infection had a significantly greater number of B. melitensis in their spleen and liver than the rats in the control group. The reason for this might be related to decreased Th1-type immune response due to ethanol consumption. Moreover, the organisms can survive in phagocytic cells and multiply to high concentrations. Besides, chronic alcoholics are thought of as ‘immuno-compromised hosts’, and infection with intracellular pathogens is prevalent (reviewed in Szabo, 1999). Thus, the differences between the number of the B. melitensis in rats' spleens and livers in both groups can be attributed to the adverse effects of the ethanol consumption and the characteristics of B. melitensis infection.

The disease spectrum of brucellosis depends on the infecting organism. B. abortus and B. canis tend to produce mild disease with rare suppurative complications. B. melitensis, the most common cause of brucellosis, also causes severe disease with a high incidence of serious complications (Koneman et al., 1997Go). However, in this study, there were no physical signs of infection observed. Furthermore, the number of B. melitensis among the ethanol-receiving rats seemed not to be affected by the amount of ethanol consumption. In the literature, the adverse effects of ethanol on intracellular bacteria such as L. monocytogenes (Saad et al., 1993Go), Streptococcus pneumonia (Davis et al., 1991Go; Lister et al., 1993Go), Legionella pneumophilia (Yamamoto et al., 1993Go), and M. avium (Bermudez and Young, 1991Go), have been reported, but that on B. melitensis has not been studied.

The animal model, which mimics human brucellosis, was developed and used to study the efficacy of various antibiotics in its treatment. The criteria for therapeutic efficacy in brucellosis animal models are a cure documented by the sterilization of the animals' spleen or reduction of viable counts of brucella cultured from the homogenized spleens (Shasa et al., 1994). Studies have employed a similar model using mice. However, studies of the effects of ethanol on host defence mechanisms in mice are difficult for several reasons. When ethanol is administered parenterally or intragastrically once before experiment, elevated blood-ethanol levels are achieved for only a short time. In addition, they do not address the adaptive changes that occur with chronic ethanol ingestion or the effects of ethanol on host defence mechanisms (Davis et al., 1991Go). In fact, the mechanisms of impaired resistance to B. melitensis infection might be more appropriately studied in an animal model of chronic ethanol feeding. Therefore, in this study rats were used. The influence of ethanol consumption on B. melitensis infection in rats was estimated by a method similar to that used to measure the efficacy of antibiotics, and the criterion for the course of infection in this model was determined to be the number of B. melitensis in rats' spleen and liver.

Despite extensive studies over the past 15 years, the optimum antibiotic therapy for brucellosis is still in dispute (Corbel, 1997Go). In chronic alcoholism, the use of antibiotics for brucellosis treatment might not be effective. In this study a new model of B. melitensis infection in the setting of chronic ethanol administration was used. Further use of this model may provide new insights into the therapy of human brucellosis.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
* Author to whom correspondence should be addressed at: Kocaeli University, Faculty of Medicine, Department of Microbiology, TR-41900 Izmit, Kocaeli, Turkey. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bermudez, L. E. and Young, L. (1991) Ethanol augments intracellular survival of Mycobacterium avium complex and impairs macrophage responses to cytokines. Journal of Infectious Diseases 163, 1286–1292.[ISI][Medline]

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Davis, C. C., Mellencamp, A. M. and Preheim, L. C. (1991) A model of pneumococcal pneumonia in chronically intoxicated rats. Journal of Infectious Diseases 163, 799–805.[ISI][Medline]

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