Experimental immunology
Natural xenoantibodies – their relation to molecular landscape of environment. Presence and possible functions of xenoantibodies in mice sera

The investigations concerning the microbial state of food or human and animal habitats are mainly focused on evaluation of the degree of pollution and their sanitary state [1, 2]. On the other hand, epidemiological and experimental studies have led to the conclusion that certain microorganisms and their components modulate the immune system and in consequence influence favorable on animal/human health [3-5]. Although numerous studies on microflora of different environments have been carried out, very little attention has been paid to the relation between the distribution of these microorganisms and their immune sign in animal/human organisms. Therefore the aim of the study was to: (I) isolate and identify bacteria of laboratory animals habitat, (II) evaluate their distribution in feed, water, litter and nearest environment and (III) perform an examination of mice BALB/C sera with isolated bacteria for presence of specific antibodies.

Materials and Methods

Mice (their sera) and the inside of animal room (its microflora) of our Institute were the objects of investigation.

Bacteria isolation and identification

Ten grams samples of each solid materials (feed- -LABOFEED H, wooden shaves, dust) were suspended in 100 ml of 0.9% NaCl. The concentrations of bacteria in suspensions and in tested water were determined by dilution plating methods. The 0.1 and 0.2 ml aliquots of sampled water and suspensions, their 10-fold serial dilutions in 0.9 NaCl up to 104 were spread on duplicate sets of nutrient agar (Biomed) plates. Air was sampled by exposition of open nutrient agar plates for 5; 10; 15 and 30 minutes. Plates were incubated (set 1) at 37°C for two days and next kept at room temperature for two weeks. Plates of set 2 were incubated solely at room temperature. During each incubation day plates were checked, arisen colonies counted and described. Sets of colonies initially differentiated on the basis of morphology (shape, color, consistence) were isolated and collected. Further differentiation and identification of isolates based on: (I) Gram staining, (II) growth on appropriate sets of prepared differentiative and selective media [6], (III) patterns of reactions with API and Lachema tests [7]. Characterized isolates were determined according to the Bergey,s Manual [8, 9].

Antigens preparation

Bacterial cells of dominated species of isolated genera were prepared in two forms, as the viable and fixed (heat-killed) cells suspended in borate buffer (pH 8.2) in concentration of 1 × 10⁷ cfu/ml.

Mice and sera preparation

Sera were taken from two groups of mice (born in situ) in five years interval (1996 and 2001); 35 including 16 young – eight to ten weeks old, and 19 older- over 26 weeks old in first stage and 16 (12 young and 4 elders) in second stage.

Antibodies specifity assays

ELISA tests were carried out according Weeman and Schurs [10]. Borate (pH 8.2), PBS (pH 7.4) and citrate (pH 4.9) buffers were employed. Wells of plates (Nunc-Immuno, Maxi Sorp) were coated by all forms of prepared antigens: viable and fixed cells at concentration 5 × 105/well. Amount of 0.1 ml of tested and conjugated sera diluted in 1% BSA in PBS for each well were used. Standard of dilution of tested sera was 1:10 – 1:100. Peroxidase conjugated goat anti mouse (I) polyvalent immunoglobulins IgG+ IgA+ IgM (Sigma Immuno Chemicals), (II) IgG+IgM and (III) IgG (Jackson ImmunoResearch) were used in standard dilution 1:5000. Incubations of plates were performed for 2 h at 37°C
or overnight at 4°C. All plates were read with Dynatech ELISA spectrofotometric reader at the wavelength 492 nm.

Results

The bacteria concentration and distribution in mice habitat is presented in table 1. It may be seen that the numbers of bacteria were basically stable for each of tested sources.
The lower numbers of bacteria cells were found in drinking water – 6.1 × 10¹ cfu/ml, more numerous in bacteria cells concentration were feed and litter – 3.1 × 10⁵ cfu/ml. There were not significant differences in bacteria concentration between samples taken in different time and growth temperature. Table 2 presents the degree of differentiation of isolated bacteria and the list of most frequent genera. More numerous in genera composition (6–5–4) were samples obtained from litter, air and dust. Number of isolated species from particular sources differ from 6 (feed) and 7 (litter, water) to 13 (air, dust). The dominating groups of cells and their systematic position are presented in table 3. As can be seen representatives of Micrococcus, Staphylococcus and Bacillus genera constituted majority of bacterial cells populations.
Table 4 shows the prevalence’s and the levels of specific antibodies (in two groups of mice sera) which recognized antigens presented in mice habitat. Positive sera of younger mice were less numerous than in older group, e.g. 18.8% v. 47.4% for antigens of Bacillus and sera dilution 1/100. Percent of positive antigen – antibody reaction in less diluted (1:10) sera were more then three times higher in comparison with sera more diluted (1:100), e.g. 43.3 versus 6.3 and 84.2 v. 52.6 for reactions with antigens of Micrococcus. The results of sera specificity spectrum are presented in table 5. As can be seen there were differences in sera spectrum specificities depending on (I) range of recognized classes of antibodies, (II) dilution of sera and (III) tested groups. Incubation of specific sera with goat anti-mouse IgGMA revealed that the higher number (12 of 35 and 3 of 16) of sera diluted 1:100 recognized three sources of available antigens, whereas 6 of 35 and 7 of 16 did not recognize presented antigens. Additionally, none of sera recognized all available antigens. Only one of the tested sera diluted 1:100 showed positive reaction with goat anti-mouse IgG.

Discussion

Bacteria and their components strongly influence on immune system [11,12] e.g. by antibodies generation or induction interleukins release [13]. Natural antibodies specific to toxins, bacteria, viruses are present in the sera of normal, nonimmunized humans or mice [14]. They are encoded by germline variable genes and their titers in mice range from 1/8 to 1/32 [14]. Most of sera of young and some of older mice presented in this work seems to posses natural antibodies; they showed positive reaction with ten times diluted sera and negative with hundred times diluted sera. Longer coexistence with antigens lead to higher than natural levels of specific antibodies and indicates that animals were stimulated by antigens (table 4). Natural antibodies are the essential part of the first line of defense and link innate and acquired immunity [14, 15]. Lack of natural antibodies may be the reason of existance of animals which did not posses in their sera any specifities for tested antigens (table 5). Explanation all of the relations between common nonpatogenic environmental bacteria and natural xenoantibodies seems to be open question for a long time.

References

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