The objectives of this joint experiment on ASTP were to evaluate components of the infectious disease process during space flight by measuring alterations in the composition of the crew and spacecraft microbial populations, the ability of each crewmember to resist infection and the ability of certain microorganisms to originate infections.
Microbial specimens were collected from each of the six Apollo crewmembers (3 prime and 3 backup) and four Soyuz crewmembers (2 prime and 2 backup) preflight, in-flight and post flight. Fifteen microbial samples were collected from the inner surfaces of each spacecraft. Samples were taken preflight on L-45 (45 days prior to liftoff), L-30, L-15, L-7 and L-0 (day of liftoff). Post flight samples were taken on R+0 (immediately upon recovery), R+7 (7 days after recovery), R+15, and R+30.
Sample sites included:
1) A 20 square centimeter area of hair (and scalp) on the top of the head
2) Right and left external auditory canals
3) 20 square centimeter area below hairline at base of neck
4) Internal area of both nostrils
5) The tonsils and posterior pharyngeal vault
6) 20 square centimeter area on right and left palms
7) 20 square centimeter area below the hairline of each axilla
8) 5 cm strip from rear to front on right and left inguinal area between legs
9) Area between the two smallest toes of each foot
10) The throat and mouth
11) A fecal sample
1) Commander couch seat
2) Flight engineer couch seat
3) Control panel in the descent vehicle
4) Surface of gas mixture regulator
5) Surface near bulkhead hatch in descent vehicle
6) Descent vehicle hatch cover under the handwheel
7) Flush side of sanitary device in the orbital module (OM)
8) Top of food locker table in OM
9) Couch seat in OM
10) Surface of food locker in OM near panel
11) Front surface of the couch backrest in the OM
12) Grid near fan in OM
13) Floor surface near OM food locker
14) Transfer hatch under the handwheel
15) Surface of transfer hatch
1) Left X-X headstrut
2) Right X-X headstrut
3) Crew couch, right stabilizer beam
4) Tunnel area
5) Right floodlight
6) Left rotational hand controller pistol grip (both sides)
7) Right girth shelf above panel 278
8) Panel 325 below window
9) Above left girth shelf (above U-3)
10) Cover plate, ORDEAL stowage locker (U-3)
11) B-6 door behind waste stowage bag
12) Inside door of food locker (L-3)
13) Top of video tape recorder module
14) Panel 251, waste management dump
15) Forward of panel 225
Preflight and post flight, crewmember samples were collected using calcium alginate swabs moistened with 0.3 millimolar (mM) phosphate buffer. Dry calcium alginate swabs were used to sample the tonsils and the posterior pharyngeal vault before collection of the gargle specimen, during which the subject gargled with 10 ml of 0.3 mM phosphate buffer followed by a repeated rinse of the teeth. Swabs were placed in 5 ml of 0.3 mM phosphate buffer for transport to the laboratory. Fecal samples were collected in a sterile cardboard container at the convenience of the subject. Sample analysis was initiated within 1 hour of specimen collection.
In-flight samples included the first six crew microbial swabs for each
crewmember and all 15 swabs for both spacecraft. Samples were obtained on
flight day 3. During flight, each crewmember used his own set of 10 swabs
to sample specified body areas. A specially developed sample collection
device was used for in-flight samples. This device consisted of a
cotton-tipped Teflon swab on a capillary tube that contained conservation
fluid to keep the microorganisms alive. Each swab was housed within an
airtight case to prevent desiccation. Groups of swabs were organized in
Beta-cloth retaining bags. One bag was provided for each of the following:
(1) Soyuz spacecraft sample swabs
(2) Apollo spacecraft sample swabs
(3) swabs used for Apollo subject A and Soyuz subject A
(4) swabs used for Apollo subjects B and C and Soyuz subject B.
All samples collected during flight were returned to Moscow for preliminary analysis and division between the U.S. and U.S.S.R. laboratories. Soyuz spacecraft samples 7-15 were taken from the part of the spacecraft not returned to Earth. Accordingly, these samples were not taken post flight.
Because in-flight samples had to be returned to the laboratory for analysis, considerable delay was unavoidable. To correlate in-flight results with preflight and post flight results, additional samples were collected from all 10 crewmembers and both spacecraft on L-0 (launch day) and R+0 (landing day). These samples were held at room temperature for 4 days to simulate the delay imposed on the in-flight specimens.
Swab and gargle samples were serially diluted under aseptic conditions and subsequently inoculated onto the surface of nutrient media. The variety of media, number of plates inoculated, dilution ranges and incubation conditions were selected on the basis of what was required to isolate and quantitate the microorganisms present in each area. A common methodology, composed of analytical procedures acceptable to both the U.S. and U.S.S.R. investigators, was used to ensure comparability of data. Following incubation, colonies on every culture plate were categorized and counted. One sample of each morphologically different colony was transferred from each dilution series to the appropriate nutrient media and stained. Following pure culture verification, one aliquot of each culture was stored by the laboratory in which it was identified (the host laboratory), and another complementary aliquot was available to the guest laboratory.
