Murine viruses in germfree mice selected for extraterrestrial life testing of Apollo lunar sample

Parker, J. C.

English

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I;br the pewiod: Septmber 1, 1968 to June 30, 1970 
Zteqxmsible Investigator: ;John C, Parker, Ph.D. 
Contractor: Microbiological Associates, Inc, 
4733 Ebthesda Avenue 
Bethesda, l.laryland 20014 
i. ... .... . <.:.; 
https://ntrs.nasa.gov/search.jsp?R=19700027157 2020-03-11T23:32:52+00:00Z
f 
The gevmfree muse oolony has been established 
virus infection, Staxting w i t h  a bre 
through the P3 generation to the expansion mlrmy no evidence has been 
found for infection wia the following viruses: p e m a  virus of mice, 
muse pneumnif-bis, palyclma, Sendai, minute virus of mice, ectronelia, 
muse admcmLrus, mouse hepatitis, lymphocytic chorimenbgitis, Riley, mouse 
salivary gland virus, thymic or epidemic diarrhea of infant mice .  Sane 
equivocal data has been obtained w i t h  regard to reovirus type 3, and 
Theiler's encephalartryelitis virus infection, however, it is likely that the 
m i c e  are not infected with either of these agents. 
throusghaut the colony wit31 muse leukemia v i m .  
There is infection 
A virus profile analysis was pxfonned on two groups of 150 germfree 
mice, each taken from a sample of 750 germfree mice used on the Apd.10 11 
and Apollo 12 missions. 
-I- 
The objective of the study was to characterize, identify8 and 
&ennine the prevalence of indigenous murine viruses which  might have 
present i n  the "gemfree" (gnotobiotic) mice used for the detection of 
actratemestrial agents by the National Aeronautics and Space Administration 
(€=A) Lunar Ibceiving Laboratpry (LRL) a 
The examples of occult murine viruses which interfere w i t h  research 
efforts are quite nunemus. While no attempt will be Made here? to enmerate 
them, it is safe to say that these viruses have caused the invalidation of 
research projects valued at hundveds of thousands or perhaps rckllions of 
dollars. Indigenous viruses act i n  an insidious manner by contaminating a 
variety of biologic systems such as: tissue cultures, transplantable -lid 
tmors, virus leukcmias, and other virus reagents. After contaminating 
these systems the viruses are then free to cause a variety of secondary 
effects. These may be and most often are interpreted by thE! investigatxx as 
primuy effects, and he is brought to the threshold of what may be, and 
often is, a vevy long, Fainful, and expensive misadventure in science- 
Exhibit A 
Table I outlines the steps leading up to the establishment of the 
Z4ASA gemfree mouse mlony and indicates the tests which were carried out a t  
each step. Table 2 arad Table 3 detail the results of the vhms monitoring 
which was carried out at  each generation step. A l l  gemfree mice i n  exhibit A 
and exhibitB were tested €or infection by the follctWing viruses: p m d  
virus of mice ( P W ~ ) ,  reovirus t y p  3 (lie0 31, Theiler's emJephalcartyelitis 
