THE ANTEBODY RESPGEQSE IN CGNVENTEDNAL AND
GERMFREE ANNALS T0 HEATED AND
UNHBJED WHOLE MILK

’a’hesis for the Degree of M. S.
MECHEGAN STATE. unwary
MYRON ESSEX; a v. M.
3.96?

“Mr-.99-

 

 

 

 

To attempt to determine if aavorely heated M1010
milk had retained immunogoniolty. conventional and gamm.
free animal: ware sensitized with whole milk autoclaved
for 30 minutes at 121 C. Rabbi“! were ifimunized with
on. of the five antigens listed below: 1) unheated whole
milk (UHWM), 2} whoio milk heatad for 13 minutes at
121 0 ‘83115’. 3} whole milk healzfid for 30 minute! at
121 G (3330’. 3) beta lactoglobulin (3L8). and 5) alpha
lactalbumin (ALA). Conventional and germfraa rats Vera
also Immunlzod with wags with and without Freund's
adjuvant or gggdatgglg Egggugsgg vaccine.

The interfacial precipitation test, and passivo
cutaneous anaphylaxis (PCA) in guinea pigs and rats
worn used to canning «11 Beta for antibody. It was
dotorminod with both tun intarfaoial and Fflh Coats that
WEBD had totainod immunosonicity for rabbits. Anti.
bodies wore not detected in the rat aura.

Antlsara produced to the @330 antigen reacted
equally well with the W330, WEE-’15, and WM?! antigens.
In. @333 antifiora reacted posit1VO17 with DLG, but
negatively with ALA for both the interracial and PCA
tests. suggosting ahnt BLG is a more heat stable

antigen than ALA.

T18 AVTIWCDY RESPOHfiK IV CUSVHfiTIOVEL
AND 833%?953 AVIfilLS TD “WaTED

AfiD UKUEATED WEOLE MILK

By

Myron Essex, D.V.M.‘

A'THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree 9!

MASTER OF SCIEflCE

Department of HleroblOIOgy and Public Health

1967

r'- L4
I]

K) (filial! :

\zfixDO

.AflfiatelEDSEHERTS

I wish to express my gratitude to Dr. Clyde K.
Smith. Departmonl of Veterinary Soloncu. Ohio State
Agricultural Exporlmont Station. for his help and
guidaneo during tho initlal phasb of thi: lnvontl-
gntlon. 1 am also indebted to Dr. navld T. Clark.
Department or nioroblology and Public Health. Mtohlgnn
scat. Univerolty. for his vnluablo advice and
nuslntanco during the oomplotlon phase of this
lnvostigntlén. and in my wafo. Dr. Elisabeth J. Bloax.‘
Michigan bapnrtmohe of Health, for hat critical roilav
and analysts at the manuscript. '

I glue wllhwto thank Mr} Gary P. Dardaa and
Dr. Glenn L. Ehxlor for thclr helpful criticisms

during the course of thlt investigation.

11

TABLE 0? COETEfiTS

ACKNOWLEQQEHENTSooooIooonoooooooooooooooooooooooooooo
LIST 0? FIQURES ARE TABLES-cooconcocoooooo-oooooooooo

INTRODUCTIONoooouoooaoooooooooooooooootoo-0.00.000...
LITERATURE RRVIEHOOIvttOtldiiiiitOODOOOiDOOOOOQUQOOOO

”11k as on Antigonoooooooooooooo-ooooooooooooooo
Antibody Production in tho Rntooooo-oooooooooooo

Th0 Germfroo “ago...noonooooooooooooooooooooooce
HATERIALS AND HgTfiGnsooooocooooootoo-coonoooooooooooo

AntigenSooooooone...cooooooooooooooo-ooaoooooooo
Anlmalsoococooooooooooooooooooooocooooaooooooono
Gormfroo ProcedHPOIooooooooo-ooooaoooooooooooooo
Bleeding Techniques and Sara: Collootlonoooo-ooo
Intorfaclal Tonto...on...cocooonooooooooooooooon
Passive Cutaneous Anaphylaxllootoo00.00.000.000.
Passive Cutaneous Anaphyloxla 1n the
Guinea Platooooooooooooooooouoooooooooooooo
Paoalvo Cntonoous Anaphylaxls‘ln the not...

“EQULTSoo9000000000t0¢00050000000otoooototoo.¢¥¢0000.
Interracial Tonto.oooooooooooooooo-oooooooooouoc
Passive Cutaneous Annphyloxls la the Guinea Fla.
Passivo Cutaneous Anaphylnxln in tho Ratoooooooo

”ISCUSSIOH-oooooaooooooooooooooooooooooooooooooooctcfi

SUflMAflYoooooooooooooooooooooooooooooooooooooooooooooo

LITERATUQE CITED-oooooooooooooooooooooooooooooooooooo

ll!

P330
11
l?

.1
3

3
33
52

59
59
60
63

6h
66

57

67
68

yo
70
71
73
7b
79
81

llgure

1o

Tabla
19
2.
3o

1!.

5.

LIST '53" YT'C-THTZS N?) TAULT‘S

filtrogonous compunonte of bovlno mllkoccooo..o

fiajcr fractiona of bovine milk proteinaooooaoo
Properties 0? rut fintibOfliflflcooaou001000.00...

nay: for feeding ot injecting antigen and
for collection of fiorum in gormrroo and

' conventional rats. first antigen was

CiVen 0“ do? 1090ooooodoooooooooooonqooooocoo.

Interracial tasks with rabbit antiooru
produced against antigens UHNM, H315. W330.

“LG. and ALAuooooooo-0.0.00uocoooooooicon-o...

PCA activity In guinea pigs nslng rabbit

Gߢ158930uooooococoon-ocoo-ooooooooooooooooooo

iv

Page

9

10

66

70

72

IKTROIIUCTION

Bocnuco of ito availability. inoxponnivoncoo.
ond nntritivo vnlno. bovino milk hao bocomc o commonly
uood food for tho human novbcrn. Bovine milk in not
without fault. howovor. ond being n toroign protein
to human beings it can indnco tho production of anti.
bodioo which in turn can initinto o hypersensitivo
otnto in the human infant. following the roccgnition
of milk hyporoenoitivity. oovorcl disease syndrome.
noro described in oooocintion with oorun antibody
titoro dovoloping in rosponoo to bovino milk protoino.
including the nnnpnylnctic 'cctodoath' syndrome
doocribod by Parish ct cl. (1960).

Soon after tho clinical recognition or milk
hyporocnoitivity, ’hoot-dcnotnrcd' milk producto bo-
camo popular on o oubotituto for nativo bovino milk
in tho diot of tho hyporscnoitivo infant. Yet.
those 'hootodonatnrod' milk products otton failed
to allovinto tho symptoms of milk-allergy: Ccnoid.
ornblc controversy nroso concerning tho iosuo cf
whothor milk could actually bo hcnt donoturcd to tho
oxtont that tho protoin fractions worn incapable of

antigenicnlly stimulating the human infant.

It was capocted tnnt because of the relatively
undeveloped gastrointoctinel environment of the germ.
{too animal. and the absence of intestinal micro-
organisms. that tho germfroo animal would lend itself
to n otudy involving the use of whole milk no an_
antigen. Thorefcro. the exporimcntel work reported
in this thosio‘uno based on the belief that the germ.
freo rut might be a suitable animal to use for study.

ing orally administered milk as an antigen.

LITtwsann QVV?VV

1% 5:3 £3. Antiénn

Hypersensitivity to bovine wiik proteins in
children was considered a possibility as early on 1916,
when Schloss and Earthen reported their suspicion of
permeation of two intestinal tracts of children by an.
digested milk protein fractions. Another early consida
oration was that of Park. who in 1920 described a
clinical ease of hypersensitivity to cow's milk in a
young child.

Anderson and Sonics: (1923) suggested that cow's
milk one cannhle of inducing an allergic state that
one in some way associatee with nutritional disturb.
encee in the human infant, noting that tho blood of
most maraSmio infants contained precipitina to bovine
milk. Merasmus in infancy in complicated by diarrhea.
derangement of the intestinal bacterial flora. do.
creased secretion of digestive enzymes, and other
symptoms of general chronic gastroenteritis. Candi.
tieno such as these in the infantile inteetine would
seemingly facilitate nbnormel absorption of unaltered
proteins (Gruskny end Cooke, 1955).

In an attempt to correlate clinical milk

hypersenettirity with tue presence of nnéifiedies,
Andersen, Santana, and "yarn (10?5) selected nine
ceildren heiqw fed hwfitfi all? 91% net knuun to have
been exflnScd t0 bevino milk nnfl HuhaeQWOHtly £94 tWGSG
children bevtne milk in vixco cf hucfin fillk. All nine
soon dcvclefied domenstrahle preeiwitins ta beviae milk.
Lippnrd, Schloaa, and Johfisgn (lfiflfii, ufling cqmfilefiont
fixetien, éewowatreted the presence or unflifieeied cow's
milk proteins in the bleed streane of nurmal infecfia
anon after a bottle feedlfig. Liphcrfl (193?} enfgegtcd
that the indiviaunl immune responfie might fllffcr in
these efiildren chewing clinical symyters cf milk
allfirry.

Quite a wide range of clinical nymytona have been
reported in essecietien with cow’s milk allergy. Sewn
of those are vomiting. diarrfica. abdcminnl pain. asthma.
rhinitis. failure to thrive, chronic ceuju, otitin
media. melena. colic. irritability, eczema. hemoptysiu.
anemia. cyanesis. shack. ufiper rccfiiratory symptoms.
and flcath (Debuts, S bless. and Andersefi, 1925: Gruekey
and Cache, 1953: nacmmen and Bees. 195?e: Crawford,
Kerrigan. nrd Arnold. 1953: Cunther et al.. 1950:
Pariah et 31., 1950: Helmet, Sears, and Kniker, 19523
Hantlnrton and Jcrzynkn. 1952: $111 and Cnombs. 1963:
Geldmen et nlg. 1943a; ficlsen, Greene, and Streup.

19$}; nelson. 1953; Sewell ct ale. 1963: Huckley and

 

Docs. 1955).

Numeroua investigators have correlated data tram
aeralogicul beats with the various symutams of cow's
milk allergy. further, Specific diseases or disease
conditione such as atepio dermatitis. celiee diaeaso.
Aldrieh's synflrome, Lacffler‘s syndrOme. puleenery
hemaaiderosis. pneumonia. unrlcr's cyndrome. congene
ital heart disease. ulcerativo colitis. mongeloidism.
and the cotadeath eyndrceo have been discussed on the
basic of clinical and laboratory cute as being ease.
ciated with hyperacnsitivity to milk (Krivit and Good,
1959: Gunther ct a1.. 1960; Reincr and Sears. 1950;
Heincr ct al.. lafiz: Perish ct 31.. 1950: Truelove,
1961; Huntington and Jettynke, 1962; Gill and Coembs.
19633 Pctcrmcfl efid Good. 1953' 391503. 19$3: Rothberg
and Farr, 1955; Withen, 1955. Boyd, 1956). Some of
these arc more relatent than other: with respect to
the current investigation, and only a few will be
reviewed.

Dubois. Schless, and Andersen {1925) indicated
an association between cutaneous hypersensitivity and
milk prccipitina in the more of normal and mares 10
infants. Freedman (1951). however. disagreed that
00". milk exerts any influence en the course of in.
fantllc catama.

Heincr ct a1.‘1952) and Immonen (lfiéjl attcmgted

ta correlato a high incidence of :rocipttins to now'a
milk and wheat gluten with the malabsorption aynfiramo
of 001130 diseaaa. Sawcll at al. (1953) noted that
ulthflrawal of milk and all milk products can be a
Valuable therapeutic measure for treatment of intraota
able diarrwea associated.vith some aastreintostlnal
disorders. Artur hating thafi many ulcerative colitis
patients improve nonsidorahly when cow’s milk is ex.
eluded from the diet. Truclovo (lfifili reintroduced
milk into the diet a! auroral at these patienta. In
every instance this was followed by a frank attack of
the disease. Aceording to Htlken (1255). seven times
as many ulcerative colitis pationts have significant
antibody titers to milk pratoins as dw narmal contra!
subjects. Ho Speculation was made as to whether the
absorption of milk proteins was a cause or a rasule
of ulcerativa colitis.

Using tha homagglutination techniqua. Peterson
and Good (1953) reported that 67% of 330 patients
with such candltions a» chronic recurrent pnanmania,
Aldrich'a ayndrame, Murlor'a syndrome. and congenital
heart disease had a aignifioant antibody titer to 069‘s
milk. nothbrrg and Farr (1955) agreed that preoipitins
to milk proteins appear mare frequently in the acra of
children with chronic puImanary 6150330 t%nn in normal

controls.

flvlaon (IDGQ) found an unusually high incidence
of hom3331utinins and precipltins to mi‘k proteins in
the Barn of monroloid childron. Eoniolnids character.
isttcally have a high anldonco of pneumonia, upper
respiratory ayrptoms. awd 1r0n dcficiency anemia.

Eilson, Hcinflr, an! Lnfivy (1953 detected higncr
levels of-milk prcclpttins in patients with iron
dcfictancy anemia than in normal nsntrol populatlnnsg
It was nut poaalble to staie whather thasc precipitins
were a caasa ar result of the iron deficiency anemia,
although the suggeatiun Haw made that One form of nunnia
might ho of a 5000n$ary nature. dapaufiing on pr1m&ry
sensitizatlun with cow's fiilk.

