Salmonellae and Salmonellosis Associated with Milk and
Milk Products. A Review/
Department of Food Science and Industries and Food Research Institute
Abstract
There has been a renewed interest in the
occurrence of salmonellae in food prod-
ucts, prompted mainly by recent disclo-
sures that the organisms were recovered
from a wide variety of foods. The 1,200 or
more serotypes of these Gram-negative, fac-
ultative, asporogenous, rod-shaped bacteria
are all considered to be human pathogens.
I n man they can cause enteric fevers (i.e.,
typhoid fever and related ailments), gas-
troenteritis, and septicemias. Treatment of
salmoncllosis is often difficult, and a lim-
ited number of patients continue to shed
the organisms for extended periods even
though they appear to be recovered.
During 1966 there were approximately
17,000 reported cases of sahnonellosis in
the United States. This is an increase
from 6,693 in 1957. Cases of typhoid fever
decreased by nearly 75% during this same
period. I t is believed by some that the
number of reported salmonellosis cases
represents from 1 to 5% of the actual
cases in the United States. Reported deaths
in the United States from all types of sal-
monellosis averaged slightly less than 100
annually during the 1957-1966 period.
Although most outbreaks of human salmo-
nellosis are attributable to such animal
products as eggs and meat (mainly poul-
ry), dairy products, on occasion, have been
reported as vehicles for the spread of this
ailment.
Sahnonellae grow at temperatures of
5.5 to 45 C, at a~ values of 0.945 to 0.999,
are retarded by acids and are inactivated if
the p t t is low enough, are destroyed by
conventional pasteurization treatments, are
easiIy destroyed by chlorine and quater-
nary ammonium compounds, can survive
1 Publication of this paper was financed by a
grant from the Dairy Industry Committee, 1105
Barr Building, Washington, D.C. 20006. Members
of this committee include the American Butter
Institute, American Dry Milk Institute, Dairy and
Food Industries Supply Association, Evaporated
Milk Association, International Association of
Ice Cream Manufacturers, Milk Industry Founda-
tion, and the National Cheese Institute.
283
E. H. MARTH
University of Wisconsin, Madison
but not grow in media with 15 to 30%
sodium chloride, and may be inhibited by
sorbic acid.
Salmonellae are known to occur in raw
milk, but the frequency and level are un-
known. Consumption of contaminated raw
milk has led to outbreaks of salmonellosis.
Pasteurized milk, too, has been responsible
for disseminating these organisms which
occurred in the product through contami-
nation after heating. Occurrence of salmo-
nellae in certain dairy products has been
demonstrated in the United States and
elsewhere. This review of the literature
further discusses the circumstances under
which these organisms may occur in dairy
products or ingredients used in combina-
tion with dairy products in the prepara-
tion of various foods.
Interest in the occurrence of salmonellae in
milk and milk products has been greatest during
two periods of time. One of these occurred
some years ago, when pasteurization was not
a widespread practice and outbreaks of ty-
phoid fever were associated with consumption
of the untreated products. Another period of
great interest began in 1966, when outbreaks
of salmonellosis were associated with ingestion
of contaminated nonfat dried milk. As a con-
sequence of this renewed interest in the rela-
tionships between salmonellae and dairy prod-
ucts, it was felt that a review of the literature
should be prepared so that concerned persons
might have available a ready source of informa-
tion on this subject. The present paper is the
result of that belief.
I t is the purpose of this review to discuss:
a) the bacteria responsible for salmonellosis, b)
the disease itself, c) how frequently salmonel-
Iosis occurs in the United States, d) some char-
acteristics of salmonellae of particular im-
portance to the processor of dairy products,
and e) occurrence of salmonellae in milk and
milk products. Major emphasis in this review
will be given to characteristics of salmonellae
with which the processor should be familiar and
to the occurrence of these organisms in dairy
products. The discussion of the bacteria, the
disease, and the frequency with which salmo-
284 MARTH
nellosis occurs is not intended to be an ex-
haustive treatment of these subjects. Instead,
these sections are included to provide the reader
with sufficient background material so that the
major areas covered by this review will be most
meaningful.
Salmonellae
Typhoid fever was studied by William Budd,
in 1856, and he concluded the disease was in-
fectious, the causative agent was excreted in
the feces of patients, and contaminated milk
and w a t e r were important in its dissemination
(135). I t was not until 1880 that Eberth ob-
served the typhoid bacillus in tissues of dead
patients. F o u r years later, in 1884, Gaffky
isolated and cultivated this organism. Another
year later, Salmon and Smith isolated an orga-
nism from cases of swine fever which they con-
sidered to be the causative agent and which
they named Bacillus cholerae suis (192). I t is
now known that swine fever is caused by a
virus, and the bacillus which Salmon and Smith
isolated was probably present as a secondary
invader. Nevertheless, the bacteria in the
typhoid-paratyphoid-enteritis group were given
the generic name Salmonella, on the recom-
mendation of Lignieres, in honor of the Ameri-
can bacteriologist, D. E. Salmon, first chief of
the U.S. Bureau of Aninml Industry (231).
I n 1888 Gaertner isolated Salmonella en.teri-
tidis from a patient who died after consuming
contaminated meat, and soon afterward Dur-
ham and de Noeble described Salmonella
typhimurium which was also recovered from pa-
tients ill with gastroenteritis following inges-
tion of infected meat (135). Loe~er, in 1892,
identified the causative agent of mouse typhoid
as a member of this group, and soon many
additional related organisms were described by
other investigators (231).
Bacteria in the genus Salmonella are Gram-
negative, asporogenous, faeultative short rods
which are usually motile by means of peritri-
chous flagella, although nonmotile forms may
occur (35a). They are easily cultivated on or-
dinary media and are able to produce acid (and
usually gas) from glucose, mannitol, maltose,
and sorbitol (35a). Generally lactose, sucrose,
salicin, and adonitol are not attacked. Fer-
mentation of carbohydrates other than those
listed is variable. Salmonellae generally fail
to produce acetoin or hydrolyze urea. They
do, however, produce nitrite from nitrate.
Salmonellae average about 2 to 3 t~ in length
and about 0.6 tL in width, but may vary in
size under different environmental conditions
J . DAIRY SCIENCE ~'~OL, 52, NO. 3
(135). Young cultures on agar may form a
predominance of cocco-baeillary cells, whereas
filamentous forms are occasionally seen in cul-
tures grown in liquid media (135). Capsules
are normally not formed by salmonellae grown
at 37 C, but most species give rise to mucoid
colonies consisting of encapsulated cells, es-
pecially when grown at 20 C.
On ordinary agar media, salmonellae pro-
duce colonies, averaging 2 to 3 mm in diameter,
difficult to distinguish from those of coliform
bacteria (135). Freshly isolated strains almost
invariably produce circular, smooth, glossy col-
onies that are more translucent and have a
more delicate texture than those of Escherichia
coli (198a). Colonies formed by strains which
have frequently been subcultured on artificial
media tend to be rough, with a granular sur-
face and irregular edge (198a). This variation,
which also occurs in some other bacteria, is
associated with a loss of virulence and of the
somatic 0 antigen.