(strah -1) muse pn@mnitis (fi) I P 1 - p  Sendai.8 mhk& of ( ~ ~ )  8 
estromelia, muse adenovirus (M.Ad.), mouse hepatitis virus (FJnrV) fymphucyt5.c 
-2- 
Table I 
1. Baseline Study - selection of breeders for nucleus colony - mice for i n i t i a l  
testing sampled at ranctom f m  each isolatxx 
Germfree Isolator €kxnbcr 
5 0 ~  7-&y101d mice for Virus \ '  I i 1 / 
50, 1~2-n&th-old mice for vi&s isolation and 
serologic studies 1 
'1 ', i 
95,  redred breeder mice- for serologic studies 
\ \  I 
48 breeders poled (24 9 24 @)and distributed to 2 germfree isolalmrs 
2. BtabUshment of i 4 i i  Colony -----I-- ' f !  
\/ 
Po generation - 48 breeders for semlcqic studies 
P1 generation - 48 7-CIay-old suckfinc~s for Virus isolations 
48 7we&-old mice far virus isolations and semlcx~ic studies 
48 breeder mice for semlcqic studies 
P2 gmemtim - 48 7-day-old su&lin;4s for virus isolation 
48 6-week-old mice for virus isolations and serologic studies 
48 breeder mice for serolcgic studies 
I 
3, Bxpansion calony at the 3rd generation - 
58 sucklings for virus isolations 
60 3-c.Jeek-old mice for virtls isolations and serolagic studies 
100 3-to 6-weeIc-old mice for virus isdations and serologic studies 
-3- 
Table 2 
1-2 m ~ .  old 0/50* 
l2-s 0/95 
P, Generation 
breeders 0/48 
P7 Generation 
b-S 0/48 
breeders 0/4 8 
0/50 
0/92 
0/46 
1/45 
0/4 8 
0/48 
0/48 
0/50 0/50 0/50 
0/95 0/95 0/95 
1/41 0/48 0/48 
1/48 0/48 0/48 
0/48 0/48 0/48 
0/50 0/50 
0/95 0/95 
0/48 0/48 
0/48 0/48 
0/48 0/48 
0/48 0148 
0/48 0/48 
0/50 0/50 0/50 0/50 
0/95 0/95 0/95 0/95 
0/39 0/48 0/48 0/48 
0/48 0/48 0/48 0/48 
0/48 0/48 0/48 0/48 
0/48 0/48 0/48 0/48 
0/31 0/48 0/47 0/46 
3-6 Weeks-old 0/64 0/57 
O/GO 
0/64 0/64 0/57 
0/60 0/60 0/60 
0/57 0/59 
0/60 0/60 
0/40 0/64 0/64 0/57 
0/60 0/60 0/60 0/60 
%. of sera positive/l-io. of sera tested 
OSee text for diiscussion of the significance of inhibitmrs 
-4- 
Table 3 
Testing of NASA Gemfree by ViruS Isolation TedmQues 
1-2 month old O/lO* 0/10 0/10 6/9 0/10 
sucklings 0/10 1/10 0/10 0/10 
P G e n e r a t i o n  -1 
7-\q&-S-Old 0/10 0/10 0/10 10/10 0/10 
sucklings 0/10 2/10 0/10 0/10 
P Generation -2 
6-weekS-0ld 0/10 0/10 0/10 9/lG 0/10 
sucklings 0/10 1/10 0/10 0/10 
mpansion Colony 
s w w s  0/6** 0/12 0/12 10/12 o m  0112 
3 -6 -~ !ek~-o ld  OA-8 0/20 0/20 4/19 0/20 0/19 
9-wAcs-old 0/8 0/12 0/12 12/12 0/12 0/12 
3Yo. p l s  vixus psitive/h'o. mls tested: each p m l  consists of 5 mice 
**lo mice p r  pol &ISGv suklings only 
-5- 
were used to detect antibody t o  11 of the indigenous murine viruses: PW, W03, 
GDVII, K, polyoma, Sendai., EKM, ectrcanelia, M.Ado, 3HV, and LCPI. 
were hdividually bled a t  a 1:5 serum dilution and tested by either the 
hemagglutination-inhibition (HI) or complement fixation (m) test, Virus 
&e mice 
isolation attempts were carried out using pols of t i ssue  extracts prepared, i n  
most case5, from 5 mice, The appropriate organ or organs were harvested and 
10% extracts prepared, These tissue extracts were inocula&d into tissw cultures 
or animals acmriiing to  established protocofs. 
An agent causing necrosis of the thymus was isolatelt w h i l e  carrying 
out the thymic virus protocol on a pol of P2 generation 6-week-old mice. $he 
gross and histologic patholqy of the thymus of mice inoculated with the 
isolate virus m s  indistinguishable from mice infected with thymic agent 
and the growth curve of the agent in suckling mice was similar to thymic agent, 
However, a diarrhea noted in the mice inoculated with the isolate has not been 
previously observed associated w i t h  thymic virus infection. 
undertaken to  determine whether the virus was infecting the PWA mice or the 
Studies were 
test mice. Attempts to reisolate the virus frcm the original mouth swab 
(a €'4SAmouse) material were not successful. Also no other isolation was made 
frcan approximately 100 mouth swab pols f r m  500 NASA axenic mice. Xouth swabs 
were taken fmn the test mice (NIK swiss mice), however no isolations were made. 