The coeqdeath syndromo is one of the max: highly
publicized aiscaao atatws to be attributed ta milk
hypersensitivity [fluutuar ct a1.. 1359; Pariah, Barrett,
and Coombs. 19593 Parisa at al.. 13Gb: Gill and Ccombs.
1933: Boyd, 1935). The flora orih.duath, or cotndoath,
ta usad wh¢a aha infant is found écaé aftar a night's
81969. with no apparent symgtauataloqy or prior $139156
condition. finiahor emu parent. phyaisian, nwr patholaa

£139 can find Valid rousun far aha again, the only

-u

canaistent lesiana bein; pulmonary edema and congestion.

F

Tho pathologist‘fl repurt oftfin cfibfia acuto ifitorfliitial
pneumonia er mechanical aflymyxia as Lain; the .arsu of

death {Huntington and Jurzynka, 1952). There cot

deaths mny account for over 20% of the mmrtality 1n
oh11dron under one year (Johnstona and Lawy. 1956).
Extensive bacteriological and virolegical fitndies havc
been conducted on untapsy samples of lung tissue, but
the Vast majority of thoaa attempts have‘baon unannoess.
ful. Johnstono and any (1955). howovcr. hava reported
lama succefis in isolation nttompts. and believe tfiat
many of these deaths are due to lower respiratory tract
tufeotions. Peterson and Good (1953} failed to detect
sigaificant Ievala of preeipitini_1n scrum samylas

from six cases a! tho orthodeath syndrome. indicating
that the hypo hosts of Parish. as mantionod in the
introduction, may not always Apply.

Before farther canslderation is given to the
way in which milk acts to induce nyyoracnsittvity. the
composition of milk in regard to its antigenicity will
be briefly mentioned.

Milk can b0 datinod as the lacteal secretion of
the mammary gland Intended for the'nourlshmcnt of young.
To moot this requirement. milk must be. and indeed In,
ono of the most complete foofls nvailab1e. The general
composition of bovine milk 15 as follows: watar 37o20§¢
{at 3.80%. carbohydrates 3.95%. protain 3.35%. and
minerals 0.?Ofi (Harrington, 1953). These figures vary
considarahly with the breed. spacing. and the time

elapsed since tho start of lactation within the

individual.

From the atandyoint of antigon1¢1ty 1n hctvrola
ogoua species only the protein fraction is significant.
Kan and Bowie (1951) break down the nitrogcnous componanta

of batine milk in the manner dogorlbod in Figure lo

100 Parts Ritroganous Compound!

I I 1

95fi’Proto£n(3.18 graml/IOO grams 5% Non Frotoln Nitrogen

 

 

 

wholc milk)
73.55 Canola (2.53) 16.5% Rilh Serum Proteins
(0:55,
35% float seabu ' 12.52% float Labno Pmtelno
Protooaonpoptono (0.13) J {0.h2)
9.2-; Albumin: (0.31) 3.3% Globunns (0.11)

Figaro 1. nitrogenous 00mponentfl of bovine milk.

10

The major fractions of bovine milk proteins and
Some of their characteristics are listed according to

Jonness at a1. (1955) in Table I.

Table 1. £330: fractions of bovine milk proteins.

 

 

Fraction from Approx. % of Melee.
Prateig £313? ififilfifflj rtlk firotnggg_ wt:
alpha casein casein 35.63
beta casein casein 19-23 23,000
gamma casein casein 3-?
beta lactaylahulin lactalhumin 7.12 35,030
alpha lactalbumin laotnlhumin 3.15 15.130
blood serum albumin lactalbumin 0.7-1.3 €5,090
euxlobulin Inctoglobulln 0.8.1.7 1S0,000
psaudoauglohulin IGOtOfiIObulin 0.5‘105 130.050

Originally the milk proteins were separated only
on differences in solubility. and werc classifted as
casein. lactalbumin, and lnatoglobulin fractions (Jeanna:
at a1.. 1955). A fourth fraction, designated ‘vrotaoao-
peptonO.‘ was isolated by Rowland (1933).

Casein is the pretax“ traction in nilk proctpi.
tatad by acifilrylng raw skim milk to pH 3.5-3.7. It
comprises 32% of the proteins, and consists of a mix-
ture of five principal components: 1) alpha-Incaseln.
2) alpha-Zooascin, 3) hota casein. h) gamma cafietn,
and 5) delta onsofin (Fries. 19$93p Those comnonanta

exist in milk as complex particle; containing calcium

and phosphate (Jenneaa at a1.. 1956; Brunnnr at 31., 1950;

11

Ian and ch10. 1951).

Approxlmntcly 17% of the protein fraction of
skim milk is the whey or milk aerum proteins. As cpv
posed to casein. this fraction 1! soluble when will in
ccldlflod to pH h.5-b.7. An additional one percent of
the total protein 15 present in whole milk only. as the
llpcprotcln designated as {at global. mambrano protcln
(Jenna‘s at al.. 19363 Fries. 1959: “runner at alug‘1960lo

Tho milk serum proteins are divided into heat
ltablc and hock labile traction: on the basis of whether
or not the protein can be rendered cold proclpltablo at
pH b.6‘by previous boiling f6! 20 mlnutco. The “protons.-
pcptonc' fraction ls that part that ls not rendered acid
proclpltablo by prior balling. and 1‘ thus tormnd heat
stable (Jcnnos: at al.. 19563 Brands: at 31.. 19508
Kon and Gavin. 1951). The boat labllo group of protelna
which is rendered practpltahlc at p“ 5.6 by previous
boiling is further classified into two fractions. tho
albumin: and tho globullnau =

One group at the heat labile milk serum proteins.
the albUmins. rcprescnt the moct algnlfleant antigen-
tron the standpoint of the Individual who 13 allergic to
nllk.v The principal comccncnts of the albumin fraction
arc alpha lactalbumln, beta-lolcctOglobulln, beta-2o
lactoglobulln. serum albumin. and peroxidauo (Fries.

1959). The globullnn. although of major importance

tron tho standpoint of lmmunlty within the lndlvldual.
are of minor significancc as an nntlgcn, and are rep.
resented as tho gamma glcbullns pseudocufllobulln and
ouglcbulln (Janncsc 0% cl.. 1955! tries. 1959).

Bloctrophorctlc analyse: hnvc revealed at least
twelve dlltlnct antlgcnlc factors asscclatcd with bovine
milk protclnc (Hanson aha Johanccn. 1959): 51: of those
arc also relatcd to bovine blood scrum proteins (Hacson.
1959,. I

Due to ac. antigenic complcxlty. Frlcc (1959)
nontlona that milk chould not ho trontcd on a single
cllcrgcn. but rather as 3 enable: of many allergens.
Tho three proteins tnac are most significant as allcr.
gens Ir. alpha lactalbunln. beta lactoglobulln. and
canola. or loose: lmportancc la bcclnc ceéum albumln.

Most ficdlctrlclana care. that come infants who
are hypersensitive to cov'c milk can tolerate «cat'-
mllk while other. cannot (ulll. 1939: Sayerstaln. 1960).
Ono reason for this is that the casein fraction 1: I
lmmuncloglcclly almost identical between the apoclos.
but that the alpha lactclbumln, beta lactoglobulln. cud
scrum albumin arc cpccicn specific (30113, 1911: “£11.
1939: irtos. 1959s Lao. 1953}. Thus. those individuals
hypcracnsltltc to thc casein: of bovine milk cunld
probably not tclorctc goat's allk. while thee. ladlvld.

unis who are hypersensltlvo to thc Mont labllc milk

l'lllflll‘ [i {If [.Illllll Ill! .n'l 1 i‘l viii!!! fl‘

13

eeru- proteine would often be able to incorporate goat's
milk into their diet without difficulty.

It is generally assumed that an antigen must be
introduced beyond the epithelial tissues of on animal
before antibody production will be ntimulotod (Carpenter.
1965). Milk has been injected into neonates for or.
perimentel purposes, however. it is obvious that o
more subtle manner of sensitization must be involved
in the prooeeo of natural induetion of hypersensitivity
(Header. 1873; uaoenstadt and neijkensjoid.V1965). it
leeet three methode for natural oeooitieation to milk
proteins have been considorod in the post. and those.
will be oioeueeod.

The first poeeible route for sensitization,
suggested by Rainer (1928). involves the passive trans.
for of milk protein antigen across the placenta from
the mother to the fetus. It is known that during preg-
neney many women have e psychie crowing for certain
(code. and the exceesive consumption of these foods.
it they happen to be milk or milk products. could than
lead to exoeeeive absorption of milk protein antigens
(Rainer. 1957). Tnese protein antigens would then be
transmitted to the fetue via the common circulatory
syetem, end thue entigonioollr etimulote the fetus to
produce antibody (flatner and Greenburgh, 1932: Rather,

Crawford. and flynn. 1955}. It would seem that the

1h

Validity of this theory aould depend on whether the
human fetus could be deolerod immunologically competent.
fieeeive maternalotetol circulatory traeemlesioo or
maternally produced antibody has also been considered
(Gunther et «1., 1959).

The sooond possible route of aonsltisation ie
aspiration of gastric contents. which in the non-breast
fod infant would usually consist of bovine milk (Parish
et 11.. 1960). Milk proteins present in the lung might
then be nhsorbod earns: the alveolar membrane into the
intoretitiol tissue and circulation with resulting
entigonic stimulation (Nelson, 1955). In support or thin
explanation are Peterson and 6004': (1963) finding!
of substantially higher milk protein antibody titore
in Children with anatomically dofoetlvo swallowing
mechanisms than in normal children.

The third and vrobobly firedomlnant route at
tensitlzation to milk proteins is intoetinal absorp-
tlon. Early investigation: by Mendel and Rookuood
{1903) were lutorprotod as indieating that protein:
oould be obsorbod intact from the intestine. They tied
off a emotion of the intestinal tract. and demonstrated
significant roduotion of e protein substance. odegtin,
that had boon placed within the segment.

Borgstrom ot al. (195?) domonstrotod that under

normal conditions up to too poroont of the dietary

15'

prototn reaches the bottom of tha small intestine with.
out being disastcd. M1113 at 81. (1923) and Ratner and
Gruéh1,(1933) beliavo that nndar normal conditions
absorption of unaltered protein occurs with great reg.
ulgrity. and that protains may enter tho blood stream
from any part of tho bouoi. including the rectum.

Three arttoric havo'boon proposed for oral sen.
situation with milk prutalnu u the protein fractions
must be in a native stato, uoluble. diniyzablo. and of
loa‘molooulmt Uflightg 2) the intestinal tract malt bo‘
particularly permeable at tho eimo the food 1! tngcstod.
and 3) the quantity ingnsted must be sufficient for
absorption ta take place (Ratnar. 195?). Holland 0%
nl. (1962) tuggastéd that the incidence 9f milk precipu
itins in the blend of infant. could be associated uttfi
increased exposure to antigon, inoronaad pcémoabllity at
tho gut to protein, or variations in host response to
phytiologio quantitlal of antigen.

Park (1951) and Nelson (196%) have suggoscad the
need for a genatia prodisgosltion to uyparsensltivaness
before sensitization to milk proteins can occur.

Tug lines of defense against tranSfllaaion of
unaiterod procaine acres. the intestinal wail ara that
protain foods are nurmaliy hydroXyncd by enzymatic
digastion 1n tho.gnstruintaat1na1 tract. and that tho

intestinal wall is usually thought to ho impcrmoablo to

collaids.(flatner. 1?5?). The failure of thcso dcfcnsas
may result {rum alteration of the intestinal wucoaa,
permitting increased abaorption of undigestad prfitcin
into the circulation (351503. lfififi). Those defenses may
fail due to pathologto candittuns. devolonmantal anew.
alien, malnutrition. convalescouco from diarrhaa, follow»
lng gastratntaatinal diaturbanoes. or increascd pcrfi
monbility in early infancy (Gruakay and Cooke, 19553
Immouen, lfiis), With ulaerative colitis patiants, 1n.
eraasod absorption of antiganie protein tram the cut
might occur far 3% least tun separate rsaaans: deprcsscd
proteolytic activity in tho uppar gastrointestinal tract.
or mare facile absorption of undigested pratcin by the
inflamed colonic mucosa (Taylnr and Truclove. 1951).

There in teascn to believe that two inte$tin31
enviranment of the germ tron animal is very mimilar
to tha intestimal tract of tho newbnrn (Smith. 1955).
This similarity is 1ar301y due to {no lack of an in.
tostlnal bacterial flora. which tha convantianal animal
acquires soon aftgr birth. This bacteria: flora 13
probably largely responsible far stimulating hifitOo
logical chmngas 1n the rnall intestine that diaoauraga
the further absorption at lmrxa moiacules (Lngorcrunts
at n1., 1955; Smith. 1956}‘

Clark (193?) 1dantiflad orally ingested buvlne

‘gamma.filobu11n and ovalbumin in tho apieal cytonlasm

16

17

of the jejunum and ileum of rats and mice that had been
fed theso proteins. Ha postulates that the coil takas
in than» particles by pinocytonis, a process or invafia
ination of the apinal 0011 mambrana to form vacuoles
containing material from the intentinal lumen. Bight.
ten days after birth. however. the columnar abaorptivo
0011. lost their ubiiity ta ingest pratains and colioids.
this being the age at which rats and mice 1030 the
ability to absorb antibodies from the intestin. in an
immunologioaliy intact fora (Clark, 1959). Anatomical
changes in th. columnar absorpiivo c011. acoampany thus.
change. in function.