Cells of salmonellae possess antigens which
fall into three main categories. The K (from
the German word Kapsel) or envelope antigens
are thought to surround the cell and chemically
are similar to 0 antigens (21a). Generally, the
K antigens are heat-labile and tend to mask
the somatic antigens of the cells, thus making
live cells inagglutinable by 0 antisera (21a).
The Vi antigen of Salmonella typhi is an ex-
ample of the K antigen. Cells of S. typhi may
undergo a loss of the ¥ i antigen, at which
time they can be agglutinated by both 0 and
Vi antisera (198a).
The O (from the German ohne Hauch) or
somatic antigens are located in the body of the
cell (presumably near the surface), are phos-
pholipid protein polysaccharide complexes, and
are heat stable (21a). Chemically, the poly-
saccharide moiety o£ the antigen is extremely
complex and consists of a variety of sugars
including : hexosamines, heptoses, pentoses,
hexoses, and desoxyhexoses (198a). About 45
serologically distinct O antigens have been
recognized. Most species possess more than one
0 antigen; thus, many species share the same
0 antigens. Species can be divided into a
limited number of groups on the basis of their
0 antigen composition, with each group char-
acterized by possession of an 0 antigen not
found in the other groups. I n this way the
majority of salmonellae can be divided into
nine 0 antigen groups, with most of those
commonly encountered falling into the first
four of these, designated as A, B, C, and D
(198a). Cells can lose their normal 0 anti-
 S A L M O N E L L A E AND S A L M O N E L L O S I S
gens and then are not agglutinable by 0 anti-
serum.
The H (from the German Hauch) or flagel-
lar antigens are located in the flagella, are
proteinaceous in nature, and are heat-labile
(21a). Many Salmonella species may have one
or the other of two sets of flagellar antigens
and are designated as diphasic in regard to
this characteristic. These sets of antigens are
known by the terms Phase 1 and Phase 2. When
a diphasic species is grown, one of the two
phases predominates and more often than not
it is Phase I (198a). Phase 1 antigens are
more and Phase 2 antigens less specific than
are O antigens. Loss of flagella by a cell is
accompanied by loss of the H antigens (198a).
The presence of antigens in salmonellae has
led to the development of the Kanffmann-White
schenm for identification of the different sero-
types. I n this schema Salmonella serotypes are
arranged in subgroups on the basis of their
0 antigens, while the H antigens represent the
type (21a). Use of these serological proce-
dures has led to the identification of more than
1,200 serotypes, all considered pathogenic to
humans. The Kauffmann-White schema is de-
tailed in the book, The Bacteriology of En-
terobacteriaceae by Kauffmann (96).
The genus Salmonella is grouped together
with the genus Sh~gella in the tribe Salmonel-
leae. This tribe, together with the tribes Proteae,
Serratieae, Erwiniea G and Eseherichieae~ com-
prise the family Enterobacteriaceae (35a).
Salmonellosis
According to Morgan (135), there are three
main types of sahnonellosis. They are: enteric
(typhoid) fever, gastroenteritis, and a localiz-
ing type with loci in one or more organs ac-
companied by septicemia. Every Salmonella
strain is potentially able to produce any of
these three clinical types of infection. Each
of these types will be described below.
Enteric fevers (135). Typhoid fever is the
classic example among the enteric fevers. The
incubation period is seven to 14 days and onset
of the disease is insidious, usually beginning
with malaise, anorexia, and a headache. This
is usually followed by a fever which, in a
step-wise manner, rises to an average of 40 C.
The pulse rate tends to be slow in relation
to the degree of fever, and nosebleeds may oc-
cur at this stage o£ the disease.
During the first week, the patient usually is
prostrate and may have diarrhea, although
constipation is even more common. Either
condition is accompanied by abdominal tender-
285
hess and distention. A cough and bronchitis
also may be present at this time. Rose spots
frequently appear during the first or second
week. Splenomegaly is common and the tem-
perature remains elevated. I n severe cases, the
patient may become delirious and show the so-
called typhoid state for which the disease was
named (typhoid fever is derived from the Greek
and originally designated a state of irrational-
ity and coma). After the third week, the tem-
perature curve shows morning remissions, and
returns to a normal level by a gradual lyric.
Blood cultures taken during the first and sec-
ond weeks often yield the typhoid bacilli, but
less frequently when taken during the third
week. The organisms may appear in stool cul-
tures from the beginning and continue to do so
until convalescence is completed. Organisms
may also be recovered from urine (during sec-
ond and third weeks), bone marrow, and rose
spots.
Relapses occur in about 10% of the cases
and probably represent a reinvasion of the
blood stream by organisms multiplying in areas
such as lymphoid tissue, bone marrow, the
spleen, and biliary system. The mortality rate
in untreated patients is about 10%, and death
generally results from intestinal hemon'hage or
perforation. I n some instances, salmonellae
may establish themselves in the tissue of the
host to produce a carrier state after recovery.
This is most likely to occur after typhoid
fever, where about 3% of the cases are found
to excrete S. typhi in their stools for over a
year after recovery from the disease. The car-
rier state is observed less frequently with Sal-
monella paratyphi and Salmonella schottmuel-
leri than with S. typhi, and its duration is
much shorter.
Other enteric fevers usually have a shorter in-
cubation period (one to ten days) than typhoid
fever and are not as severe. Fever and malaise
are the dominant symptoms and they usually
last from one to three weeks.
Gastroenteritis (69). This form of salmo-
nellosis has an incubation period of from 3 to
72 hr, with most outbreaks occurring within
12 to 24 hr after the organisms have been
ingested. The principal symptoms of a Sal-
monella gastrointestinal infection are nausea,
vomiting, abdominal pain, and diarrhea that
usually appear suddenly. Their occurrence may
be preceded by a headache and chills. Addi-
tional symptoms often associated with the dis-
ease include watery, greenish, foul-smelling
stools; prostration; muscular weakness; faint-
ness ; a moderate fever; restlessness ; twitching;
and drowsiness.
J. DAIF.Y So~lq'oE VOL. 52, NO. 3
286 MART~

Severity and duration of the disease vary
with the amount of food (and hence salmonel-
lae) consumed, the kind of Salmonella, and the
resistance of the individual. Intensity varies
from slight discomfort and diarrhea to death
in two to six days. Usually, symptoms persist
for two to three days, followed by an uncom-
plicated recovery. I n some instances, however,
symptoms may linger for weeks or months.
Some patients (0.2 to 5%) become carriers
of the Salmonella organism which caused their
infection. The mortality rate is generally less
than 1%.
Septicemias (135). Septicemias caused by
salmonellae are characterized by a high remit-
tent fever and blood cultures which yield the
causative organism. Intestinal involvement is
usually absent in adults, but in children the
septicemia may occur as a complication of
gastroenteritis. Organisms may localize in
any tissue of the body and may produce local
abscesses in the perineM and pelvic regions,
cholecystitis, pyleonephritis, endoearditis, peri-
carditis, meningitis, arthritis, or pneumonia.