H o w e v e r  since the virus could not be reisolated from the iW% inice and since the 
thymic agent is prevdlent in conventionally reared mke it is likely that the 
virus was an accidental oontaminant of the isolation system and i s  not present 
in the NASA gumfree muse stocks. 
-6- 
illtizougil Reo 3 and GDVII liemagglutination inhibitors 'weye present 
in  the germfree muse sera it is likely that they are nonviral i n  nature 
and represent nonspecific iniiibitors. Ektmsive observation and testing 
of the inliibitor for Reo 3 revealed the following infomation: 1) The 
inhibitor was usually of low titer, was never associated t 4 7 i t h  CF antibody 
and did not stain Reo 3 i n  the indirect  irnnunofluorescent anl5body test 
(Table 4) 
oompletely by chmucal treatment with the enzyme RDE. 3) epidaniohgy 
of the inhibitor was not typical of a reovirus infection. 4) V i r u s  was never 
isolated from any of the lJASA mice. 5 )  A similar nonspecific hemagglutination 
inhibitor is sometimes observed i n  other colonies of mice which are not 
infected w i t h  the virus. 
the infection would have spread, clinical disease would have become apparent 
and the nutiber of sero-positive animals ~.x>uI.d have increased; this did not occur. 
W i n g  the course of the contract changes w e r e  made i n  several 
2) In most instances the inhibitor, unlike antibcdy, was destroyed 
6) If reovirus infection were present i n  the isolators 
of the isolation techniques. 
were changed from the Coi.lUL to  the XC test; a tissue culture cytopathic test 
developed by Drs. Hartley and m e ,  NIH. 
of choice for the followhg reasons: it requires only 1 4  days for ccmpletiori 
me mth& used for leuk&inia virus isolations 
%le XC test became the tecAnQue 
cxanpared to 42 days for the CXXlUL, it is a technically sinpler test and is 
less espnsive to prfoxm than the CCMULJand the XC test is as sensitive 
or perhaps mre sensitive than the Ct24UL test. 
the source of mice utilized for several of the isolation techniques. 
a spntaneous diarrhea of unknam etiology was found to be associated 
w i t h  igational Laboratory Animal Company mice (RlN) the colony could not be 
used for ED321 isolations. 
mice were selected as the alternative source since they v7-e sensitive to 
C;rmnges also were made i n  
When 
Area 68, Charles Piver Zreedinq Laboratories 
-7- 
Properties of a Reovirus m e  3 &magglutination Inhibitor in the 
P2 Generation Breeder Germfree :.louse Sera 
Serologic Test 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
160"" 
80 
80 
80 
80 
80 
40 
40 
40 
40 
40 
40 
40 
,+ 
20 
20 
20 
20 
lr20 1:20 1: 5 1:10 Initial Serum Dilution 
*&ceptor Destroying Enzyme 
+iwative a t  <1:20 serum dilution 
**bz ipca l  antibody titer 
8 A l l  sera were heated at 56OC €or 30 min. prior to test 
-8- 
E33I.i-i infection, were readily available, had heen screened for their virus 
spectrum, and had been observed for several yeaxs without any evidence of 
diarrhea. 
sought for use in LCTI and Riley virus isolations. 
available i n  sufficient quantitites to f u l f i l l  contract obligations. 
follawing colonies producing ICR and Swiss 'CJebster mice were tested for LC5I 
and Riley virus sensitivity; Charles Ptiver Breeding Laboratories (area 681, 
i~ficrobiological Associates, fnc. , Cmberland View Eixms and Ercokside Farm. 