Pérticloa absorbed by the epithelium of the small
intastino can be picked up by tho intcutinai lymphatics.
and transported to thc blood stream. Ingestion by no.
lunnar absorptivo calla uppsars to bo a nonasoleativa
machnnisn for faking in whatever happana to b. in the
onvironnont. Laboiiod proteins absorbed in this manner
can be recovered in tho biood within nin¢ty minutes
trou.tho timo of oral ingeation (Clark. 1959).

Farr. Dickanaon. and Smith {1960) (ed bovinc
torus albumin ta rabbits in tha drinking water. and
found that t 0.0025§ aolution for thirty days. or a
6.25% solution for one day an: able to élioit the pro.
duction of a lufficiont antibody titer to b¢ detectable

with the ammonium sulfat. teehnique. Antibody an:

18

detected as early es seven days otter the firet feeding
of e 0.1% eolutien. Intravenous injection of e non.
antigenie dose of 0.006 mg. of bovine serum albumin.
however. resulted in e higher blood level of bovine
serum albumin after seven day: than did the feeding of
o 0.25% eolutien at any time. Thus it is expected that
immunisation by the oral route in initiated by lymphoid
tissue associated with the intestinal tract. where
higher oonoentrotiono of bovine serum elbuoin are avail.
able (rerr, Diokoneon. end Smith. 1960). These eenei.
tired lymphoid cell: could then be transferred to

other lites of antibody production each on the spleen
and lymph nodes (Farr and Diekeneeu. 1961: Holland et
ai.. 1962). Intestinal permeability is greater in the
newborn. and iomonolegieeily oompetent oe11e_ef the
newborn lie in the gut wall. making then quite to.
toolihio to antigen: in the gut (Hinkie. Bong. and lost.
1951).

Rothborg, Kraft. and tort (1966,1957) thawed that
antibodies produced to either orally or parentereiiy
administered bovine eermn albumin were qualitatively
similar when tested by radioimmoneeieetropheresie.
passive hemegglotinetion, zaneroeptoethenel treatment.
detection of antibody in acetone density gradient
fractions of serum, ammoniuo oulfate test. antigen

precipitating capacity, and density gradient

19

ultraoontrifugetion.

Soon otter the advent of commercially prepared
eraporatod milk, it became evident to pefiiatricians that
heat donnturation of milk proteins significantly reduced
their antigenicity.. This loss of antigenicity we. I
probably due to at least two factors! 1’ actual destruo-
tien of the antigwnie portion of the molecule by host
donoturation. and 2) increased susceptibility to eosyo
motio breakdown in the gut with consequential reduction
in protein available for intestinal absorption.

Denaturetion of a protein implies any change in
physical and chemical preporiios from the native state
including especially the loss of solubility at the lone
electric point for that protein. but else changes in
electrophoretio mobility. and reduction of sulfhydryl
groups (Mauro! and Scidelborger. 1951; Loreen and
Rolleri. 19553 Seporstein and Anderson. 1962). These
changes can be broeght about by e large variety of chem-
ical and physical agents including ensymeo. irradiation.
snide. alkaline. alcohol. soetono. sslts or heavy
metals. dyes. hoot. light. and pressure (Rainer. 1957i
Grogan and Crawforo. 1961). The stages of denaturetion
are not necessarily all or none with many proteins. and
some stages of thermal donaturntien are reversible.

Denaturetion of native proteins often result.

in lees or diminution of specific immunologic properties

becaunn, due to their physical structure. coagulatod
proteins arn delayed in fun gastrointestinal tract,
thns giving morn time for enzymatic digestion (Levin
nnfl Hayden. 1932; Rainer. 195?}. Also. as evidenced
by the effects of trypsin on proteins in vitro. discs.
tive anzymes are nor: effective per unit time on
partially'donaturod pratoins than on native proteins
(Wallonahnwronqe ann Koch. 1930). This rnsults in.a
marked lass of ability for the milk protnina to anti.
gnnically sensitize the indiviaual by thn oral-route
(Rntncr and truchl. 1935).

Denaturnd proteins may act as aimplo or nonpicx ‘
bantens. to block a reaction batwnon antibody and
native antigen without proflucing a visibln rnnntion

Superstein and Andarsnn, 1952). Same dnnntured
proteins may act as antigen: to induce production of
antibodies to the denaturad protein as wall as to the
unaltered protein.l Haboob and Borelia (1965) found
that the donatnration of bovine serum albumin in which
the disulfido bonds ware broken resulted in.n loss of
reactivity bntween antibody pronuced against the do-
naturnd bovine serum albumin and undenatured bovina
serum albumin.

There 15 congidernblc diascnsion in the litora.
tare regarding the issue of vhctfinr a heated protein

Iantigen has retained sufficient stfangth and npncificity

to act as an alinrgcn when orally ndministorrd. In

the case of allorfir to bovine milk, curtain rascarchert
fool that individuals hyperscns itivo to miik could
tolurato-a boat denatured produce (Rainer. 19573 crawford,
1960. Crawford and Grogan. 1961; Hinklo. flung. and west.
1961). Oihor investigators. in contrast, boiiora thai
donatnrod milk is not a safe oubotituto for milk in in.
41!. of hypersensitivo indivifiuala. and that instead
Iuohproductn as any milk and moat base milks should be
alod (Saporutoin and Anderson. 1962; Rainer. Sears. and
Knikor. 19623 Ln: and Todd. 196k). Saporstoin and
Anderson (1962) have suggested that the term heat do.
unturod milk can b. nbandoned as a designation imply.
inc nonoantigenioity or non.a11erganicity or the
proteins in those products.

Rainer and his asnoointes beiiaro that the
unjoriiy of children hyparnonnitivo to milk can safely
consume heat treated milk. and thnt 103; than on.
{burth actually requiro n milk rubstituto (antnor,
Grantord. and rlynn. 19563 Ratnor. 1957: Rather oi n1..
1958.}. According to nntnor. the lack of a favorabl-
responao to a host treated milk in this latter 251 in
an. to n casein hypersensitivity. Superstein (1960)
'b-liovos that no evidence has been given for casein
hypornenoitirity. and that suspected canes ware due

to incorrect diagnosas and. with casein tune was

contaminated with lactoglobulin or lactalbumln. It
cannot he Gisputod that lncompicto purificurinn of tho
milk protoin fractions hag resultad in man misleading
conclusions. and early studies were carried out with
Canola fractions contaminated with lactalbumin and
1ac€6310bulia. and lootalbumln fractions contA£lnated
with lacioglobolin and serum albumin (Ratfior at 31..
1958a; Cola and Laos. 1953).

It would seem that txo newly acquired Specificity
of in. antigon should be considered as woll as the
original quantity of nativa protein remaining VHO"
trying to determine the value of a heat denatured milk
product for uao in tho diet of hypersensitive individ.
null. Synthetic aolypoptidos of low molecular weight
are antigenic in rabbits and guinea pigs. auggoating
that severely denatured proteins could still be anti.
gonio (Hooter. 19fi2. 1963; Hauror. Gorulot. and Pinonuok.
1953; Lavina. 1965a.b). To be considered also in the
fact that the ability of a substance to act as on anti.
son based on classical serologionl techniques of anti-‘
body detection is not necessarily indicative of tho
ullorgonlé provortiOI of that substance (Hanson and
”museum, 1951). ‘

Turth (1925) noted that the heating of horse
Larum or crystalline egg albumin to 169 C oroatod a

new antigenic specificity. intisora obtainod by

N
b!

injecting antigen heated to 103 C procifiitcted strong.
1} the antigen used in its pro notion, hut canned only
weak procipitntlou of unficntod proioiu. Guitar (192?)
found that tented whey proteins wore more reactiVO in
animals annuitixod with hontod milk than in animal-
lonaitisod with raw milk. Louis and Haydon (1932)
found that boating altered some antigens so that anti-
Iorn prodacad against them worn morn or less specific
for the hentofl antagons. the soucnllofi cocLa-antigens.
Included in this group was bovine silk that had been
heated to 115 C for 15 minutes.

‘Hirntn and Suasdorf {1955) found that tho partial
donnturntion of bovine sorum albumin created a non
antigenic specificity. Wkon £11: partially donnturod
bovinn scrum nlhumin was lujootod into rabbits. it

resulted in higher nntibofiy titora and n larger number

of rooctorn than did native bovine serum albumin when
oach antigen was tostod with its homologous antiserum.
Peters and Cootzl (1955) state that appreciable config.
nrationnl changes can be induced in the bovino serum
albumin molecule without markedly altering its antigen-
ic prozeriioa, and that immunologic detorminanta an.
reactivo in the nativo molooulo may be uncovered by
manipulaticms Hf'xich do not break the {mythic c’mirl.
(dromntngrnphy of a tryptic hydrolysnto of beta lacto.

globulin by fling. Gusdon,.nnd fitnvitaky (1955) yielded

22.-

30700 active peptide haptans. These inhibited the
hamngglntinatian. precipitation, and passivu outaneeua
anaphylazia reactions in a beta lactoglobuiin anticbeta
lactoglobulin system.

Although it is usually accepted that casein is
tho most heat stable protein in whole milk. considerable
disagreement exists concerning the comparativo heat
stability of the more heat labiio whey firoteins. Larson
and Rolleri (1955) conciudcd that alpha lactaibumin was
the moat boat atahiu a! tha whey proteins. Cravford
and Grogun (1951}. Colitas~¥illiamo (1962). and Lu:
and Todd (196%) maintain that alpha lactalbomin in the
more heat labile, and that beta Inocogiobnlin in tho
most heat stable of tha whey protein:. -Hanaon £1965),
believe: that serum albumin in tha must beat stable or
the serum proteins.

Wells (1903) Itated that crystaiiino egg albumin
and bovine mitt do not ioao their sensitizing power
for anaphylaxil than heated tn 100 O for 30 minutes.
Attempts by flutnar at .1. (19580) to orally sonoitiuo
'or ohnilongo guinoa pigs with hunt denatured milk were
invariably unsuccessful. Lu: and Todd (1965) found
that milk that had been haatcd to 235 I for 19 uinutos
showed only slight difforonooa in antigenicity from
unhantcd milk when hunted by the Prnuunitznfinstnor

techniquo. Hiero.0uehterlony toohniqueu show that

25

heating causes a loss of precipitablltty with the native
protein. but in this case such a loss is not necessarily
Iquivalent to a loss of antigenicity or allergenlalty
(Lu; and Todd. 196:»).

In 1929. Cutter failed to detect any stgnttlcant
difference: in the degree of anaphylaxis ollct$ed with
casein: from pasteurinod OR*¢ommerciully available
evaporated milk that had boon exposed to 220 r far 30
minutes or 200 f for 20 ntnntoa. nouiand (1937) boltcvod
that danatnration of alpha lactalbumin and beta lactoo
glébulin was complete in tire to ten minutes at 100 c.
EWOIn. Anrand. and Hobart: (1961) found that paotanra
{tatton canned nogllgiblc milk protein denaturntion.
bu. antoalavtng at 250 I for 15 minutes denatured all
tho who: protoinl. Ancarding to Hanson (1953) canotn
can withntand 120 0 for at 1.39% 15 minutoa. and beta
lactoglobultn 100 o for gt least 15 m£nucea. nausea
and fiansaon (1961) using immunoo1ootrophorouia could
not demonstrate the prasonoo of whey'proteinn from milk
heatod to 120 c for 13 minutes.

Batnor at :1. (1958b! on the basis of syutamio
unaphylaxln touts. state that the allersanlolty of
tlpha lactaIbumln was eliminated in a heat denatured
milk product. and that even nasal» evidenced a degree
of boat lablllty. cal. and Boo. (1963) claim that

beta lactoglobulin in milk oxyosed to evaporation 1|

26

only partially denatured and still able to clicit.cna.
phylaxis when guinea pigs are sensitized with unheated
beta locioglobulin.

Snporstcin (1969) found that both alpha loct.
albumin and beta lactogicbclin in commercial evaporated
milk products were ctill nntigcnically intact. In I
later study. Sapcrctcin and Andcrcon (1962) concluded
that hoot treatment had some affect on alpha lactclbunin,
but did not reduce the antigenicity of casein or hot:
lactoglobulin. Vith precipitation tests. milk products
heated for ton minutes at llo O or 30 minutes at 99 c _
all gave positive precipitntcs with bctn lactcglcbulin.
alpha lactclbumin and casein. Three hour heating at
99 c did not inactivctc thc alpha lnctalbumin. bot.
lactcglobulin or coccin no tested by pan-ire cutanccul
cnaphylcxic (Superstcin and Anderson. 1962).

According to trio: (1957. 1959) allergy to ccw’c
milk is not as frequent as uncritical cvnlnntion would
makc it snow, and many disturbances induced by milk
should not bc categorized as allcrgic in naturc. The
only satisfactory way to diagnose milk allergy in to
chow that symptoms arc alleviated with removal of milk
from the dict. and that thcy return anon milk feedings
are restored (Gold and Rubbing. 1965).