Salmonella choleraesuis is one of the most com-
mon organisms found in this type of infection.
The mortality in Salmonella septicemia ranges
from 5 to 20%.
Treatment of salmonellosis (135). Formerly,
treatment for typhoid fever and other Salmo-
nella infections was largely supportive and
consisted of maintaining the fluid balance and
nutritional state of the patient. 1Vfore recently,
sulfonamide drugs have been found beneficial
in the treatment of certain Salmonella infec-
tions, but their use to treat typhoid fever has
been disappointing. A combination of sul-
fonamides with larger than ordinary doses of
penicillin has been found to be of limited thera-
peutic value.
Streptomycin, although active against sal-
nlonellae in vitro, has not produced beneficial
results when used to treat typhoid fever. Oral
administration is accompanied by a marked
reduction in number of typhoid bacilli in stools,
but the bacteria reappear when streptomycin
is discontinued.
Chloramphenicol is effective in treatment of
typhoid fever, but patients do not become
afebrile until about the fourth day after ad-
ministration of the antibiotic is started. Pa-
tients may become carriers in spite of adequate
therapy with this antibiotic. Treatment of
other Salmonella infections with chlorampheni-
col has been even less satisfactory. I t is thought
that the intraeellular location of the typhoid
organisms accounts for the slow response of
this infection to antibiotic therapy.
Incidence of Saimonellosis
The number of reported salmonellosis and
typhoid fever cases in the United States during
1957 to 1966 is summarized in Table 1. An
inspection of the data reveals: a) the number
of typhoid fever cases declined from 1,231
in 1957 to 378 in 1966, b) salmonellosis cases
increased from 6,693 in 1957 to 16,841 in 1966,
c) salmonellosis cases was nearly constant (ap-
proximately 17,000 per year) since 1964, d)
the reported incidence in 1966 of seven diseases
was greater and that of 22 diseases lesser than
the incidence of salmonellosis plus typhoid
fever.
TaMe 2 records the number of deaths in the
United States attributable to salmonellosis and
typhoid fever during 1956 to 1965. F r o m the
data it is evident that: a) the number of deaths
attributable to typhoid fever decreased gradual-
ly from 54 in 1956 to six in 1965, b) a slight
increase in number of deaths from sahnonellosis
was noted during 1963 to 1965, when compared
to data from 1956 to 1958, c) more deaths than
from satmonellosis plus typhoid fever were

TABLE 1. Reported cases of salmonellosis and typhoid fever in the United States during 1957 to 1966
(217).',b,°
Disease 1966 1965 1964 1963 1962 1961 1 9 6 0 1 9 5 9 1 9 5 8 1957
Salmonellosis (exclud- 16,841 17,161 17,144 15,390 9,680 8,542 6,929 6,606 6,363 6,693
ing typhoid fever)
Typhoid fever 378 454 501 566 608 814 816 859 1,043 1,231
Total 17,219 17,615 17,645 16,956 10,288 9,356 7,745 7,465 7,406 7,924
a Data from Alaska included since 1959 and from Hawaii since 1960.
b Diseases with a higher reported incidence (1966) than that of salmonellosls plus typhoid fever: in-
fectious hepatitis, measles, rubella, streptococcal sore throat and scarlet fever, tuberculosis, syphilis,
and gonorrhea.
c Diseases with a lower reported incidence (1966) than that of salmonellosis plus typhoid fever:
ameblasis, anthrax, aseptic meningitis, botulism, brucellosis, diphtheria, encephalitis, leprosy, leptospi-
rosls, malaria, meningoeoeeal infections, whooping cough, poliomyelitis, psittacosis, rabies, rheumatic fe-
ver, shigellosis, tetanus, trichinosis, tularemia, typhus fever, and venereal diseases other than syphilis
and gonorrhea.
J. DAIRY SCIENCE VOL. 52, NO. 8
 SALIWONELLAB A N D SAL~ONELLOSIS 287

TABLE 9.. Reported deaths from salmonellosis and typhoid fever in the United States during ]956 to
1965 (217). ~'b
Disease 1965 1964 1963 1962 1961 1960 1959 1958 1957 1956
Salmonellosis (exclud- 87 67 72 62 64 82 72 65 60 62
ing typhoid fever)
Typhoid fever 6 14 21 ]5 17 21 22 23 34 54
Totat 93 81 93 77 81 103 94 88 94 116
~Diseases with more deaths (1965) than from salmonellosis plus typhoid fever: encephalitis, hepa-
titis, measles, meningococeal infections, rheumatic fever, shigellosis, tetanus, tuberculosis, and syphilis.
b Diseases with fewer deaths (1965) than from salmonellosis plus typhoid fever: amebiasis, anthrax,
botulism, brucellosis, diphtheria, leprosy, leptospirosis, malaria, whooping cough, plague, poliomyelitis,
psittacosis, rabies, streptococcal sore throat and scarlet fever, trichinosis, tularemia, typhus fever, gon-
orrhea, and venereal diseases other than syphilis and gonorrhea.

associated with nine notifiable diseases in 1965,
and d) fewer deaths than from salmonellosis
plus typhoid fever were attributable to 19 no-
tifiable diseases in 1965.
The frequency of salmonellosis was greatest
during the last one-half of I966 according to
data in Table 3. The incidence ranged from
1,369 to 1,818 cases per month during July
through December, and from 951 to 1,462 cases
per month during J a n u a r y through June. A n
appreciable increase in number of typhoid
fever cases was observed during the warmer
months of July, August, and September.
Table 4 provides data on the distribution of
salmonellosis and typhoid fever cases among
the different United States geographic areas.
I n general, highest numbers were associated
with areas which had highest concentrations
of population (e.g., Middle Atlantic, South At-
lantic, and Pacific).
Some observers believe that the reported in-
cidence of salmonellosis represents from 1 to
5% of the actual number of cases (67). I f
this is true, then the actual incidence might
range from 340,000 to 1,700,000 per year.
Should the former of these figures be true, then
the incidence of salmonellosis would be greater
than that of all other notifiable illnesses except
streptococcal sore throat and scarlet fever. I f
the latter figure is more nearly correct, then
the incidence of salmonellosis is much greater
than that of any other notifiable disease in the
United States. It should be mentioned here
that although some outbreaks of salmonellosis
have been attributed to dairy products, the
largest number continue to be associated with
eggs and egg products, and meat (mainly poul-
try) and meat products (14a, 19a).
Characteristics of Salmonellae Important to the
Dairy Foods Processor
The processing of dairy products utilizes a
variety of heat treatments, low temperatures,
drying, and fermentation. Frequently, sub-
stances such as salt, fruits, acids, eggs, or
other products of animal origin may be added
to dairy ingredients in the manufacture of
certain products. These additives may con-
tribute to the Salmonella problem by adding
organisms or by creating conditions either fa-
vorable or unfavorable for the growth and sur-
vival of these bacteria. Sanit~zers and cleaning
compounds, hopefully, will aid in the control
of salmonellae by inactivating the cells. The
relationships between salmonellae and the
processing treatments listed above, as well as
others, will be described. Data on destruction
of salmonellae by heat or other agents cited
in this review are those reported by the in-
vestigators who did the work. I t must be
recognized at the outset that many of these
investigators failed to give adequate recogni-
tion to the logarithmic nature of death of orga-
nisms. Furthermore, some of these workers
did not have the benefit of present-day experi-
ence in recovery of salmonellae. Nevertheless,

TABLE 3. Reported eases of salmone]losis and typhoid fever in the United States during 1966 by month
(217).