T4ie claries River Breeding bboratories, area 6 8 ,  mice tere chosen as the 
alternatiw source to National Laboratory. 
An alternative sowm of mice to  Uational Laboratory also was 
ZJLW mice often were not 
%e 
Wiibit €3 
EJASA'~ decision to advance the Z4pllo mission dates necessitated 
an addendan to the contract. 
established to release mice for use i n  the LlXG vertebrate (mammalian) 
protocol for analysis of lunar samples brought back by PpSlo 11 and A p l l o  12. 
A decision was made to use mice frcm back-up germfree breeding isolators 
k i n g  maintained at CM3L for use in such a situation, however mice 
E Y ~ I  these isolators had not been test& for virus profile. 
600 weanling mice? frm the back-up isolators on July 1, 1969, and again on 
October 29, 1963. 
agents required that a virus profile analysis be completed on a group of 
150 weanling mice randcanly selected frcm the same isolators as the 
Prelinximry tests were cmpleted idthin one month on tile sme 16 indigenous 
murine viruses monitored for in the ?%%%gemfree muse colony analysis. 
isolation and serologic tecluriques used were the same as for the I W A  germfree 
colony. 
The "new" gemfree colony was not yet sufficiently 
L K  received 
U s e  of these mice for testing lunar samples for extraterrestrial 
sample. 
The 
REsults of the serologic tests are presented i n  Tables 5 and 6, A l l  
-I& 
-9- 
Table 5 
Virus Serologic Testing of 150 Awllo 11 Xke 
Serologic Test 
i%, of C0IWl-t 
Germfree Sera Hemagglutination-Inhibition I;li;ration 
Ssolator No. Tested PIIN Reo3 GDVII K Polycma Senctai fM! Ektuamelia il.Ad. i4EW Lxs4 
4A 8 
6A sr 
1lA a* 
18A 0" 
28A ON 
3 Q 9  
33Md 
2A T 
%A d 
25A ? 
27A $ 
34A d 
10 
10 
10 
LO 
10 
10 
10 
10 
10 
10 
LO 
10 
10 
10 
LO 
*Negative 
-10- 
Table 6 
Virus Scsrolcgic Testing of 150 Apollo 12 &ice 
Serologic T e s t  
No. of (xmplmwt 
Germfree Sera Hemagglut in;ition-Inhibi tion Fixation 
Isolator i4o. 2'ested PVI Reo3 GDVJI K PolYom Sendai iJWl Ectromelia !i.Ad. :'EN ILXi 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
LO 
10 
-11- 
sera were negative for antibdy to the test viruses, %e virus isolation 
results are presented i n  Tables 7 and 8. kliti? the exception of mouse leukemia 
virus a l l  virus isolation attempts were negative. :louse leukemia was 
isolated from 15 of 30 Zip0110 11t i ssue  extract spechens and 18 of 30 ApLlo 12 
tissue extract specimens e 
-12- 
“able 7 
A t t e m p t s  to Derrronstrate Latent  V h s  in 
a Sample of 150 Apllo 11 liice 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
LO 
10 
10 
10 
10 
Total. 150 I - ... 9** - I 
*%I& negative is a test on 2 wols of 5 mice each 
**Leukemia virus was isolated f m  n ice  f r o m  9 isolators. A total of 
15 pools, consisting of 5 nice each, yielded viruse 
-13- 
Table 8 
Attempts to  Cemonstrate Latent V i r u s  i n  
a Sample of 150 A p l l o  12 l i ice 
_- 
10 
10 
10 
10 
10 
LO 
10 
10 
10 
10 
10 
10 
10 
10 
10 
-I- 
+ 
4- 
f 
+ 
-I- 
+ 
4- 
f 
-I- 
f 
.. 
... 
- 
- 
*Each negatiw! is a test on 2 pols of 5 mice each 
**Leukemia virus was isolated f m  mice frm 11 isolators. A total of 18 pols, consisting of 5 mice each, yielded virus. 
-14- 


Study of the murine viruses present in 'germfree' mice Final report, 1 Sep. 1968 - 30 Jun. 1970

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