Recorded incidenccn of clinical symptoms of

dzow's milk allorjy in randomly selected children has

varicd from 0.31 to 7.0fi (Cloin. 195a. 1952; Collins-
Williams, l?fi£; Glaser, 1956; nachman anfi Docs, l?5?c}.
finned on clinical symptoms; reportod incidences of
ccncltlvity to cow‘a milk in allergic grouya varied
from 5.0% in ngthmctics to 55% in allergic children
undcr four years of ago (Davidson. l9$2x Fries. 1957’.
In allcrgic patients under two years, tho lacidcnco

of hypersensitivity to cov‘c milk is nigh, bcing
proscnt in oyproximatcly one in three infants (Bockman
and Docs, 1957b)¢

matnor ct at. (1953a) and CollincoWllliams (1962)
attribute the greatest allcrgonicity to milk proteins
to thc beta lactoglobulin fraction. Fries (19??) and
Cole and Boos (1953) stoic that sensitization to milk
is usually to tho lactclbumin fraction. Golénan ct a1.
(lgfijo) orally challenged children known to ho allergic
to milk with the milk grotoin fractions. Tho
frcquoncioc of rcactionc tc‘tuc various fractions
were: casein 57$. bcvinc scrum albumin 51%. beta lactca
globulin 65¢, nna alpha lactalbumin 53?.

Botcctablo antibody titers to bovine silk in
both healthy nnl allergic infants arc concidnrahly
more canman than clinical signs of milk ollcrgy.

Tfic vrccipitation test. as performed by the gel
diffusion or capillary tube technique. ccnsistcntly

~dctccts only tho high titorod scro. Tynntyufivc

23

percent of 170 normal and sick infants had precipitins
ta oow‘o milk by tho 591 diffusion technique (Peterson
and 606d. 1953). Prooipitlnr are more frequently found
in sort of children 31th chronic pulmonary disease or
iron deficiency antmla than_in normal centrolr. The
incidouoo an dotarmlnad by 301 diffusion in irpn
dotioiont infant. fad cov‘rmilk prior to diagnasla
in. 75% to 80% (Hilton, Rainer, and Laney. 1952).
Gunther at :1. (1960) found that 95% of 286
rerun sampler takon from normal infants had hamagglu-
tinin titorl fiptn 1090 with a mode 9f 6“. Similar
lamploo from hoalthy adult. and newborn infants boftra
uxpoaura to bovino milk uhouad titers of rare to tour
for the moat part. but samples from infant! aorou to
97 rock. old had tit-rs of two to 128 with a nann of
16 to 32. Supersteiu at :1. (1963} and Collinsnwilliamu
and Salama {1965) feund similar pnttornn of homagglug
tininl for normal children. childrun allargio t0 milk.
and allergic children that were not illargie to milk.
Those finding. disagrvo with than. of Gunther 0t :1.
(1962) and Parish at :1. (195a: that high hemagglntinin
titers to milk proteins are correlated with suscepti¢
bility to anaphylaxit in infantr. Poternan and Goad
(1963) found that 67% Of 283 sarum samples from both
healthy and diseasod children had a milk protain

haaagglutinin titer of ten or more. The highest titer.

29

of hemagglutlnins were obtained from the acra of
significantly disabled children, All sera with hem.
agglutination titnra over 1,230 had proclplttns also,.
but most aura shaving hemagqutinating activity in low
titers only did not have precipitlfis.

Skin testing techniques did not correlate with
either hemnaglutination or precipttin titers. giving
negatlvotests with earn showing the highemt positive
in vitro titers by hamagglutinatton or precipitation,
and aura with lower homagglutinin or precipitln titer.
ocoaaionally giving positiva skin taste (Patnraon and
Good. 1953). Antibody specific for bovine serum
albumin by the ammonium sulfate technique was in no
way related to positive skin tests for milk or haviné
aerwm albumin (Rothbarg and rarr. 1952). flatner.
Crawford. and Flynn (1955) and Goldman at 31. (1953b)
report that strongly positive skin tests with purified
mtlk protains have considerablo diagnostic valua-

annthar at 619 (1950), using tha homaggluflnation
technique. Found antlbofiy ageoiflo to casein. “thfi.
1actalbum1n.'and bovine serum albumin in the serum of
normal infants. no untibodf was found specific tt bots
lactoglobulin. In contrast, Peterson and Good (1953)
found precipttlnn to all milk protein fractions except
casein 1n narmul and allergic children. In thaao Itud108.

the most common antibody to a milk protein fraction was

anti-Leta lactogiohulin.

Using tau axmonium sulfate teat, Rothbar; and
Farr (1952) found circulating antibody to bovine aarum
albumin in serum sample: from 73% 9f the cuildren and
17$ of the adult: tasted. Thirtyuscven perccnt of the
children and saven yeroonc of the adults had antlhofiy
to alpha lac‘fialbumin.‘ Truss... althou:_i§h tf'xcre 5.8 com-11:1.-
eraaly mora alpha lactalbumin than bovine serum albumtn
in bovine milk. the bovine serum albumin aoumed to not
as a more efficient antigen. However, it should alga
be considarodthut bavina serum albumin would be present
in any beef censumod. whereas alpha lactnlbumin would
not (Farr. 9393.

Groups of healthy and fiisaasnd children showed
no diffrrences in incidenco of antibodies to havino
aarum albumin or alyha lactalbumin (flothberg and Farr,
1365). It was concluded that when an immediate type of
hypersensitivity was associated with the prosonco of
circulating antibady. tRmt antibaey is necessary but
not alona sufficient ta cauac gyrwtums on ro~GXposure
to the antigen. Other factars such as Variatian of
tfle tyye of antlbndy produced. and organ auflceptibtlity
and reactl?ity, permeability of the blaod gut barrier.
and 4030 of antigen or raucxposure a1! determine whether
an individual will have an untaward reactign.

The possibility of'n cross raaciion between

31

antibody produced against bovine milk protein antigens,
aspcclally bovine serum albumin. and other heterologous
antigens has been ¢on51dcred by $31519 (1952). «01319
and MoConahcy (1962). and nothbcrg and Farr (1985}.

Ho sharod antigenicity wan detected between bovine

corn. albumin and other antigens to which the penulntion
might be Iroquantly expo-ed; such as oralbumtn. polio
vaccine. influanza vaccine, tetanus toxoid, diphtheria.
ana portussta vaccinc; In vivo reactions between

rabbit antiobovtng serum albumin and ten crass reacting
mammalian ulbumina were capable of producing systemic
anaphylaxts, passive cutaneous anaphylaxis. and the
Arthun phenomenon in sensitized guinea pigs. When

fidual amounts of precipitating rabbit antlnbovlna serum
albumin were used to passively sensitxno the animals.
sum. of the cross ruaoting albumin: produced as never.
symptoms as the homologous bovine aurun albumin when

a sufficiant amount of antigen was usod in the challenge
dos. (E61510. 1962).

Saperstoln It .1. (1953) concluded that the
passive cutaneous anmphylaxiu test was more Valid than
tannad 0011 homagglutinatian‘techniques. double gel
diffusion. or akin testing techniques for determining
tltorc of milk protein antibodies in the zeta of
allergio‘ohildren.

Hinden. Reid. and Farr (1955) studied the

31

relative sensitivity of several uerological tests for
their respoctlvu unpacity to detect circulating anti.
body to bovine serum albumin in human aura. Those
studied were douhlo gel diffusion, homagglutination,
Prnuanitz~fiustnnr. passive cutaneous anapbylaxla.
syueomto annphylaxis. radioimmunoelectraphoresls.
spontaneous precipitatlan. and the ammonium aulfato
tent. The only consistently positifu tests for
detection or antibody worn the ammonium aulfato and
radiotmmunoelectrophoreats techniquos. Seventy-fit.
percent or the sera were posltlvo by hemagglutinatlon.
and 50% were positive by spontanaous practpltntlon.
The remaining tests were considerably less sensitive

in this atudy.

33

aptnwig ”gel-“347M. :31 13 :32 3.33.

Tha r28 hws ta b3 cansidcrod n wosr afitibofiy nro¢
ducer When exyofied to commonly usafl nntisaua “r cwni
vanéional matfioés (339(01. 1951; (abut and mayor, 1951;
Bancvttx and T?apnfi1, 1355). Althauffi this wank rasnonao
becomes especially eviaant when trying to induca ana-
phylaaia in this egjcics, rats are also reiatively poor
producers of precipitins and agglutinins (Lipton, Stano.
and Frennd, 1955a wimcbright and titan. 1952: Cody and
Coda, 1953). Anaphylaxis in also difficult to produce
in the mouse, aitfiougfi this species is unually not as
unrcactive as the rat (Kind, 1937). This réaigtanco to
anayhylazia is prabahly aaaocfiatcd with the fact that
both the rat and mouse are extremely tolerant to the
effoctc of histamine. being unaffected by a done at least
fit. hundred times as large as that which will kill the
guinoz pig (Drill. 1958).

Doapito the difficulties involved, early workers
Parker and Parker (1923). Deliauny (1930), and
Hochwaid and Rackenann (1926) reported ouocoas in
attempt: to produna anmphyiaxis in rats. flare recently,
a vifia variety of techniquoa have boon cmploynd far
inoroaaing the rat's ability to respond to sensitization
and nubsoquont challenge with foreign proteins. Tfioso
inoluda hypoyhyaootamy. fasting. insulin induced hypo.

glyoomia. tha adminiatration of aacarbio acid. thyroid

hormones. zymosan, Rrucella abortua vaccine. Pround‘n
adJuvant, Bogdoto;12 gogtusstg'calla. and nqrdetoLLg
Bertgggig vaccine (Buchwald and Raokomann. 19363
Parfantjov and Goodlino. 1938; Halkiel and nargis.
1952a.b; new: and Code. 1953: Kind. 1957i Sanyal and
Host. 1958: Spencer and Host. 1952; Hanna. 1953;
Adamkiovios. Sacra. and Ventura. 1963; ninaghi and
Bonacorrat. 1963b)-

A partial list of tho protein antigens used in
fin. 1ucoosafu1 antigenic sensitisation of rat. Include:
human serum. hora. iorum. egg albumin. bovine serum ti.
bumin. human lorum albumin. human gamma globulin. bovine
gammn_giobultn. fibrinogen. the flagella! antigen from
Salmggglgg as . and the 0011 oxtrnat from
W 2ra$§§cng§g (Hoohvald and Rackcmann.
19363 Sanyal and East. 1958] Uta-bright and Fitch. 1962:
Binaght and Bonaaerraf, 1963a; Vilaon. Bionenatock. and
Bloch. 1966). Hot: (196ha) found that egg albumin was
3 nor. offeetivo antigen than human serum albuwln. rat
gamma globulin. horlo gamma globulin, human gamma glob.
ulin. or having gamma globulin for ch; profiuctioh of
antibody in the rat an hosted by the passive cutaneous
«naphylnxln and homagwlutinatlad tochnlquos.

- Various route. of antigen injection have been
umployod 1n the rat. including intraporttoneal. sub.

cutaneous. intravenous. and intramuscular routes

(Eota. 19533}. Using ton microgram $0555 of antigen,
Einebright and Fitch (1952) found negligible differences
betwnon the offectlvcnosa of intr‘yeritonoal. intravo¢
nous. and subcutaneous routes of injection for tfie
primary production 6f aggluttnins to the particulate

a

   

flagellnr antigen of Salmone typhosa in normal and
aplonoctomizod rats. With a amaller dose of antigen,‘
0.001 utorngaml. tho intravenous or Lntrnperitonenl
routes were more effective than the subcutaneous route.
Rota (1953) reported that sensitization by the intra.
peritoneal route is more effectivo than the suboutanaoua
rout. for producing must 0011 disruption and tha appear;
anco 5f detectablo blasma histamine. Binazhi and
Bonacorraf (196kb). Mata (1963c). and Binaghl. oettgen,
and Bonncorraf (1966) report that subcutaneous injection
or antigen into the footpnd, along with Freund’o edjn-
want or Bordetclla gortussi: organisms is the most
effective proceduro for the productinn of rat anaphy.
lactic antibody. I

Tho twu most common methods of nensitlnlng rat.
for the production of anapuylaotio antibody includes
the injection of the antigen; 3) with Bordetolga
aggtugslg organisms or vaccine. or b) with campleto
tround'. ndjuvant. '

Rats immunised with bovine serum albumin and

complete Freund'a adjuvant produce high precipitin

k.)

u

J

36;.
titers. watch Ligtcn, Stone, and Frocnfi (133$) believe
to be correlated with ausccptihility to Lygtemic aha“
phylflctio death. Mata {18533) fannd that two rat pro-
duced a high titer of hang: lutinatlng antibody when
lnjoctcd witfi ton milligrams of ca; albumin with com.
plots Frcund's-cdjuvant intramusculcrly chry wcck for
two month: plus two injections of alum precipitatcd ogg
albumin intramusculcrly at a two sock interval. This
cams proceduro rosaltcd in no dctoctablo passive cutan.
sons annphyloxls antibody titer. Hunos (1953) reported
that susceptthility to fatal anachylaxis in mice was
equally cnhanccd whether complete Frocnd's cdjuvant or
Bordcselgg goggp§§ig ccEII were used with a large dosc
of onttgcn; but with c small deco of ontigcn. Frcund's
adjuvant was more effective. nincghi and Bonacorrcf
(19653) concluded that for the production of antibody
demonstrating activity by the pacsivo cutaneous can.
phylaxls test. the use of pertussis vaccine as an ad.
Juvant resulted in an earlier primary response than
did the use of tround's sdjuvcnt. By tho eleventh day,
scrum titers of antibody. as measured by the passive
cutaneous ancphylcxis tact, were essentially equal.
shothcr pertussis vaccino or trauma“: cdjuvanc had boon
Omplcycdo

