A note on the influence of microgravity on the microbial endoecosystem of Japanese quail

Japanese quail are commercially farmed for meat and eggs in Southern Europe , Asia and India. Due to small size, low husbandry costs, short generation interval and adaptabil i ty to a wide range of husbandry condit ions they are also popular laboratory animals. The Japanese quail has also become an object for space research (Boda, 1979) because it may help to solve dietary problems connected with space flight. Recently, the effect of microgravity on endocrine functions and adapta t ion processes (Jurani et al., 1988), embryonic development (Boda et al., 1991) and productivity of Japanese quail was studied. The present work extends this research to the microbial endoecosystem and its biochemical and physiological properties after exposition of birds to 7 days of microgravity on board of the orbital station. This study may contr ibute some information on the microbial status of Japanese quail under space flight condit ions and help to select bacteria which could prevent microbial dysfunction of the digestive tract dur ing flight.


INTRODUCTION
Japanese quail are commercially farmed for meat and eggs in Southern Europe, Asia and India.Due to small size, low husbandry costs, short generation interval and adaptability to a wide range of husbandry conditions they are also popular laboratory animals.The Japanese quail has also become an object for space research (Boda, 1979) because it may help to solve dietary problems connected with space flight.Recently, the effect of microgravity on endocrine functions and adaptation processes (Jurani et al., 1988), embryonic development (Boda et al., 1991) and productivity of Japanese quail was studied.The present work extends this research to the microbial endoecosystem and its biochemical and physiological properties after exposition of birds to 7 days of microgravity on board of the orbital station.This study may contribute some information on the microbial status of Japanese quail under space flight conditions and help to select bacteria which could prevent microbial dysfunction of the digestive tract during flight.

Experimental procedure
The experiment lasting 9 days was carried out on twelve Japanese quails aged 15 days divided into three groups of 4 birds each.The birds in the flight group were sent into space on board of the orbital space station MIR.The birds in the synchronous group were kept under simulated flight conditions.All the birds were fed the same standard pelleted diet.The quails from the flight group were killed after landing on Earth, quails from both synchronous and control groups were killed in the laboratory.Crop and caecum contents were sampled from each bird, mixed with a glycerin-phosphate buffer (1:1) and frozen on dry ice until further processing.

Resistance to antibiotics
Resistance to six antibiotics was studied using commercial Sensi-La-Disks (Lachema, Brno).Agar plates with disks were incubated at 37°C according to the

Determination of bacteriocin-like substances
Production of bacteriocin-like substances was studied according to Skalka et al. (1985).The indicator bacteria strains used in the experiment are presented in Table 1.Staphylococcus aureus CB 44 (Veterinary University, Brno) was as the positive control.

Lactic acid production
The quantity of lactic acid produced, expressed in mol.L _1 , was examined using the precipitation method as described by Pryce et al. (1969).Urease activity (E.C. 3.5.1.5.) was measured by a spectrophotometric method according to Cook (1976) and expressed in nkatm -1 .
Total bacteria counts expressed as log 10 ± SEM Urease activity and lactic acid production are arithmetical averages + SEM.

RESULTS
The largest total counts of enterococci and enterobacteriacae were found in the caeca of Japanese quails in the flight group (Fig. 1).The total counts of lactobacilli, streptococci and staphylococci were the largest in caeca of the synchronous group.The difference between the flight and the control groups was significant (P < 0.05) only in respect to the enterobacteriacae count.Higher counts of enterobacteriacae, staphylococci and streptococci were found in the crop of birds from the synchronous group (Fig. 2) in comparison to the flight group.According to the identificational scheme (Buchanan and Gibbons, 1984) the strains were allotted to the species Enterococcus gallinarum (EGIO.EG 12), E. avium (EA7), Staphylococcus gallinarum (SG2) and S. aureus (SA7).All of these strains were resistant to penicillin and strain EG10 was also resistant to streptomycin.All the strains were sensitive to all of the other antibiotics used.Bacteriocin-like substances produced by strain EG 12 inhibited the growth of all bacteria species used.The antimicrobial activity of the bacteriocin-like substances produced by all the strains inhibited the growth of at least one of the nine indicators used.The average diameter of the zone of inhibition ranged from 4 to 6 mm and the majority of these zones were clear (Table 1).Urease and lactic acid production ranged from 0.39 to 3.7 nkat/mL-1 and from 0.720 to 0.186 mol/L-\ respectively (Table 2).

DISCUSSION
In general, the microbial population of Japanese quails was not affected by microgravitation.The occurrence of facultative anaerobic microorganisms was lower than reported by Barnes et al. (1972), Fuller (1977) and Koniarova (1991) in the caeca and crop of chickens under normal conditions.The enterococci  species found were the same as those described by Devriese et al. (1991) as associated with normal poultry gut flora.
The majority of the screened strains were monoresistant with the exception of strain EG 10 which was resistant to two antibiotics.The strains, isolates from the crop and caecum, produced bacteriocin-like substances with a wide antimicrobial activity not only against Gram-positive, but also against Gram-negative bacteria.The strain EG 12, the producer of a very active bacteriocin-like substance may be used for producing the preparation capable of adhering to the intestinal wall epithelium and supressing endogenous bacterial infections.This may be important because the normal bacterial flora present in faeces (e.g.Clostridium sp.) may multiply under the stress conditions of space flight and cause entheritis.The inhibition of pathogenic strains with bacteriocins produced by lactic acid bacteria, has also been described by Lewus et al. (1991).

Figure 1 .
Figure 1.Total counts of bacteria isolated from caecum of Japanese Quail

Figure 2 .
Figure 2. Total counts of bacteria isolated from the crop of Japanese quail.

TABLE 1
Production of bacteriocin-like substances by tested strains