Disease Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.
Salmonellosis 1,144 951 1,013 1,115 1,462 ],369 1,361 ],800 1,723 1,613 1,446 1,818
(excluding
typhoid
fever)
Typhoid fever 19 19 23 28 27 30 53 46 46 37 23 23
Total ],163 970 1,036 1,143 1,489 1,399 1,414 1,846 1,769 1,650 1,469 1,841

J. DAIRY SOIEI~CE VOL. 52, NO. 3

288 MARTH

TABLE 4. Reported cases of salmonellosis a~d
typhoid fever in the United States during 1966 by
geographic region (217).
Ty-
Salmo- phoid
Area nellosis fever Total
New England 1,488 13 1,501
Middle Atlantic 3,362 62 3,424
East North Central 2,548 47 2,595
West North Central 836 37 873
South Atlantic 3,345 65 3,410
East South Central 574 43 617
West South Central 1,049 38 1,087
Mountain 872 18 890
Pacific 2,767 55 2,822
the data can provide valuable guidance on the
behavior of sahnonellae under a variety of
conditions.
Temperatures for growth. Although under
laboratory conditions most salnmnellae grow
best at 37 C, some of these bacteria can grow
at temperatures of 5.5 to 45 C. Matches and
Liston (117) determined the minimum growth
temperatures of ten serotypes by means of a
temperature gradient block. Salmonella heidel-
berg and S. typhimurium were found able to
grow in a liquid medium at 6.6 C but not at
6 C. The p H of the substrate influenced the
minimum temperature at which growth was
observed. Lowest growth temperatures were
associated with p H values of 7.0 to 8.0. Length
of incubation is another factor which must be
considered when the organisms are held at low
temperatures. These same investigators found
that the minimum temperature for growth of
S. heidelberg was between 6.0 and 7.0 C
when observations were made after seven days
and that it dropped to between 4.0 and 5.7 C
after 15 days. Comparable results obtained
for S. typhimurium and Salmonella derby at
seven days were 8.5 to 9.0 C and 8.2 to 9.0 C
and at 15 days, 7.8 to 8.2 C and 6.6 to 7.0 C,
respectively. Initiation of growth by salmo-
nellae at low temperatures can be important
when foods are stored for long periods at tem-
peratures of 4 C or above. Deotto (54) ob-
served that cells of S. schottmuelleri, after
exposure to a temperature of --2 to --3 C for
20 to 40 min consumed 200 to 500% more
oxygen in their respiratory process than did
similar cells held constantly at 38 C.
Growth of S. enteritidis in a variety of
foods at 22 and 37 C was studied by Segalove
and Dack (180). They inoculated cans of as-
paragus, spinach, string beans, tomato juice,
peaches, shrimp, salmon, corn, and peas. Du-
plicate cans of each food were incubated at
22 and 37 C. Growth was observed at 22 C
J. DAIRY SCIENCE ~r0~. 52, NO. 3
in all foods except peaches and at 37 C in
all but peaches and asparagus. I n nearly all
instances, growth was greater at 22 than at
37 C. Recently Subramanian and l~arth
(202) compared the growth of S. typhimurium
in skimmilk at 22 and 37 C. They observed
numbers approaching 1 X 10° per milliliter
after 12 hr at 37, with little additional growth
during tile next 4 hr at the same temperature.
I n contrast to this, at 22 C numbers slightly
in excess of 1.0 X 10~ per milliliter were at-
tained after 16 hr of incubation. Undoubtedly,
higher numbers would have been obtained at
22 C if the incubation period had been ex-
tended beyond 16 hr.
Growth of S. typhi at 45 C has been reported
by severaI investigators. Spencer and Melroy
(193) noted that this organism grew well for
36 transfers at 45 C and for at least 148 trans-
fers with alternating temperatures of 45 and
37 C. According to Ware (229), growth of
S. typhi at high temperatures was enhanced
by adding certain amino acids to the culture
medium. A simple glucose-salts medium sup-
ported the growth of S. typhi at 37 but not at
40 C. Growth occurred at 40 C when the medi-
u m was fortified with L-arginine, L-glutamic
acid, thymine, or L-lysine. Addition of L-glu-
tamic acid plus L-arginine and thiamine or L-
lysine permitted cell production at 43 C in
24 hr. Elimination of L-arginine was accom-
panied by cell multiplication in 48 but not 24
hr at 43 C.
Moisture requirements for growth. The mois-
ture requirements for growth of salmonellae
can best be expressed in terms of water activ-
ity (a~). Christian and Scott (46) studied
the a~ requirements of 16 Salmonella sero-
types at 30 C. Growth of 15 motile strains
occurred in liquid media at aw values between
0.945 and 0.999. I n foods, the lower limit for
growth was slightly less than in culture media.
Anaerobic growth was only slightly less than
aerobic growth at each a~ value. The single
nonnmtile strain grew more slowly over a
smaller range of a~ values.
The a~ requirement for growth of Salmo-
nella oranienburg in liquid media was investi-
gated by Christian (44). He adjusted the a~
of 0.25-strength brain heart infusion broth,
nutrient broth, and a casamino acids-yeast
extraet-casitone broth with a salts mixture
(NaC1, KC1, and Na_.SO,) or sucrose. I n all
instances growth was observed at an a~ value
of 0.95 but not at 0.94. Substitution of a
glucose-inorganic salts broth for the media
listed was accmnpanied by multiplication at an
a , value of 0.97 but not at 0.96, regardless of
 SALMONELLAE AND SALMONELLOSIS
the substance used to control the water activ-
ity. Christian (44) further noted that the mini-
mum a~ for growth in the glucose-inorganic
salts medium could be reduced to 0.96 by addi-
tion of five amino acids including methionine,
histidine, proline, serine, and glutamic acid.
Addition of eight vitamins (thiamin, ribo-
flavin, biotin, folic acid, pyridoxine, calcium
pantothenate, nicotinic acid, p-aminobenzoie
acid) plus the five amino acids was accompanied
by a further reduction in the minimum a~
permitting growth at 0.95. Addition of the
vitamins without the amino acids had no effect
in reducing the am required for growth.
I n other experiments Christian (45) used
sucrose, glucose, glycerol, NaC1, and KC1 to
control the a~ in the glucose-inorganic salts
medium and tlmn studied the behavior of S.
oranienburg in these media. Use of glycerol to
adjust the a~ of the medium permitted growth
of the organism at 0.96 in comparison to the
0.97 required when other compounds were
added. Respiration of cells was inhibited less
by glycerol than by the salts or sugars used
to control the a~. Control of a~ by addition
of glucose or sucrose was accompanied by ac-
cumulation of potassium (but not sodinm) in
S. oranienburg cells. Accumulation of potassium
was greatest at an a~, value of 0.975. When
NaC1 served to adjust the a ~ accumulation of
potassium was small and none was concen-
trated when glycerol replaced NaCI.