Probably the most widely acccptcd tccnniquc for

inducing ancphylaxis¢ or tho production of nnaphylsotto

37

type of {:1:.-;3.?.-m’-y 3.11 t‘w rat. or mum), is £313 mminlu-
tratirm of "-:‘r:"~-€;a711_:1 {firtmvig cells or VAC-31.118 with
tfie antlfien. Rico injrctod witfi pcr£u2315 vaccine are
more susceptlhlo to histamine. serotonin, pfisaivo and
activa anaphylaxls, the gram negative bactvrixl vhcolnes.
and infection with living bacteria: althnugh in Chase
03535 the mousa 13 ”wt in a debilitated confiltlon (Klnd.
1953,.

nepause histamine and serotanln are the usual
mediators of anaphylaxia in wany species; this has led
to the 1nvfilid assumption that increased sensitivity t9
these compounds in the rat and mouse is rcspodsltle for
increased SHfiCEUtlbllity to anaptylaxts. Acoarding to
sanyal and Refit (1953) the paécntlating action of
‘EfirdfltflTln gertzsgts vaccine in the enhancement of and.
phylaxia could be due to: l) increas d antlbOdy formation.
2) increased production and rdlaasg of tha amines. or
3) increased tissuo aensltivlty to tha amines. King
(1958) has hascd the aohsillting prepertias of £3-
ggrtusglg oh two assumptionsf 1) that sensitivity to
hlstamlno. serotonin. anaphylaxls. and endotoxins is
due to a defect in steroid metabolism. and 2) tfint in.
creased susceptibility to the lethal offsets of endo-
toxins. aflath1&318, peptanca, and other agonts 1: due
to the rolanse of histamine and gorotanin. Th9 action

of‘2, nertwssis on steroid metabolism may be cnfiancemont

33

of destruction of these compounds or the ability to
lncroa o tioono rooulromonts for tho stora‘fls. E,
portnsols also olthor elicits a hypoglycemic response
by the animal, or orovonfis tho dovolonoont of compon.
notary hypoilyoomla (Pieroni and Levine, 19fl7).

Runes and Bergman (1966) and Ploroni, Broderik.
and Levine (1965) have-ouccossfully characterized the
histamine sensitizing factor of‘fip nortuooig. A: a
result, an entirely now theory has amorgod regarding
the ability of‘g, pertussis to enhance nnaphyloxts in
rodent. (Lnulor at 31.. 1965; Keller. 1955). Rumorous
invostigatora nova domanotrotod that.§, Egrtusgil does
ploy a sfigntfloont role in blocking adrenorgtc receptors.
than interfering with the action of epinephrine (Gozy
and Kate. 19623,b; norgmon and Munoz, 1965; gone: and
Borgamn. 1965; Keller. 1965). If this theory is accepted.
the onhonoomont of onaphylootlo shook by odrouoleotomy
could be oXplalnod on tho basis of loss of tho adrenal
medulla. rather than the loan of the odronal cortex.

Sanyol and float {1953) noted that too: days oftor
treatment with £9 ggxggssgg'vacclno, rots woro five
times more sensitive to histamine. and by the tenth
to twelfth day wore three timoa more sensitive to sore.
tonln. This information lod those investigator: to
support tho theory that the potentloting octlon of

pertussis vaccine is dquto the induction of increased

-I~

tlfifiUU saw itivlty to 22933 ruiusx. Tunas-(1,53) and
Mata (19555), haxfivnr. flirahrcr, an? Liixtain that
futinwin: srnaieizafisn, 11cr0;2 4 sgdnltivl?y to rna¢
phyluxts la still présfint at a tine V203 hygfrsofisitlvlty
to histnmtnc e21 serotonin fins diSZFQGTTCd. In afidition,
tho adginistrction of antifilstarines 11d anti-scratanin
¢OMP31ndQ could not fully prntect rats from anaghylactlo
éaath.

Recent work mjyports the theory that tVo most 1m~

portant actinn of H. nvrtnzwfig in sensitizing tfla rat

n W'— '—

to annpfiyiaxis is the enhancement of anapuylaotzc anti.
bofly produchian. Althoqgh tfla mechanism by ”314%
partussia organiums anhunco tho production of anaphy-
lactic antibady is still thonrized, the fact that passtvo
transfer of the indwcoi hypntaonsttivity 13 p033 hla
with serum or puriflad antibody r3193 out the possibility
that pertussis organisms ware only modifying the
physiological responsn of the animals to tfie mediators
involved in annphylaxts (nlnagfit uni Bcnacorruf, 1963a).
Munoz (1963) £901: thit two main mochanism by
which E, pfirtgjgjg_1ncrcascs nuaccptih11tty of nice to
activcly induced mnnpfiyiaxis is by stimuIating thu anti.
body response to the sensitizing ancigrn. That portucsla
vaccine enhances the firoduction of agglutinina in mic.

hAI boon demonstrated by Kind (195?). However. unlike

othar phenomena induced with partusuis vaccine. the

' ho

agglutinin response coouro only when pertussis vaccine
is administered at the some timo and by tho same route
as the antigen. noon (1963a) boliovoa that 5;. mrtugsgs
vaccino in the rat enhance. tho produofiion of on anti.
body typo different from the prooipitin. having an
affinity for tho mast cell. Sollidoy. Rowloy. and

titan (1967) state thotwg, Eggtugsgl vaocino onhanoos
tho antibody resounso in tho rat by inducing proliferation
of tho antibody forming 0011.. raehor than by affecting
the antigen or the initial antibody rosponoo by tho
immunooompotont coils olroady present.

Nata (196kb) concluded that onophyloxil in rot.
in a result of a mixed rooocion in which both immodioto
and delayed hypersensitivity are present. and that'
actively sensitised rats are fully susceptible only
when both immediate and delayed hypersensitivity or.
fully ootabliohod. Anophylootio shook in tho rat can
uauully only ho produood botwoon the tenth and trout!-
firsi days after sensitisation. Sonya! and root (1958)
found the twelfth day to bo tho most successful for
producing severe ahaphylaxis. When the ronotion to
fatal. death usually occurs several hours attor antigen
injection (Mata, 1953b).

fiyntomio anayhylaxio in tho rat is a progressive
oiroulatory oollonse with the small intestine as the

target organ (Sunyol and Host. 1958). With sovero

In

anaphylaxls. the first nymptoms soon in tha rat are
rufflnd hair. loss or reaction to painful stimuli.
falling of blood prosanro. and Iowerlng of body tompnr—
nturo. hcao are fallofibd by wankness. cyanosia.'
IOVQrO proatration. viophthalmoa, convulsions, and.
ofton death. This 3330 ganeral syndroma 18 seén in
rats following the injection of anapuylatoxin. a toxic
principle produced by incubation of certain antigen.
antibody complexes in normal rat serum (fiata, 1959).
once oonsitisatton has occurred [at tfie devel.
0pmont of systemic anayhylaxis in the rat. tfia
I0§0r1ty of shock can fie depresacd with such procedures
as splodootomy, thymoutomy. or the administration of
antihlotamines. anti-5-hydroxytryp8awinns, unfl gluoo.
cortloolds (Spanner and vast. 1952; Cody and Cudo,
1953’. Thompson (1955) reported that anaphylaxis and
histamine shock arc depressed during tha {all season}
Although the roasan for this is unknowfl. this phanom.
anon 1! probably afisociated'with mbalances in fiulfur
metabolism»(Perora and flangar. 19S5).

Active cutaneous nnnpuylaxis has alao boon preo'
duccd in the rat. by injecting bovine serum albumin
and complete Freund'a adjuvant. Far vroducing the
cutaneous type of anayhylaxis. ghocklng doses of
diluted antigen are repartodly more affectivo than

highly concentrated doses of antigan. The

#2

histOpathological changes in the sltns of int¢nso skin
reactions were similar to these seew in the Arthus
reaction of the rabbit (Lipton, fitoae, and Freund.
1955’.

Both passive cutaneous and passive systemte aha.
phylaxls have alga been suaoéssfully praducod in the
rai KRota, 1962). Reports on the value of‘fl, Eartu§sig
for use in passive anaphylaxia havo been contradictory
and confusing. ’

Attempts to prnduoo passive systemic anaphylaxis
in rats. using sarum from rats sensitized with antigen
and fly pertusaig organisms have been largely unsuccops.
tul (Mata, 1961g'Blnagh1 and nonuoorraf. 1953a). Coulson
and Stein‘(1965), houflver, successfully produced passive
systemic anaphylaxis in rat; uatfig rat antisgra produced
by inoculation of agg albumin emulsified in Freund'a
adJuvant into the footpad with an additional intra.
poritonaal injection of.3, pggtnssig organisms. Sanyal
and Vast (1958) report that passive transfer of rabbit
or rat antibody into rats that had received an injmotion
of fly Ecgguggts vaccine twelve days before resulted in
susceptibility to ansphylaxis.' flats (1962) disagrees.
stating that pretreatment of tha acrum recipient with
39 gortusagg does not influence either passiva nnnphy.
laxic. or tum anaphylactoid reaction induced by the

injection of soluble unttfiannantibady oomplcxas.

33

Passive cutaneous nnnphylaxis has bnon proiuced
in rats using rat, rabbit, and human antlkody (Ovary.
1952: Hota, 19§3a| Sonnctag and flarcns, 1953; Lovett.
Movat. and Wardlav, lgfifi), While avary (1952) reports
that a positive passive cutanoowa nnaphylnxis reaction
can be obtained for only a few hnwrs after injection of
antibody in tho gkin, Binaghi and ncnaoorraf (1953a)
raport that sensitization was maximal at sixteen hours.
and positive {cautions persisted for many days without
significant change.

Tue rat serum titers of passive cutaneows anaphy.
laxis antibody are rarely very high. although Wilson.
Biononstock. and filoch (1956) obtained titers up to ona
to one thousand using thnggfirnQfiylgg.bra11 en‘ a3
the antigen. Connall and Sfierman (1955) report that
tfio titer of hamngglutinating antibody in rat serum is
directly correlated with the ability to induce pnésivo
cutaneous nnaphylaxls. but that the titer of skin sen-
altiaing antibody bears no relation to the capacity of
a Icrum sample to induce passive cutaneous anaphylfixisu

Oettgon. flinaght, and nenacerraf (19?5) rcpurtad

hat treatment of rats with cortisone or diethylstll.
boatorol resulted in increasad serum conooncratlon of
anaphylnctic antibody.

Recent findings by Mata (195%, 1951, 1952) and

Keller (1957) hnvo stressed the importance of the

mast cell in producing anaphylaxls in tha rat. Tissue
mast cells show qualitatively similar alterations in all
types of shack. suggesting twat t;eso cells and damage
or dcétructlon of them is assoolated with histamine
liberatlen (Riley. 1953).

Histamine and serotonin. two ccupoundg tkought to
b. important in the shock syndrome, are found in high.
out cohocntratlon 1n the mast cells. and rfilfiaflfid into”
the extracellular fluid following tho action of a
ohymotrypsiuollkc enzyme upon the mast cells (Rota,
1953: Keller and Hanger, 1953; austen at 31.. 1955).
Though antihistamines protect rats against anaphylacelo
shack. they do not prevent mast cell disruptian and the
reloasa of hiatamlne. Plasxa histamine normally reaches
a maximum five minutes after injection of antigen (flota,
1957);

I Binaghl and Bcnacerraf (lfifiha) found that when
tissues from actively sensitised rats were incubatfid
with labeled spacific antigen, the ugtako of antigen
was much higher for tissue: from animals in which mast
cell damaga could be induced than for tissue: from
sensitized animals not showing mast cell damage or from
normal untreated animals. yuan mesontory from normal
rats was annsitiaed in vitro with anaphylactlo anti.
body, auhscquont Contact WitJ {we antigen predacod

extanslva mast cell damamo with extrusion of the granules.

’35

.- D

Both pasfilva extantuufi anagkylnais and acnsiuézmti n

p

of mesamtert gzva aLailar rcmalbs in l air of

‘fcci on
mast cells.

Altfiough unaphylaxis in éhu mouse saiws similar
in many respects to annphylaxis 1n the rut. Sinkinder
‘19fl31 fanla that tpa mdflt cell plays na essuntial role
ln mouse unughylnxis. Further. tissues cantnining the
most mast cells ura nnt necessarily the $133103 showing
aha greatest histOpathological arrests following ana-
phylactlo shock.

Accorfiing to Rota (19533) seasitizatinn to ana‘
phylaxia in tha rat involves the prodxction of two
dl3tinct1y diffcrent bygo» of antibody. The first type
13 the classical vraeigiiin; and tho second tyye is an
antibody 3131 an affinity for mast cells which is termed

mast cell lytlo antibody, mast cell annuitizlag antln

H
r.
‘r".
r— J
'5‘
U
'u

body. reaginio antibady, or rat anaphylaotie ar’
In this tucsifi. the term rat raagin will ha usad.