The U.S. PI was responsible for identification of all filamentous fungi, nonlactose-fermenting Gram-negative rods, and members of the genus Haemophilus. The U.S.S.R. PI was responsible for the identification of yeasts and yeast-like fungi, Gram-positive cocci and lactose-fermenting Gram-negative rods. The responsibility for identification of Gram-positive, catalase-positive cocci was shared by both laboratories. In addition to these microbial identifications, the responsibility for specific tests was also divided.
Saliva from the parotid gland was collected from the 10 crewmembers on L-45, L-30, L-15, L-7, L-0 and R+0. In addition, prime crewmembers were sampled on R+7, R+15 and R+30. The special saliva collection device was attached by pressure to the inner mouth surface over the opening of the parotid duct. Parotid fluid was collected for 10 minutes with the flow being stimulated by a sour candy drop placed in the subject's mouth. The resulting saliva was frozen at -40 degrees Celsius and stored for future IgA and lysozyme analysis. No saliva was collected in-flight.
Venous blood was collected from the antecubital fossae of backup and prime crewmembers preflight on L-30 and L-15, and from the 5 prime crewmembers post flight on R+0 and R+3. From each sample, whole blood was smeared onto slides and treated with Wright's stain for differential leukocyte counts. The remaining blood was allowed to clot for serum separation. Serum was used to determine humoral antibody levels and bactericidal activity.
No significant difference was observed in the number of viable bacterial cells recovered from the skin sites of the five prime crewmembers, indicating that Apollo and Soyuz crewmembers retained the same microbial load on their skin despite the geographical and cultural differences involved. Data also showed that values obtained immediately after space flight were within the preflight range, demonstrating that the population of aerobic bacteria (measured in terms of total viable cells) inhabiting the integumentary surfaces was stable following space flight, and no medically important alterations occurred.
Analysis of the bacterial loads recovered from the nasal and oral cavities of ASTP crewmembers were likewise determined. Upper respiratory tract samples obtained from the Soyuz cosmonauts yielded lower and more variable counts than those obtained from Apollo astronauts. Even with this variability, the data show that space flight had no demonstrable effect on the total bacterial load recovered from the oral and nasal cavities.
Most sample areas within the Apollo and Soyuz spacecrafts were not sterilized before flight affording a potential source of crew contamination. Contamination within the Soyuz was increased on R+0, a response similar to those previously reported for other space flights. Apollo contamination was considerably decreased post flight, possibly a result of exposure to dinitrogen tetroxide during landing. However, this gas did not measurably influence individual crewmember microbial loads because of the short exposure time of the astronauts to the gas.
The quantitation of Gram-negative rods was too variable to establish any flight affected alteration. This is to be expected because these species are mostly transient to humans rather than being true autofloral members (except in the lower gastrointestinal tract, which was not evaluated in this study). There was, however, an increase in the number of cosmonaut body sites from which Gram-negative rods were recovered upon return from the space flight. Bacteria spreading to previously uncontaminated sites probably resulted from the altered hygiene regimen used onboard the Soyuz.
Escherichia coli is generally accepted as the most reliable evidence of fecal contamination. Outside the intestinal tract, under certain conditions, it often produces diseases. This species was repeatedly recovered from the groin of Apollo subject B and occasionally from the upper respiratory tract of the other two Apollo prime crewmembers. In all cases, neither the recovery pattern nor the quantitation indicated medical significance.
Enterobacter aerogenes occurs in the large intestine. The pathogenic significance of E. aerogenes is similar to that of E. coli. This species was always carried in the nose and mouth of Apollo subjects B and C and was frequently isolated from two backup Apollo crewmembers. This organism did not spread to Apollo subject A or to other areas on the carriers during flight. Also, there was no post flight increase in quantitation. This is an excellent example of a potential pathogen, carried by two Apollo prime crewmembers, apparently being unaffected by the conditions of space flight.
Proteus mirabilis, a microbe associated with secondary infections of the middle ear, mastoid process, meninges, wounds and urinary tract, was carried in low numbers in the nasal passages of Apollo subject A. The ASTP mission had no detectable affect on the qualitative or quantitative presence of this microorganism.
Other Gram-negative rods present were Mima polymorpha, 4 species of Haemophilus and members of the genus Moraxella. Members of the genus Moraxella are parasites of human mucous membranes, which can cause diseases such as conjunctivitis. Although carried in the oral cavity of Apollo subject C for 45 days preflight, no discernible alteration was demonstrated following the mission. M. polymorpha was isolated from multiple sites on Apollo subject B. It did not contribute significantly to the load of Gram-negative rods recovered from ASTP prime crewmembers.