The influence of amino acids on a~ require-
ments of S. oranienburg was examined further
by Christian and Waltho (47). They observed
that a reduction in the a , value by adding NaC1
or sucrose induced a lag and then decreased the
rate of glucose oxidation by the organism. A t
a relatively low value (0.970 a~) addition of
amino acids such as proline, aspartic acid, as-
pargine, glutamic acid, glutamine, and eysteine
caused an appreciable synergistic increase in
respiration rate. Proline was the most stimu-
latory of the amino acids tested and at an a~
value of 0.960 only this amino acid and its
analogue azetidine-2-carboxylic acid gave ap-
preciable stimulation. Proline was also stimu-
latory when glucose was replaced by pyruvate
or succinate. These authors believe that proline
is stimulatory by increasing the amino acid
pool of the organism and, hence, decreasing
internal water activity.
Effects of acids on gro~vth and survival. Most
investigations on the relationship between sal-
monellae and acids have dealt with destruction
of the organisms. Only relatively few experi-
ments have considered the effect of different
289
acid concentrations on the growth of these orga-
nisms in a variety of substrates.
Stearn and S t e a m (194) conducted tests to
determine the effect of p H on colonial char-
acteristics, morphology, motility, and staining
behavior of S. enteritidis. When grown on nu-
trient agar adjusted to p H values in the range
of 5.16 to 6.0, colonies appeared opaque, dis-
crete, granular, iridescent, and had raised cen-
ters which flattened and appeared lysed and
in which were formed secondary colonies with
toothed margins. A t p H values of 6.1 to 6.3,
colonies appeared circular, glistening, white,
moist, smooth, and entire. Normal translucent
colonies developed at pI-I values in the range
of 6.3 to 7.6, whereas at p H values between
7.8 and 8.4 colonies appeared very thin, spread-
ing, and translucent. Changes in the p i t of a
brotll medium were accompanied by morpholog-
ical changes in the cells of S. enteritidis. Dur-
ing the first 24-hr incubation period at p H
5.15 to 5.6, chains and clumps of cells were
evident and individual cells varied greatly in
size and shape, ranging from coccoid forms
to long, curved rods. A t p H values of 5.6 to
7.0, cells were generally short and plump and
exhibited nmrked bipolar staining. An in-
crease in p H to 8.4 was accompanied by a
greater tendency toward evenly stained, slender
rods, which appeared Gram-variable. This
organism lost its motility when cultivated at
p H values below 6.0, but regained it after sub-
culturing in a neutral medium. A t acid p H
values, cells stained a deep pink with safranin.
The color was lighter at a neutral p H value
and cells became Gram-variable when the p i t
entered the alkaline range.
The effects of lactic, acetic, and hydrochloric
acids on Salmonella aertryclce were studied by
Levine and Fellers (110). When hydrochloric
acid was added to nutrient broth, growth was
not inhibited until a pI-I value of 4.0 was at-
tained. Inhibition with lactic acid also oc-
curred at pill 4.0, whereas with acetic acid it
was observed at p i t 4.9. The organism was
destroyed during a 48-hr incubation period
when the medium was adjusted to p H 3.1 with
hydrochloric acid, p H 4.0 with lactic acid, and
p i t 4.5 with acetic acid. Subramanian and
Marth (202) determined the effect of citric,
lactic, and hydrochloric acids, when added to
milk in increments over a 16-hr period, on
the growth of S. typhimurium. During incu-
bation at 37 C, a slight reduction in growth be-
came evident after 8 hr, regardless of acid
added, when a p i t value in the range of 5.05
to 5.35 was attained in the milk through
gradual addition of acid. Additional incuba-
J . DAIR'g SCIENCE WC~OL. 52, NO. 3
290 ~A~T~
tion was accompanied by further inhibition,
citric acid being most inhibitory, followed in
order by lactic and hydrochloric acids. In-
hibitory effects of all acids were greater at 22
than 37 C, with citric being most active, fol-
lowed in order by lactic and hydrochloric acids.
I n addition to the work on destruction of
sahnonellae, cited above, additional studies with
lactic acid were conducted by Schillinglaw and
Levine (175). They suspended centrifuged cells
of S. typhi in a 0.02 N solution of lactic acid
at 30 C. Less than 1 hr was required to de-
stroy 99.9% of the cells. Violle (225) also
studied the effect of lactic acid on S. typhi
and noted that the cells were killed in less
than 1 hr by addition of 1% lactic acid to the
cultm'e and in less than 24 hr by adding
0.5% of the acid. The combined effect of lactic
acid and NaC1 on Salmonella breslau was de-
termined by Vizir (226). According to this
investigator, five to ten days of exposure to
0.90% lactic acid was required to destroy the
organism. Addition of up to 15% NaCI did
not enhance destruction of the organism by
the acid.
Limited trials on the destruction of S. typhi
by citric acid were conducted by Schillinglaw
and Levine (175). Cells of the organism were
centrifuged from their growth medium and
resuspended in a 0.02 ~- solution of citric acid.
Ten hours of exposure were required to kill
99.9% of the added S. typhi cells; whereas, 32
hr were needed before a similar Escherichia coli
population was destroyed.
Since acetic acid is commonly used in the
preparation of mayonnaise and salad dressings,
and since these products can become contam-
inated with salmonellae from eggs, numerous
experiments have been conducted on the sur-
vival of these bacteria in acetic acid at various
concentrations. Wethington and Fabian (233)
added 1 nil of 24-hr cultures of various salmo-
nellae to salad dressing and mayonnaise and
held the inoculated products at room tempera-
ture and 37 C. A t both temperatures, the sur-
vival in mayonnaise (0.48% acid, p H 3.80)
was 12, 12, 6, 6, l , and 6 hr, respectively, for
S. schottmuelleri, S. typhimurium, S. paratyphi,
S. enteritidis~ S. choleraesius, and Salmonella
pullorum. I n salad dressing (1.10% acid, p H
3.20) survival was 6, 1, 6, 1, :l, and 1 hr, re-
spectively, for the organisms in the sequence
listed above.
When mayonnaise was made to contain 0.15%
acetic acid ( p i t 5.0), survival was 144, 144,
156, 156, 156, and 132 hr, respectively, for the
organisms listed above. Tests using salad dress-
J. DAIRY SOIE~CE VOL. 5~, NO. 8
lugs with 0.4% acetic acid ( p i t 4.4) yielded
survival periods of 144, 120, 24, 96, 96, and
96 hr, respectively, for the salmonellae in the
above sequence. Comparable observations to
those just summarized were also made by Kint-
ner and Mangel (100), who studied survival of
S. typhimurium, S. enteritidis, S. pulloru.m~
Salmonella gal,linarum~ and S. choleraeslus in
salad dressing.