The rat praclpitating and reagiuio types of anti.
bodies have many important contrasting characteristics
(Bonacorraf flfid Rabat. 1089; n ta. 1961; meta. 19533,¢3
Blnaghl at 31.. 19533 Ovary. Bloch, anfl Banacerruf.
19543 Stanworth and Kufins. 1975). The most impartant

at these ara listed in Tabla 2.

i:-
Cd

Table 2. Frown. \, (:3 0: rah}; aatth_I.-.:z-;Ticvs.

1) ya; gfnclyitgg

circulating tyre of Lnilhody

sedimentation canstant usually 73

unlike human 3?in sensitizlng untihodion
ruadlly detectable with in vltro techniques
stable at 55 C for 30 Linuamg

passive cutaneous anaphylaxis reactions inaneod
with this antibody lnat fur anly a fnw hours
antihistamines have no affect on ahlllty to
produce {massive cutmmous anghyl 3213‘;

clear increased after multiple antigen
injections

Fround'a adjuvant most effective for

I713". OC'L 1.1011053

produce no annaroat mast cell damage and
histzzzwlzw relomae

usually a gamma-Enalobulln

53,-" 7. 3’0 :3: A": n
m

a cytoyhillc type of antibudy

sedimentation constant greater than 75
similar to human skin mmsltlsflng antibody

detectable only by in viva techniques

dcatroycd by 55 C for 30 minutes

passive cutaneous anap“ylaxin reactions induced

with this an;lhaay last for seweral in; 3

an1;1h13tanilnas aboliz;h aEJlllty to produce

pnsslvc Cutnnaous nnaphylaxia

titer docraascd attvr multiplo antigen
injectlans

ndjuvant Bordnbella pertu53;;,mogt offectlvo

for proluciiou

produoo mast cell damage and histamiao
release

probably a gammacluglobulin or a betaaz.

;,lo!;ulfi.n

1‘?

Lonacone (1932) was the first to suspect the
possibility of an antibody other than the proclpltln
as being rosponslbla for anaphylaxls in tha rat.
Kallaway (1930) dléagrecd, maintaining that tfie lntcr.
action of precipitating antibody fixed to amoatk
muscle cells and Specific anayhylactia antlficn was
rnaponllblo for annphylaxls tn the rat uterus.

While many investigators have reported that rat
antlsorum is ineffective for paaalvo sanslttaatian or
rat tissuns. otwers have successfully produced this
—roaotlon. Much of the discrepancy 10 due to the pron
domlnunt type of nntlbofiy present in the acra used for
9053170 Icnaltlnation. which in turn depends on the
typo of ndjuvant and method of antigen injection used
for production of the antiserum. Thus. rats receiving
maltlplo injections of the antigen emulalflcd in
complete Frcund's udjuvant would be expected to have
a high titer of preclpltlns. but a lower titer of
rousing. Conversely. rats receiving a atnxlo injection
of adtlgon plua‘g, pgrtusgi: vaccine or organisms would
be expected to have a hlgh titer of ranging. and a
law titer of preclpltlns. For some unknown reason,
aultlplo injections of antigcn results in tHe appear-
nno. of a factor ln scrum that is able to reverse an
already established sensitization for the productlan

of ronglnle antibody (Esta. 1953h). Also. mast coll

h8

damage and histamino release can be expected to occur
only when rats have boon sonsitiscd with a single
injection of antigen plus‘fip gcgtngsig (Hots, 1961).
flota (lOStc) uses three criteri. to estimate the
content of rat remain in a givon scrum sample!
1) histamine index a the percentage of niotcmino re.
locscd in a given uyatcm of rat mcoontory and antigen
following addition of the antiscrum to be tested.
2) moat coll indo: - tho percentogc of most coll:
damaged cftcr the addition of antigen following incu-
bation of rot moaontory with tho unknown antiserum,
and 3) passive cutaneous cncphylaxil titer . the high.
out dilution of ontisorum able to induce passive
cutaneous cnnphylcxin in rat: ocvonty~tvc hours after
antiserum inJootion. Since precipitating antibody
does not induce passive cutaneous cnophylcxis after
lcvcntyntwc hours, thin reaction is considorcd specific
for rcczinic antibody.

Binnghi and noncccrraf (19533.b) concluded that
only a minor fraction of the circulating antibody of
tho rot possesses nnophylactic proportion, but that
the vcry low serum titer of the rennin can't be taken
as an indication of tho lcvol of its synthesis if one
concidcra that this antibody probably has a high
affinity for certain rat tissues. Scrum titers of

significance may only be found after those tissues

”9

or. loturotod.

sensitisotton to rot roaginio antibody can be
60:00:04 ono olnuto after incubation of the tissue
with antloorum. Reagins can persist in skin for at
least on. month, the time of porslltonoo being related
dirootly to the amount at antibody injectod. Nonspecific
gamma globulin: hovo on inhibitory effect on passive
Ionotttsotton.1ndnood with rat roogln. but considerably
not. gamma globulin is necessary to produce this offoot
than to noodod for passive outanooua onaphylnxla in
tho guinoa pig with rabbit antibody (onunt, HaIDorn.
and Binaghi. 1959). Rat coaxial. antibody to unotoblo
in ooruuw booauno exposure at room temperature for a
(OUVhouro. or at h o for several days roanltod in n
oonploto loan of onaphylootlo activity. A: with most
typo. of antibody, curtain 1nd1v1duoi rain are oonoid.
orably not. prone to producing roogino than othora.

Roagtnio ontibodioo in rat. were shown to bo
lutormodioto in molooulor size. nod: noting between
the 70 and 190 globaltna to nor; from both singly and
multiply injected animals (Jonas and 0511710. 1967).
I! t. possible that the rufi roagin may exist as a
polymer of 7. component: (Binnghl 0% a1.. 1953).
ElootrOPhorotlooliy. tot ronain migrates with the fast
immunoglobullnl. but does not seem to be related to

tithe: the IgG or tho 13A rat immun03lobu11fls (Janos

50

and 0311719. 195?).

'fiota (1955) noted that although splonoctomiaed
rats responded with vory much diminishofl production of
other typos of antibody. their production of rousinic
untibofly was aimilar to that of nonusploneccomizod
animals. This suggested that tho aploen plays little
or no rolo in tho proéuctton of rat teasinic antibody.

The rat roagin appoars to have proncrtioa similar
to the human reagln and to too skin sensitizing anti;
body produced by tho dog (Gottson. Binaghi, and'fionacorraf,
1955; Schwartomann and Rockoy. 1955). All are prosont
1n scrum in only trace amounts as compared with the
concentration of gammauzoantibody, all unpoar to remain
attached to tissues for extended period: without loss
of sonoitizing properties, and all aro'hoat labile.
Those roaginic antibodies also produoc their reactions
in their own species only. It has not boon ponoiblc
to passivoly aonsitlzo the skin of rats with human
rcagins (Einaghi ct a1.. 195%; Ovary, filoch, and
Bonaccrrof. 1953). The rat and huwan reagina both
have andimontaticn conotants greater than ?3.

In contrast with tho-rat ranginic antibody, tho
guinea pig anuphylactic antibody exists in high con-
oontraticns in scrum, is hoot stable. and has a
sedimentation constant of 7s. The guinea pig antibody

docs not attach to tissues for as long a period of

51

time as rat or human remainic antibody (fiinaghl ct «1..
195’” .

not antiserum has boon tnoffoctivo 1n passivoly
sensitizing gulnoa pig tissooa for general annphyloxis.
passive cutaneous anapfiylaxis. the Schultzafialo reaction,
and mast'coll degranulation in Vitro (flunghray and
Mata. 1959; Binagui anfl flcuacorraf, IQCha). Antigen
1abclod with 1131 showed thnt antibody adsorbed on
guinea pig masontory could combine equally well with
antigen, whotncr the antibody came from rabbit or rat.
although tho unuphylactio rosyonno foiled fio tonult
(Humphrey and Meta. 1959). Meta (lflfiha) has reported
successful pasaivo transmission of rat rousinic

antibody to the mouse.

2:32. Scmzi‘rem u
The histarical aSpQOca of anatohiolcgy hava bean

adequately roviawed hy noyniera (195?) and Lackey (1951).
The gormfree animal may be defined as an animal reared
in tha absence of damonstrahla living bacterial, myco.
19310. protozoan. and macrOQaragitic associates accord.
1H3 to tha test procedures routinely Onoloycd. Suffla
clont studies havo not been canuuctod with pleura.
pneumonia-1tke-organiems, rickettsta. and viruses to
dacido whether they should be included in the list of
organisms known to be absent from the onvironnont of

he garmfroe animal (Hostmann and aardon, 1930). no-
cause of the many varied and cumbersome techniques
needed for isolation and identirlaazion of the many
known group: of viruses. and the possibility of the

ncrmfrao

U

preaenca of as yet anidantificd viruses,
animals are not likely to he provon virus-free for
a long period of €150.
Althou;h coma an mals, such as chickens. grow
Just as $911 undcr germfreo conditiona as under con-
ventionnI canditioua. this is not so with the rat.
Kormal growth 16 $11_ht1y rotérded in this $900165
when reared under germfree conditions (39rdon, 1959).
Animals raised under garmfroo conditians can be
considered to have much 1&33 contact with antigenic

materials than animal: rul5nd 1a a nwrmal environment.

53

although a complete lack of contaat with antigenic
materials such as {and cnnatitnonfs, dead bacteria,
flust canstltuants, and viruses cannwe ha excluded
(Thorbcckn. 1959).

Lagercrantz et a1. (1955) found tfint antigenic
material of flintary erfigin in tke gastrointestinal
tract of man an} conventional rats was capable of in»
hibiting tho pafislvo hanagglutinatlcn reaction between
tho nnttrentc intestinal material invgorwfrce rats and
two homologans antiserum to this material. In contrasfi.
tho intentlnal antigenic mntnrlnl from garmfree rats
was not canahla of inhibiting the rmactlon between
dietary antigen from conventional rats or ran and
hamologoufi antl3¢rn tram thaea aawn canvcutional rats
or flan, tfius indiflafiing that although antlgfinlc matarl.
a1 is present in the gut af aarmfrea rats, there 18
still additional antigenic material preacnt in tho
intestinal contents of cowventlonal rats or man.

Lodinnvn, Jouja, and Lana (19$?) rcnort that
newborn human infants rcociva mxtcrnnlly predicad duct.
badges against fiscfinrinfiia cal and otfinr intestinal
bacteria across the placanta. If thlg is the case
with lower animals. thoso ocsnrenn darivod gmrnfreo
animals would be expected to have highar levels of
antibody against the intaatinal bacteria than wowld

those garmfraa animal3 born to garmfreo parants.

5’:

Excluding the ocean. the intestinal tissue in
gormfroe animale weighs less than a oomparablo section
in conventional animals (Gordon. 1959: Smith. 1955).
Thie ie largely due to e decrease of lymphoid tissue
and moisture content of the intestinal tissue in germ-
free animals. In some inetancce the liver weighs lees
in gcrmfrce animale than in conventional cnimnle. but
the adrenal glands ere often heavier in gcrmfree rate
than in conventional rote. The organ: normally free
from bacteria in normal life ere similar in eiee in
germfree or conventional animals. whereas the organs
normally in contact with haeterie ere reduced in also
in gcrmfree animals. with the exception of the cocoa
which ie enlarged in germfreo rodents. although not in
germfree chickens (Gordon. 1959).

Adult gormfroe rate have e five to seven fold
increane in cecal weight over conVentionel rote
(Woetmann and Brucknoruxardoee. 1959). decal enlarge-
ment in germtroe rote start: during the eooond week of
life. end reachoe the degrce of dietoneion soon in
edule germfree rate by tho twenty-fifth any. Upon ex.
poeore of the garntroe rat to e normal mieroflere, the
cocoa shrinks to normal eiee within e few veeke (Gordon.
1959). Hoetmnnn end Bruckner-Kardoee (1959) suspect
the enlargement of the ocean to be due to the absence

of stimulation by the normal intestinal organisms

rather than duo to a nutritional imbalance. In other
epocloe. especially tho guinea pig and rabbit, it is
euepoctod that tho enlarged cocum has on adverse
effoct on reproduction duo to ovarian degeneration and
physical obstruction.

In all epoclce investigated, on undordovclcgcd
immunological dofcnee system is proscnt under gormfrce
conditions (Miyakeve. 1959a Vcetmann and Gordon. 1950).
1&0 gormfroc state it choraotoriscd by a low number of
roticuloondothelial cello. and a decrease in lymphoid
tieeze. especially in arch organs that in conventional
enimcle harbor or are in close association with an
ebundont bacterial flora. such as the intestinal tract
(Jostmann and Gordon. 1950).

In conventional animals. lymphoid tissue actively
concerned with defense against invading bacteria or
other antigens gives rise to secondary nodules. The
cells in the secondary nodule! probably do not produce
any antibody, but give rise to reactive nodules. which
are areas of basophilic bloat cells or plusnncytio
cells. The baeophlllc blast coil: or placmncytlc cello
edjnoont to the lymphoid nodules probably produco the
antibody (Thorbocke et 31.. 1957! Thorbccko, 1959:
Gordon and Wootmnnn. 1950: 3011 and Fancy. 19Sfi)o
Although rarely eoen in gcrmfroc animals. reactive

nodules have been observed in tho lymphatic tissue of

56

germfree rats (Emith. 1966).

Plasma cells are found in the spleen. moecnteric
lymph nodee. and the mucoeel lining of the intestine
of gormfree rats, but usually only in lmnll numhvrs ee
compared to conventional or exagormfree rate (Thorbccke.
1959). Conventional mice reportedly have three times
as many bloat cello and potential antibody forming cells
as germfree mice (Olson and Vostmann, 1965, 19653).