Haemophilus influenzae is found in lesions and the upper respiratory tract. It is commonly a secondary invader in influenza and pertussis cases. This species was isolated from each crewmember before flight in quantitations ranging from 10,000 to 60,000 viable cells/ml. The post flight loss from 2 prime Apollo crewmembers and the quantitative reduction in contamination of Apollo subject A is contraindicative of a spaceflight-mediated increase in infective potential. The overall quantitation and incidence of H. haemolyticus and H. parahaemolyticus decreased following the ASTP flight.
Previously discussed observations support the view that Gram-negative rods underwent unfavorable dysobacteriotic changes following space flight. Therefore, those few instances in which such changes have been reported should be considered to be coincidental with, and not a function of, space flight.
The yeast, Candida albicans, has long been recognized as a normal component of man's autoflora, and has been recovered from the crewmembers of previous spaceflight and space simulation missions. Data analysis indicates an intracrew transfer of C. albicans to Apollo subject A during flight. No such transfer occurred with the Apollo backup controls, although C. albicans was routinely carried in the mouth of one backup crewmember. C. albicans was recovered from only one crewmember (Soyuz subject B) on days L-33 and L-15.
Although Staphylococcus aureus is a common skin and nasal contaminant, all strains are potential pathogens. Several space flight simulation studies have indicated increases in the toxigenic activity, virulence or pathogenicity of this species with stressful confinement of the human host. ASTP data show that each prime and backup crewmember carried a different strain, each of which is expressed as a numbered bacteriophage type. One type (52, 52A, 80, 81), carried by Apollo subject B, was transferred to Apollo subject A during flight. This type was not isolated from Apollo subject A preflight, but was recovered on R+0. Apparently, colonization did not ensue because this strain was not recovered from this subject after R+0. Transfer of this microorganism did not occur between crews.
A post flight increase in the incidence of S. aureus has been reported in previous studies. A measurable increase in the number of S. aureus cells recovered from Soyuz crewmembers was present immediately post flight. Although recovery incidence and quantitation of this medically important species was unusually high, no disease events were reported.
There was no demonstratable decrease in the incidence of medically important microorganisms recovered from the ASTP crewmembers on recovery day, a finding that supports earlier studies. Accordingly, no post flight diseases were reported.
Parotid fluid of sufficient quantity and quality for detailed analyses was not received for study from U.S.S.R. cosmonauts. Among the U.S. crewmembers, the collecting device could not be used on Apollo subject B because of an anomaly in the opening of the parotid duct. Samples collected from prime crewmembers the day of return from space flight are generally lower than the preflight mean for the rates of flow and concentrations of IgA and lysozyme. However, because similar variations occurred in the control population (backup crew), none of these changes were shown to be space flight related. Therefore, there is no reason to suspect that changes in the oral autoflora during space flight were influenced by changes in the IgA or lysozyme content of parotid fluid. No significant alterations in the measured parameters were observed post flight, with the exception of an increase in IgA in Apollo subject B on R+15. Medication treatments given to the astronauts in response to dinitrogen tetroxide were capable of effecting a reduction in some measured parameters, although no significant decreases were observed.
Blood serum from U.S. and U.S.S.R. crewmembers was reacted against antigenic preparations of Staphylococcus aureus, Streptococcus sp., Haemophilus influenzae, Escherichia coli and Candida albicans. All species were isolated from the crew autoflora. In addition, preparations of Salmonella typhi "O" antigen and Bacillus abortus antigen were likewise reacted against crew serum. Data indicate only two significant post flight changes. With S. aureus, the serum titer of Apollo subject A increased from 1/64 preflight to 1/8 on R+0. Likewise, the titer of anti-E. coli antibody in the serum of the Soyuz subject B was increased from greater than 1/8192 to 1/1024 post flight. Because these were unusual isolated incidences and because all other titers remained essentially unchanged, it must be concluded that this space flight did not effect a demonstrable change in the presence of species-specific antibodies. In addition to the considerable variation among individuals, there was a general trend for lysozyme levels to be elevated the day of return from space flight. However, the magnitude of change was erratic and could not be correlated with the actual space flight. Therefore, these data alone cannot be used as evidence that blood lysozyme levels are affected by space flight.
In conclusion, the proposed simplification of medically important
microorganisms and the theorized post flight microbial shock could not be
supported by the results of this study.
|Mission||Launch/Start Date||Landing/End Date||Duration|
|ASTP||07/15/1975||07/24/1975||9 days, 7.5 hours|