Jandl (94) found a freshly isolated culture
of S. typhi to be more acid-resistant than old
laboratory cultures. I n the presence of milk
higher concentrations of hydrochloric acid
than occur in the stomach were required to
kill the organism. This was not true when
broth or water served as the substrate. This
author concluded that viable salmonellae in-
gested with milk will pass through the stomach
and i n t o the intestines.
Since carbon dioxide forms carbonic acid
when in solution, its effect on salmonellae will
be considered at this time. Schillinglaw and
Levine (175) exposed S. typhi cells to carbon
dioxide (42 psi) and observed that 90% of the
bacteria were destroyed in 6 hr at 30 C. F o r
comparative purposes, 28 hr were required to
achieve a similar reduction in number of E.
coli cells. A pigmented, nonpathogenic strain
of S. typhi was isolated by Sokolov (190).
He exposed cells of this organism to an at-
mosphere of Co.~ and found that they lost
their ability to form pigment and also became
pathogenic, with virulence which approached
that of conventional S. typhi strains.
The effect of high p H values on survival of
S. typhi and Salmonella montivideo was stud-
ied by Riehl et al. (158). They reported that
at a p H of 11.0 to 11.5 and a temperature of
15 C most cells were destroyed in 4 hr. Addi-
tional tests indicated prolonged periods of ex-
posure to alkaline water, regardless of other
components, killed many of the bacteria.
Thermal destruction of salmonellae. Read
et al. (154) isolated Salmonella anatum~ Sal-
mon~lla bi~tza, Salmonella cuban a~ Salmonella
meleagridis, Salmonella new-brunswick, and
Salmonella tennessee from dry milk. They then
studied these organisms and Salmonella senf-
tenberg 775W for their heat resistance, to de-
termine if they vcould survive pasteurization
of milk as recommended by the 1965 U.S.
Public Health Service Pasteurized Milk Or-
dinance. Thermal inactivation tests were made
on washed cells of test organisms which were
resuspended in sterile whole milk. Excluding
S. senftenberg, D values ranged from 3.6 to
5.7 sec at 62.8 C, from 1.1 to 1.8 sec at 65.6 C,
and from 0.28 to 0.52 sec at 68.3 C. Similar
 SALMONELLAE AND SALIVION]~LLOSIS
values for S. senftenberg were 34.0, 10.0, 1.2,
and 0.6 sec, respectively, for exposures of 05.6,
68.3, 71.7, and 74.0 C. Results of these tests
suggest that the present recommended pas-
teurization processes are adequate to inactivate
all seven strains of salmonellae studied, pro-
vided the initial concentration does not ex-
ceed a calculated 3 X 101~ sahnonellae per mil-
liliter of milk.
Tests on the destruction of S. typhi in milk
were conducted recently by Evans and Litsky
(62). They inoculated milk to provide an in-
itial concentration of 10~ cells per milliliter and
then processed it at varying temperatures, using
a commercial plate-type pasteurizer which had
a heating time of 14.5 see above 37.8 C, to
produce the desired temperature, a calculated
holding time of 0.6 sec, and a cooling time of
5.0 see from process temperature to 37.8 C.
Data obtained indicate that strains of S. typhi
studied showed a thermal extinction tempera-
ture of 73.9 C. These results suggest that milk
can be freed from salmonellae by the use of
rapid heating and cooling rates, with no in-
tended holding time at process temperatures
readily attainable in present continuous flow
pasteurizers.
The z values (degrees F required for passage
of a decimal reduction time curve through one
log cycle) for rough and smooth variants of
S. senftenberg 775 W in various media were
determined by Thomas et al. (207). They
found the rough variant had z values of 11.494,
10.989, 10.638, and 10.204 in 0.5% NaC1, skim-
milk, beef bouillon, and green pea soup, re-
spectively. The smooth variant yielded values
of 10.753, 10.417, and 10.753 in 0.5% NaC1,
skimmilk, and green pea soup, respectively.
Elliot and Heiniger (61) determined the D
values of S. senftenberg 775 W and S. typhi-
murium at relatively low temperatures by means
of a temperature-gradient incubator. They re-
corded a D value of 24 for S. sen~tenberg at
55 C and of 8.5 for S. typhimurium at the same
temperature.
Numerous studies have been conducted on
the thermal resistance of salmonellae in various
poultry products. No attempt will be made to
review the literature in this area. However,
a few recent and pertinent references will be
discussed below. Bayne et al. (25) determined
the heat resistance of S. typhimurium and S.
senftenberg 775 W i n ground chicken muscle
heated to four temperatures in the range of 55
to 75 C. Multiple 1-g samples of meat contain-
ing 3 X l0 s cells of S. typhi~nurium, after
exposure for 5 rain at 60 C, contained no viable
cells. The more heat-resistant S. senftenberg
291
775 W required an exposure of 10 to 15 min
~t 65 C to kill an equal number of cells.
Destruction of salmonellae on eggshells dur-
ing washing was investigated by Bierer and
Barnett (30). They found that S. pullorum, S.
galli~arum~ and S. typhimurium on eggshells
were killed by washing the eggs at 65.6 C for
3 rain. This procedure, however, resulted in
slight Mbumen coagulation on the inner sur-
face of the shell. Coagulation did not occur
when eggs were washed at 65.6 C for 1 rain,
although salmonellae were recovered from one
egg out of 600 that were washed. The salmo-
nellae used in this study were destroyed in
wash water at 53.3 C. Data just cited may be
applicable to destruction of salmonellae on
equipment surfaces by use of wash water.
The times required to free hard-boiled eggs
from salmonellae was calculated by Licciardello
et al. (112). When raw eggs were placed di-
reetly into boiling water, they indicated that
5.6, 8.4, 8.7, and 9.4 nfin of exposure are re-
quired to free small-, medium-, large-, and
jumbo-sized eggs, respectively, from S. seften-
berg. Yalues for S. typhimurium were found
to be 4.5, 7.2, 7.3, and 7.8 rain, respectively,
for egg sizes as listed above. Exposure periods
of 14.1, 16.0, and 20.6 rain are required to
destroy S. senfte~berg in medium, large, and
jumbo eggs, respectively, when they are placed
into water at 20 C which is then brought to a
boil and simmered. Yalues of S. typhimurium
under the same conditions are 12.6, 14.8, and
18.8 rain, respectively.
Destruction of sahnonellae in dry products
requires a higher treatment temperature, a
longer exposure time, or both. Data on destruc-
tion of these organisms in dry dairy products
seem to be missing, but such information is
available f o r other materials. Rasnmssen et al.
(153) utilized modified thermal death time
tubes to determine the heat resistance of sal-
monellae in naturally contaminated meat meals
containing 8 to 10% moisture. A temperature
of 82.2 C for 7 rain was sufficient to consistent-
ly destroy all salmonellae in these meals. A
third meal containing 13% fat required an ex-
posure of 7 rain at 90.6 C to consistently free
it from viable salmonellae.
I n dried egg white the resistance of salmonel-
lae to thermal destruction is 600 to 700 times
higher than in liquid egg white, according to
data cited by Prost and Riemann (150). They
also reported that seven days of heating at
49 to 50 C was required to eliminate salmonel-
lae from naturally contaminated dry egg.