Yet otter stimulation with the antigens human gamma
globulin and Salmonella tzdhggg vaccine. germfroe mice
aunt‘s proportionally greater increase in the production
of pleemacytic cells. bleet cells, and large lymphocytes
than do conventional mice. indicating that these cells
are probably less committed in gormfree animals due to

s lock of previous antigenic stimulation (Glenn and
ertmnnn. 19553).

Although immunologic activity may be retarded in
gormfree animals as compared to conventional animals.
the production of specific antibody following antigenic
stimulation is still evident (Olson end Icetmonn. 1965).
Boson. Hokinodan. and walburg (1966) claim that growth
of the spleen and the primary antibody forming potcn.
tiel of spleen cello is the some in conventional and
germtree animals. They concluded toot orpcsure to
neturelly occurring antigens, excluding those each on

food that acrmtrce animals would have contact with.

57

is not a prerequisite for normal developcont of the
immune potential. Lerner (1963) reported that follow-
ing injection of gormfroo and conventional guinea pig
with ovalbumin. the gcrmfroe animal: produced high
levole of circulating antibody, nna showed considerable
hypertrophy of lympi'xoid tissue.

Although the serum of gcrmrrcc rats contains the
some major antigenic components as the serum of conven.
tional rote. certain conetituonts. especially those of
low mobility. are present in such small amounts tnnt
detection of them is difficult, ovon with tho moot
sensitive precipitation methods. Gormfroo rot gaoma
globuline show a relatively hixh mobility. and injection
of gormfroe rat sorum into rabbits cocoon production
of the entire spectrum of anti-gamma antibody othereiee
found in rabbit anti-conventional rat scrum (Grabar.
Couroon, and Woetmann. 1962).

In gcrmfroo animals. eopoololly tho rat. the
eerum fractions alphauzuglobulin. beta globulin. and
gamma globulin are lower than in the conventional
animal. Therefore. all three of these fractions could
be rogardod no poeeibly harboring antibacterial enti.
bodice. When tho gormfroe rat is exnoeod to e conten-
tionel flora. an antibody titer to many of the intee.
tlnol organisms oooure in about two nooks. and this

is rougnly paralleled by e riac in beta globulin.

V!
(23

The lower cantcnt of immune globulin: 1n the serum of
germfraa animals-18 compensated for witfi a higher
albumin content, resulting in a similar total serum
protein contant for garmfree and conventional animals
(Wastmann and Gordon, 1950: Asofshy, 1955: Asofsky.
Iknri, and Hylton, 1956).

Vostmann (195?) reported that hifiher levels of
serum gamma Llohulinn are present in germfraa rats
fed milk than in garmfroo rats fad other diets. Smith
(1955) has noted twat germfrea lambs and coats immune-
logtcally respond to orally udmtntstergd sterile bovine
milk.

Coatcs and G‘Donoghuo (1957) found that infant
germfrea rabbits given bovine milk in tha dint often
manifested an anaphylaetic type of allergic raactian
shortly after each feeding. The diet was sterlllned
by ultra high temperatura heat treatment for short
periods. a process whiah resulted in little 103' of
nutrient‘value. and prnbably also a angliglblo loss
of the milk's ability to function as an antigen. T51!
allergic condition seemed similar t0 tho”cot-doath*

syndrome described in human infants.

N .35.??? RI 3L3 AW D METRE-SI} 3

Ant S. If en 5‘.-

Tha antigens cmyloyed ware whole paahaurtzod
homogenized milk. bata lactuglobulin1 (HLG). and
alpha lacta.humin2 {ALA}. All the antigens were of
bovine origin. The whale milk was usea as an tntigon
without heating beyond the original pastourtsatlon
pruoess (UHWH). and after autoclavtng nt 121 C for
Humans (was) and 30 minatai (man! u 15 pound.
pressure in glass two liter Squat. Pak flasks).

Incomplete Frcund's ndjuvanch and Bordoggllg
aggtuasis vaccine, were used to onhanoa the effective.

nos: of the whole milk as an antigen.

_. L-

1. Nutritional Biochomtoali corp-o Cleveland. Ohio
2. Nutritional Bioch¢mlcnls Carp., Clevaland, 0310
3. American Storiltaor 00.. Erie. Pennsylvania

8. Dirac Laboratorias. Dotroit. Michigan

5. Hlohlgan Department of Health. Lanstng, Htohlgan

59

60

An ma

Adult convontlonol How Zonland white rabbits. and
adult conventional and adult gormfroo rats of the
Fischvr strain were usod in this study-

Tho rabbits wore allotod into 31: groups of two
ouch, groups fiI-RVI. and the rabbit. were troutod at
throo to {our day intervals in the foltouing manner:

Group Ri- goouintrnmusculur injections of 3.0 m1

Group RII- Ton intramuscular injections of 3.0 m1
E315

Group REIIu Ton intramuscular injections of 3.0 at
W333

Group nzv. Eight intramuscular injections of
15?.) ":3; “LG suspended in 3.0 m1 stun-110
physiological oalino solution
Group RV. Sovon intramuscular tnjoottona of
150 mg ALA suspended in 3.9 m1 Itortlo
physiological aaltno solution
Group RVIu Controls. no treatment
The germfroo rats were purchased as uoanltngss.
and roared in plastic isolator. until a weight of 200
to 300 gm was reached. at which ttuo they were used for
the oxporimont. The germfroo rut. were red Purina
50.3007. 3 Special diet dosignod to contain additional

hoot labile ingredients. so that after storiltaatlon

 

‘ AZ.

6. .Tho Charles River Breeding Laboratorios. Brookltno.
.xassachusettu
7. Purina Laboratorloo. St. Louis. Missouri

61

he reoclting diet will be nutritionally the some as
Purina Laboratory Chen. The principal protein used to
supply the amino acids ie soybean meal.
The germfreo rate vere ellotod into groups
£1.MIV according to the treatment receivndt
Group MI. Seven gormfree rote ted wnao es the
only source of fluid for a period of
62 days. at which time the experiment
was terminated
Group M11. Seven germfree rate given subcutoneone
injections of 1.0 ml unzo at {our to
It: day intervals. with each receiving

e total of eight to ten injectione

Group MIII- Si: gormfree eontrol rats receiving
no treatment

Group HIV. root germfree rote. each given e
einglo intranetitoneel injection of
1.0 m1 W330, and at the same time e
eingle incroperitoneal injection of
1.0 ml gerontollg Egrtuesig vaccine
The conventional vets need for the experiment
were born and raised in a conventional environment.
but were second and third generation descendante of
exogermfreo parents originally obtained from the eame
eeuree an the weanling germfree rote. Tfieee rate were

fed Purine Laboratory Chews. The conventional rate

were elleted into six groups Hv-nx.

“‘fl

8. Purina Laboratories. St. Louie. Missouri

 

Group
Group

Group

Group

Group

Group t

NV-

MVIu

1VIIe

MVIII.

MIX"-

62

Six conventional rats subjected to
the same treatment as two gormfree
rate in group MI

Sovon conventional rote subjected to
the same treatment an the gormfree
rate in group R11

Fourteen conventional control rate
receiving no treatment

Five conventional rats receiving the
same treatment as the gormfreo rate
in group NI?

rive conventional rats receiving 3
einnle intramuscular injection of
1.0 mi EH30. and a single
intraperitoneel injection of 1.0 ml

Egrdotoils gorSuggig vaccine

Five conventional rate receiving e
single intrnperitenoel injection of
1.0 m1 W%30 emulsified in 1.0 m1
incomplete Fround‘e adjuvont

In every instance. the only milk preparation

used for all etuoiee. with both conventional and germ.

free rate. won fiMBQ.

53

Gorafrec Progedure:

The general procedures so described by Allen
(1963) and Heroes (196%) were used for establishment
and maintenance of the isoletors. Plastic film
isolators. similar to those described by Trexler and,
Reynolds (1957). measuring 2% in x 2% in X 36 in vere
used. Roch isolator contained two or three plastic
cages with two to four rate per cage. Hater. diet.
bedding. one other supplies were sterilized by eutea
oleving in stainless steel drone at 121 c and 15 pounds
pressure before being transferred into the isolatoreb
An aqueous solution of 2.0% perooetio sold9 and 0.15
lseoonollo, an alkyl aryl oulfonote. woe needles the
disinfectant for sterilisation or the plastic transfer
chambers used to connect the stainless eteel supplying
drums with the germfree isolators. Drinking water
was sterilised in two liter volumes by eutoeloring '
st 121 C and 15 pounds pressure for 30 minutes.

The general proceoure used for determination
of germfreo status was tent of Wagner (1959}: Animals
were checked for bacterial contamination each time
the isolator system was supplied. which was every two

to four weeks. and again just prior to termination of

 

__ ‘ .‘Z‘.

9. Rocco Chemical 60.. Buffalo. Rev York
10. .Notionel Aniline Division of Allied Chemical Corp..
Row York. Row York

the experimnnt. Tubes of liquid t“ioglyoollato medium
325511 were inoculated with fecal samples and then
incubated at 25 C. 37 C, and 55 C for three weeks.
Glass slides were smeared with the focal sgccimono

and stained by Gram'u techniqge. Thom: slides were
then thorouehly examined for the presence of beetoria

using the oil immersion lens of a light microscope.

eohnimx as

   

 

glooding 5nd ggélecgégn‘gg'Sorum

The rabbits were bled by inoiaing the dorsomodial
artery of the our no loss than three days after the.
last previous injection of antigen. Each rabbit
received seven to ion antigen injections at intervals
of several days. From each rabbit. 25-50 ml of blood
were collected a minimum of six times. at approximately
four day intervals. ‘ho blood was allowed to clot. and
the resulting serum was clarified by low ageod centri.
fugntion. The serum we: then stored at .13 C.

All rats were bled from the venous plexus located
caudal to the eye by puncturing the medial canthus of
the orbit with a Pasteur pipette. From the original
technique describod by Riley (1960) for bleeding mice.
several rodificntiono were used to facilitate tho

bleeding of germfroo rats in isolator: and conventional

"” 4

ll. Difeo Laboratories. Detroit. Fiohigan

55

rats outside of isolazors.

Snvaral minutes hcfare #ha hloedtng procedure.
the rats Hora given n tranquilizing dose of 23 mgfkg
sodium pentobarbttal intrnpnritanoatlylzg During the
biaeding process the rat was hntd 1n QLQ left hand
with.the hona of tho rat toward tWO flour. and with
the thumb and index fififfi? of the left hand being
poattianad to pinch off the jugular rain. The rats
var. helfi in this ponittan to increase roman: pressure
in the area of the orbit. Sawplos of fozr to 51! m1
of blood could bu taken from adult rats withcuc undue
risk for the life of the animal. Gain: this techniquo,
up to saven serial b1ond aneclmcns Hera taken from
oach rat.

The rats rocaiving antigen warn bind-at least
five days after tWeiprovlous nntiaan injactlon.

Table 3 lists tHa days when additional tnjactions of
‘antigon were gi?en and wfien bland was 001:0ntnd for

ouch group.

h; A A...‘ A M

*w-vwv—

12. Halatala Jenscn~8nlsbcry Laboratories. Kansas City.
Hianourt

66

Table 3. Days for feeding or injecting antigen and
for collection of oorum in gcrmtroo and

convontionol rats.
on day 1.

First antlgon was given

 

95223 W gnjeotmn (day) Serum ggllooeiog (any)

MI oral antigen only“ 18. 23. 30. 35, #6.
continuous 53. 6h

xx: 1. h. 7. 10. 17. 21. 2%. 29. 33. 50.
25, 31 so. 62

”III OOfltrOI 1. 7. 13. 20. 31.33

HIV 1 7o 12

RV oral untigon only- 18. 23, 28, 33, #5,
continuous 57. 59

av: 1. h, 7. 10. 23. 13. 23. 28. 33. us.

NVII control 1. 5 11. 16, 23.

39. £5

MVIII 1 7. 12

MIX 1 7. 12

Fix 1 7. 12

Eggozfaogg3 Toss

At loost one serum sample from each animal was
loroonod for prooivlbins with the interfacial toot.
Too proceduro according to Compboll at al. (1953) was
followod. The antigen. omployod wore UHWfi, Hal}. UMBO,
unheated BLG. and unheated ALA. For uso 1n the tutor.
facial test the antigens UHWH. W315, and WMJO wore

adjusted to contain 0.033 mg/ml protein antigen and

{A
V

ELG and ALA were adjusted to contain 0.3033 malnl
protein antigen.

Undilutod and tenfold dilutions of rabbit anti.
torn from at least one of the last three bleeding:
were tested with tfio interfactal test. Undilutod
samples of tho lost sora preparod from the last blood.
ingo of rats in group: MI-HII! and groups HV5MVII
not. .130 tasted. For rats in group: ”IV. MViII. Eli.
and MI. samploa taken on days seven and twolvo were

tested.

Posgtgg ggtahooug no In:
For tho passive cutaneous onophylaxil (PCA)

toato, ho proooduroa described by Ovary (1953). and
Ovary and our (1953) were followod with minor mat.

ficotiono.