Stokes et al. (199) were able to recover
viable cells of S. paratyphi B from sludge
J. DAIRY SCIENCm. "~r0L. 52, NO. 3
292 MARTH
(sewage) after 27 but not after 41 days of
drying. I n other tests S. typhimurium was re-
covered from sludge after 180 days of drying.
Effect of chlori~e compounds. Stuart et al.
(200) studied the effectiveness of commercial
chlorine-type germicides on S. pullorum. They
noted that a higher concentration of chlorine
was needed to disinfect a clean inanimate sur-
face bearing a given number of S. pullorum
cells than was required to kill the same num-
ber of cells suspended in a liquid. Commercial
sodium and calcium hypochlorites, ehloramine
T, and 1,3-dichloro-5,5-dimethylhydantoin were
equally effective in the disinfection of previous-
ly cleaned surfaces. Alkaline chlorine-type ger-
micides were less effective and, if large num-
bers of bacteria were present, failed to destroy
all of the organisms. Sodium hypoehlorite so-
lutions with an acid reaction were found to be
more effective in destroying S. pullorum than
solutions of similar strength having an alka-
line p H value. Smith (188) observed some
strains of S. typhi to be more resistant to
chlorine and chloramine compounds than the
coli-aerogenes bacteria.
According to experimental evidence presented
by F r i b e r g and HammarstrSm (70), at 6 C
and p i t 7.2, a 1,000-fold reduction in number
of S. typhi cells required 0.025 to 0.05 mg
free available chlorine per liter. S. typhimu-
rium was somewhat more resistant and, hence,
0.10 to 0.15 mg available chlorine per liter was
needed for its destruction. Bacteriophages ac-
tive against S. typhimurium were more sensi-
tive to the action of chlorine than were the
host cells. Kabler et al. (95) found that water
containing 0.22 to 0.23 p p m chlorine required
0.5 to 1.5 hr at 25-26 C and 2 to 15 hr at 1.5
to 3 C to kill cells of S. typhi. These investi-
gators also observed that freshly isolated strains
of S. typhi required approximately 2 hr of
exposure to 0.22 to 0.23 ppm chlorine for de-
struction, but the same cultures, after a year
on laboratory media, required only 0.9 hr of
exposure for destruction.
The effect of p i t and temperature on the
destruction of S. typhi by chlorine was deter-
mined by Butterfield et al. (39). According to
their data, at p t I 7.0 and 2.5 C, 100% destruc-
tion was accomplished after 10 rain by 0.02
ppm, after 3 min by 0.03 to 0.06 ppm, and
after 1 rain by 0.08 ppm. An increase in p H
to 9.8 at the same temperature was accom-
panied by 100% destruction after 60 rain by
0.15 ppm, after 20 rain by 0.40 ppm, after
10 rain by 0.74 ppn b and after 5 rain by 1.0
ppm chlorine. The concentration of chlorine
required for 100% destruction at 20-25 C was
J . DAIRY SOIEI~CE "~rOL. 52, NO. 3
somewhat less at each time period than noted
above.
Effect of quaternary ammonium compounds.
The phenol coefficients of several quaternary
ammonium compounds when acting against S.
typhi were determined by Lane (103) and
Croxall and Melamed (52). They reported phe-
nol coefficients of 335, 75, 310, and 345 for N,
N-his (trimethylphenylpentcnyl)-N, N-dimeth-
ylammonimn chloride, (4-hydroxy-2-butynl)-di-
methyl (oetylbenzyl) ammonium chloride, (4-
hydroxy-2-butynl) (dodecyl-methylbenzyl) am-
monium chloride, and (4-hydroxy-2-butynl) all-
methyl (pentadecylbenzyl) ammonium chloride,
respectively.
Goetehius and Grinsfelder (76) exposed S.
typhi for 10 rain to various concentrations of
alkyl tolyl methyl trimethyl ammonium chloride.
Their results indicated 50, 80, 90, 95, 98, and
99.5% destruction by concentrations of 6.25 ×
l f f ~, 7 . 6 × 1 0 ~, 8 . 4 × 1 0 -~, 9 . 2 × 1 0 4 , 1 0 . 2 ×
10 -5, ;i0.8 × 10-~, and 11.5 × 10-~%, respective-
ly. Stedman et al. (196, 197), working with
S. schottmuelleri, found that a 1:2,000 concen-
tration of di-isobutyl phenoxy ethoxy ethyl
dimethyl benzyl ammonium chloride destxoyed
99.99% of the organisms in 10 rain and that
a 1:1,000 concentration was necessary to do
the same job when organic matter (serum) was
present. I t was further observed that concen-
trations of 1:500, 1:100, and less than 1:25
were required to destroy the organism on the
surfaces of stainless steel, asphalt tile, and li-
noleum, respectively. Similar tests on a mix-
ture of alkyl dimethyl 3,4-dichloro benzyl am-
monium chloride and alkenyl dimethyl ethyl
ammonium bromide revealed that slightly high-
er concentrations were needed to accomplish the
same task. I n other experiments Stedman et
al. (195) observed that quaternary ammonium
compounds brought about flocculation of bac-
terial cells, but no germicidal activity was
associated with this phenomenon. Flocculation
of S. schottmuelleri was attributed to altera-
tions in the charge at the cell surface produced
by adsorption of the chemical. The relative
adsorption of different quaternary ammonium
compounds varied, depending on differences in
hydrophobic, polar, and other properties.
According to Ross et al. (162), quaternary
ammonium compounds with 12 to 14 carbon
atoms in the alkyl group showed maximum
antibacterial activity against S. typhi. Incor-
poration of more polar substituents in the
benzyl group increased the germicidal action,
provided the alkyl group contained less than
12 carbon atoms. I f the alkyl group contained
14 or more carbon atoms, antibacterial action
 SALMONELLAE AND SALMONELLOSIS
decreased. These authors postulated the fol-
lowing relation between structure and anti-
bacterial action. The compound is more
strongly attracted to the bacterial cell as the
chemical becomes hydrophobic with an increase
in carbon atoms in the alkyl group. When the
number of carbon atoms exceeds 14, the critical
micelle concentration becomes so low that the
number of free molecules available for adsorp-
tion on the bacterial cell is too low for strong
activity. Polar groups increase the critical
micelle concentration and the concentration of
available compound and, therefore, increase
the biological activity except when the number
of carbon atoms is high. I f the latter is true,
then the extra bulk of the polar groups pre-
vents normal packing on the bacterial surface.
E]ect of hydrogen peroxide. Satta (170)
added oz. typhi (30,000 cells per milliliter) to
milk and treated it with hydrogen peroxide
in an attempt to destroy the bacteria. Use of
0.25 to 0.30% peroxide destroyed the salmo-
nellae in 4 to 5 hr at temperatures of 17 to
32 C, whereas, a 0.2% concentration of the
chemical required 8 to 9 hr to kill the bacteria.