£95 1.3 m m 533 tour hundred to flvo hundred

‘grom albino gulnoo pigs were used for testing at least
on. serum specimen tron ovary rabbit and rat uood in
tho experiment. Ono day before the PCA test. the hair
on the onttro back of the guinea pig was clipped. and

a depllatory agent!) was applxod. A total volqmo of

__ 4_ M ”I.

 

13o Sargon. Crookes-Barnoa Co.. wayno. New Jersey

0.65 ml of undiluted antiserum was injected intro.
cutaneously in each of at: sites on the back of the
animal with a 27 gauge noodle. For challengo, 5.0 mg

Evans blue <1on1‘

and 33.3 mg of protein nntigon in a
singlo 2.0 m1 VOIUmo woro givon intravenously in tho
sophonous vein. 36 reactions were road 15 minutoa
after challenge.

At least one of the 13;: three sora collected
from each of tho rabbits in groups RI-RVI was hosted
with five undiluted antigen. (UHWM. Wfils. WEBB, ELG.
ond ALA). For the guinea pig PC‘ tests using rabbit
sort, o three hour incubation period was employed.

Tho last oorum sample takon from each of the
rat. in groups “I‘M!!! and HYBMVII. and samples taken
on day: seven and tvolvo from groups HIV and $711!.
‘HX was tooted for POL activity in guinea 9138. A11
Dora were used undiluted, and only the ontigono Uflwfl
and 3330 voro used for challenge. Incubation periods

of three hour: and 16 hours were used.

mm fig EM Conventional rota of tho Fischer strain
weighing 200 gm were also usod for tho PEA toot. The
proceduro for PflA as doocrlbod for tho guinea pig was

followed with thooo exceptions: 1) tho rat was nubjootod

 

.7 “:w‘“

15. Hathooon Scientific Inc.. Detroit. Michigan

69

to light pontobarhital anesthesia. a dose of 23 mJ/kg,
'whtlo tho intraoatanooua injections of antisera were
givon, 2) for ohallongo. o doao of 3.75 mg Ewans blue
dyo and 15.5 mg milk protein antigoo woro given intro.
vonoualy’in tho tail vein. 3) only a three hour
incubation period was used. and #) only UBVM and VMEO
voro used on challengo antigens.

flabbie aoro from each rabbit in groups at and
fill! (those rabbit: which recoivod UHWH and EH30) cor.
tostod by the PCA test in tho rot.‘ Only tho rabbit
antlnora that had already given positive reactions on
tho interracial toot and the Pan test in tho guinoa
pig: wore used. 1

Soto from terminal bleeding: of at loast two rats
in each of tho groups Mlofix wore also tootod for PCA

activity in tno rot.

RESULT3

lntorfaoga} m

The results of thointorfaoial hosts with rabbit
ontlaora aro shown in Table 3.
Table 3. Interracial tent. with rabblt anttlorn pro-

duced against antigens UHWM. W315. wuao.
me. find A”.

Anti-eta Antigens
Group Dilution UHHM VH1} U330 BLD AL‘
RI 0" o o o o o
(untic 10 o o o o o
onyx)

IRI! U! o o o o o
‘antio 10 9 O O o o
VKIS’

R!!! U! o o o o .
(anti. 10 4 O O I. u 0
am)

R11‘ 01 o o o o .
(antto 10 O O o O 0
8L0)

RV ' UN 0 Q o a o
(anti. 10 O o a ~ 0 9
Au)

‘Undtlutod

In a few instances only one of the two rahbtts
within the group roaotod poottivoly when two sorum
was diluted tenfold, although both reacted positively

yo

71

fihon the serum woo undlluted, .In those cases a posltlvo
roootlon for that group la recorded in Table 3 for
tho tenfold dilution.

It should be noted in Table 5 that WM39 rotalnod
sufficient nntigonlclty to sensitize rabbits for the
production of proclpltins. Further. thoso preolpltina
roootod with oltfior W330, W313, or unwu nntlgon in vltro-
Antluora produced by injecting WMJO had no dotoctohlo
ontlbody to ALA antigen in tho lntorfoolal toot. Ton.
fold dilutions of WMBO antisoro did not react with
8L0 ontlgon. but undllntod 9330 ontlaora did.

The rat ontlscro wore invariably nogatlvo for

.proolpltlns with tho interracial toot.

P333170 gutonooug Ananhzzaaga
m the galnog m

The results of tho PCA toot ln gulnoo 9153 using
rabbit ontiaora aro shown ln Table 5. Only those
roaotlona shoving o well dlfforontlntod deposition
at tho blue dye vlth 1 diameter or at lease 3/16 in

not. considered positive.

72

Table 5. FDA activity in guinea 9135 using rabbit
antisera.

Antlsera Antigens

Group Haws VMIS wage “LG ALA
RI 0 e o e e
‘6” t‘-

UHVM)

R11 9 O O O o
‘Bfltio

415)

R111 9 o 0 e -
‘Bfltto

Hugo)
RI? 9 e e e, 0
(anti. I

8L6)
R? e e . . e
(anti.

ALA}

Table 5 shows that sufficient activity remained
in U%15 and WM}? antigens to sensitize rabbits for
antibody production. These results coincide with the
results obtained with the Interracial test. The alpha
lactalbumin cantained in the HMBO antigen seems to b.
severely altered by the thirty minute heat treatment
as evidenced by failure of the VH3“ antigen to produce
detectable antibody to this component when tested 81th
the ALA antigen.

A. with the interfacial test. the rat antisera

were invariably negative for PCA activity in guinea pigs.

9assive Cutaneons
Lg She Rafi

Only the rabbit antisera from graup RI. the anti.

 

UHWH aera. showed any possibility of reacting ponitivoly.’
and this reaction was quite diffuse and indistinct shun
compared with the clearly interpretable Pin reactions
obtained when using the same corn in guinea pigs.

All the rat sore tested were again negative.

DISCUSSION

The result: of this stony clearly snow that
whole milk subjected to severe heat treatment remains
antigenic. Thin was demonstrated in rabbits using
whole milk that had been autoclaveo for 30 minutes
at e temperature of 121 c. Tues. reeulte are in agree.
ment with those of Lu: and Todd (196%) end Separatein
and Anderson (1962), but do not support the result.
of Retner et el. (1958b.o) end Crawford (1953). It
is difficult to compare these publications with toe
- reeultl presented here. because most of the work in.
waiving the antigenicity of heated milk is based on
eyetemic anophylaxie in guinea p13: or clinical skin
test: in hypersensitive human patientl.

The results presented here offer no eupport to
the hypothesis originated by Burth (1925) that the
heating of a milk protein createe a new antigenic
specificity which in turn will produce antibody
epeoific for the heated antigen. Hy results indicate
that anti.wn30 sore reacted at least as well with
the UHWH antigen as with the H330 antigen.

It is realized that although the two liter

volume. of milk were heated in on autoclave for

7h

75

15 or 30 minutes at 121 C and 15 pounds firessuro, the
actual temperature in the center of the flank probably
did not reach 121 C for the entire 15 or 33 minute
time period. This is expected booouoo sterility checks
showed the two liter Volumes of milk wore not sterile
until heatod for 25 a 23 minutes at this temporoturc
and presoxre. Since smaller volumes of milk could be
sterilized by autoclavina for only eight to ten
minutes at 121 c and 15 pounds pressure. it is expecta.
ed that the actual effective heat treatwont on the
milk protein antigens was considerably less than 121

C for 15 or 30 minutos.

It must also be considered that if it took longer
for the two liter volume of milk to reach and maintain
a temperature of 121 C. than logically it must also
take longer {or this volume to cool down to room
temperature after removal from the autoclave. Addi.
ticncl denaturotion of the protein antigen would then
take place during the cooling period.

It is expected that the antigenicity of at
least two of the milk proteins. beta lootoglobulin
and alpha loctalbumin vac considerahiy reduced as
indicated by the interracial test. The entinwujo
serum from only one rabbit reacted positively with
3L0. and neither of tho wx30.anttsorn reacted with

ALAe

76

Both the intnrfncial test and tha PCA test
indicate that the anti-W330 Born possessed mars
activity for tho BLG antigen than for ALA. Those
results'agree with those of Crawford and Grogun (1961).
Collins«w1111nma (1952). and Lu: and Todd (1953) in
that BLG is more heat stable than ALA. It in expeofiu
ad that the more heat stable casein would play a
significant part in the total amount of undanaturod
protein reraintng in the H330 antigen.

Failure to detect antibody In tha conventional
and germfrea rats esposod to UM3O could be explained
on the basis of on. or several pannibilltiest'l) that
tho UHSO did not retain sufficient antigenicity for
the rats to elicit the productien of antibody: 2) that
proolpltating antibody was produced but was precant
in nueh low quantitIOI that the antibody could not b.
detectea with the relatively insenstttva interfactat
and PCA techniques. whereas it a more sonaittvo
technique such as rudioimmunoolactrophoresta or tanned
coll hamagglutinntion had been employed, antibody
would have been detected: and 3) that a rnzginia type
of antibody was produced. but because of its cyto»
philio nature was not detactablo with the in vitro
or paxclvo recipient testao If the first mentioned
possibility was reaponsiblo for the absence of

dptoetable antibody in rats. it must ba.aasumod that

77

tha rat would require a larger dose of effectivo
unmigcn for antibody stimulation than did the rahbit,
since the rabbit readily produced precipitins to the
'VM3Oo

The fact that severely heated milk (wage: is
still antigenic suggests that heat denatured milk prod.
'uotn for use in the diet of milk hypersensitive ififants
in highly questionable. Besglto the probabilltf that
conlidorabio donnturation of the milk proteins may
tnko place upon heating. it is evident tfiat a alight
dogrto of antigenicity romains. This antigenicity could
be crucial in circumstances wharo the shocking dose of
‘ antigen is provided to a milk hypersontitivo human
infant potentially sngnnptibie to the 'cotodoath'
syndrOmOo

Gormfroo animals offer many aGVantagoc o'er
conventional animals for curtain types of immuno.
losical investigation. and in some instance: the germ»
fro. :nimnl is almost a naceasity for obtaining valid
ranultu. Unfortunately. the tat and mouse. which or.
rolativaly easily raarcd under cernfroo conditionn. are
poor aubjectu from the standpoint of humoral antibody
production. In contrast, the rabbit in a gcod producer
of precipitina. and the guinea pig an excellent chute.
for nnaphylaxis testing, but both of those animals I

are very difficult to rear under gormfreo conditions.

Thus. the question concerning tho intestinal absorption
of antigen. and antigenicity of milk in gurmfrco

animals ro aims largely unanswered.

78

S“ 311"? 331’

live groups of conventional rabbits were immunised

with five antigens: 1) unheated whole milk (UHHN).

2) uholc milk heated for 15 minutes at 121 C (VMIS).

3) whole milk heated for 30 minute. at 121 c (VHBO).

3} beta lnctoglobulln (8L0). and 5) alpha lactalbumin
(ALA). Each group received only one of the five antigens.

After several antigen tnjccttcnu, 111 groups had
specific serum antibodion to tn. antigen injected,
whcn tested with the interfacial and passive cutaneous
cnaphylaxls tasks. In ndditlcn. =ho UMIS and WfiBO
antigen: cross-precipitated tha ”HUN anticcra. and the
UHWN antigen also cross-precipitntcd tho VH1} and VMBO
nntlsorao

The BLO protein was less severely donntured by
heat than the ALA antigen. becauso the UMJO antinorc
reacted pcsltivcly with 8L0. but negatively with ALA
for both the interraciai and passive cutaneous annphy-
13:13 tests.

Passive sensitization of the conventional rat
with rabbit anti-VMBO sore and rabbit anti-unwu car.
van unsuccessful. Actlvc sensttlxation of the garmfrcc
or conventional rat using v.30 was also unsuccossfulo

79

80

Several methods were employed in attempting to sensitiso
the rats. These included varying the route of nntigcn

injection as well as tho use of Freund'a incomplctc

ndjuvant and Barficggza§ Ecgtgggig vaccina.

LITERATUHR CITED

‘dwt.'1.‘. 7.3.. ng. 8.0“. .nd J. veritur‘. 196”.
Glyccmic stats. and tho horse rerun and egg whit.
cncphylactio chock in rats. J. Immunol. 92l3~7o

Alton. L.B. 1963. The cffccta of orally administered
enterococci on certain plasma enzyme lovcll and
antibody titers of Ironic. gnotobtctlc. and can.
vontionnl chickona. 8.5. tnesie. ulcfiignn fitate
University». E... Lansing. '

Anderson' A.F.. and O.H. Schlcsc. 1923. Allergy to

can a milk in infants with nutritional disorders:
a preliminary report. Amoro J. Dis. Child. 25:
#51-h7b.

Anderson. A.F., 0.x. Schloss. and c. Hyera. 1925. The
intestinal absorption of antigenic protein by
ncrwal infants. Proc. Soc. Exptl. Biol. Med.
238180.182.

Anarchy. R. 1965. Immunoglcbulin formation by lymphoid
tinauca from gormfrcc mica. Federation Prcc. 2b:
502.

A.0:'ky. R0. N.S.Ikari. ”d 51.33. “fltafig 1966. Immu-
noglcbulin uynthosis in gormrreo mice. Federation
Proc. 25:537.

Austcn. K.F.. K.J. Dicch. A.R. Bakcr, and 0.6. Arnnaon.
1965. In vitro sensitization of rat peritoneal
mast cells by rat annphylactlo antibody. Fedora.
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