Raw milks were inoculated with S. typhi,
treated with 0.2% hydrogen peroxide, and in-
cubated for 14 to 24 hr at 20-22 C and 28-30 C,
in experiments by Monaci (133). When com-
pared to untreated controls, peroxide caused
a reduction of 7~ to 96% in number of bacteria
present in incubated milks. S. typhi was re-
covered from one sample.
I n contrast to the results cited above,
Roushdy (164) was able to destroy oZ. typhimu-
rium in milk by exposing it to 0.03 and 0.075%
hydrogen peroxide for 4 and 1 hr, respectively.
Sadilek and Stepanek (167) inoculated milk
with S. enteritidis~ warmed it to 57 C, added
150 or 200 mg hydrogen peroxide per 100 ml,
and treated the milk with catalase. Neither
level of peroxide destroyed the 0~ganisms un-
der the conditions just described.
Effects of salts. The behavior of fresly iso-
lated salmonellae in the presence of sodium
chloride was described by Bergmann and Seidel
(29). They cultured the salmonellae on agar
fortified with 5 to 10, 15, 20, and 30% sodium
chloride and in broth containing 5 to 10, and
15% of the chemical at 37 C for one and at
room temperature for three days. ¥ i s u a l in-
spection showed that 7 to 10% sodium chloride
in agar and 8 to 10% in broth caused impaired
viability, or killed salmonellae. The authors
also observed that a few strains of salmonellae
recovered after they were inhibited by 15 to
30% sodium chloride in broth. Somewhat com-
parable results were reported by Severens and
293
Tanner (183), who isolated strains of S. pul-
lorum, S. typhi, and S. schottmuelleri able to
grow in the presence of 8, 6, and 8% sodium
chloride, respectively. Vizir (226) observed
that S. breslau was resistant to the effects of
high sodium chloride concentrations. At 12 to
16 C the organism remained viable for at least
45 days in milks with 5, 10, and 15% sodium
chloride and in broth with 5 and 10% of the
chemical. The organism was inactivated after
24 days in broth containing 15% sodium
chloride. Addition of lactic acid failed to in-
crease the sensitivity of this bacterium to
sodium chloride. Recent experiments by Goep-
fert, Olson, and Marth (74, 75) demonstrated
that S. typhimurium was able to initiate
growth in skimmilk adjusted to p H 4.9 with
lactic acid and fortified with 3% sodium
chloride. Raising the salt content to 4.5% in
the same medium was associated with slow
death of S. typhimurium when held at 7.5 or
13 C for five weeks. A further addition of
0.1% acetic acid to the medium enhanced death
of the organism.
T a r r (204) demonstrated that addition of
0.02% sodium nitrite to fish digest broth
at an acid p H caused inhibition of S. typhi.
According to data by Severens and Tanner
(183), selected strains of S. pulloru~n, S. typhi,
and S. schottmuelleri were able to grow in the
presence of 1:800, 1:800, and 1:600 solutions
of copper sulfate, respectively, and 1:25,000,
1:50,000, and 1:25,000 solutions of mercuric
chloride, respectively. Normal strains of these
organisms were inhibited by a 1:4000 solution
of copper sulfate and a 1:300,000 solution of
mercuric chloride.
Effect of streptomycin in ndlk. Nagel (139),
in studies on the use of antibiotics to preserve
milk, found that concentrations of streptomycin
up to 800 rag/100 ml failed to completely in-
activate S. paratyphi B and S. enteritid/is in
market milk held at 37 C for 10 hr. I n other
experiments, 3 mg streptomycin per 100 ml
failed to destroy as few as 30 cells of S. typhi,
S. paratyphi B, or S. enteritidis per milliliter
of raw milk held at 4 or 22 C for 24 hr.
Effect of sorbic acid. Doell (56) demon-
strated that at p H 5.0, 0.1% potassium sorbate
provided a concentration of sorbic acid both
baeteriostatic and bactericidal for salmonellae
(as well as staphylococci and psendomonads).
When the p H was increased to 7 to 8, a 10%
concentration of the chemical was ineffective
against the same organisms.
Effect of lysozyme. Complete lysis of S.
typhi and partial lysis of S. ente~'itidis were
obtained by Huerta (91) when he treated the
J . DAII~y SCIENCE VOL. 52, ~O. 3
294
bacteria with a 1:64 concentration of lysozyme
recovered from donkey milk and egg white.
The optimal p i t for enzyme ~ctivity was 4.6.
Lysozyme appeared to be inactive against S.
paratyphi, S. schottmuelleri~ S. typhimurium~
Salmonella anatis~ S. derby, Salmonella thomp-
son, Salmonella newport, Salmonella pensa-
cola, and Salmonella adelaide.
Effect of ultraviolet radiation ~nd ozone.
Cathcart et al. (43) found that an exposure of
8 to 45 min was required to inactivate air-
borne cells of S. enteritid~s when they were no
more than 24 in. from an ultraviolet light.
These authors also noted that exposure of the
same organism to an atmosphere of 4 ppm
ozone for 60 min had little effect on its survival.
Effect of antioxidants. The antibacterial
properties of propyl gallate, ethyl protocatechu-
ate, and nordihydroguairetic acid were studied
by ttirose et al. (88). The mininmm concentra-
tions of antioxidant with inhibitory activity
against S. typhi and S. typhimurium were 6.0
and 0.8%, respectively, for propyl gallate;
6.0 and more than 10.0%, respectively, for
protocatechuate; and 0.006 and 0.4%, respec-
tively, for nordihydroguairetic acid.
Effect of ethylene oxide. Winkle and Adam
(235) were able to fumigate 50-kg packages of
dried egg or shredded coconut with ethylene
oxide without changes in product quality or
appearance. A 4,000-liter kettle was loaded
with bags or cases of infected food, evacuated
to 40 ram, 1.0 g ethylene oxide per liter ad-
mitted and circulated for 10 to 15 min by
means of a pump, and the container again
evacuated after 2 to 4 hr, if bags were treated,
or after 6 hr when the foods were in eases.
Tests on the treated products revealed the
absence of salmonellae and a reduction in
total number of bacteria.
Effect of bcta-propiolactone. Bl"ueh and
Koester (37) treated liquid whole egg or egg
white previously inoculated with S. sen/ten-
berg or S. typhimurium~ using from 0.05 to
0.3% beta-propiolactone. Egg white containing
10 ~ cells of S. senftenberg per milliliter was
sterile after 12 hr at 10 C when 0.1% lactone
was used, and after 2 to 3 hr when 0.3% was
added. Liquid whole egg inoculs~ted with l0 s
ceils per milliliter of either species of Salmo-
nella was sterilized in 4 to 5 hr at 10 C with
0.2% lactone or in 2 to 3 hr when 0.3% was
employed. A mild heat treatment (15 rain at
37 C or 1 rain at 55 C) reduced the exposure
time required for sterilization.
Effect of leeithip~. According to results ob-
tained by Levin (108) and Levin and Olitzki
(109), addition of 0.2% lecithin to broth was
J. DAIICY SClENC~ VOL. 52, N0. 3
MA~TH
responsible for the loss of virulence by S.
typhimurium after 250 dMly transfers in the
medium under aerobic and after 230 daily
transfers under anaerobic conditions. When
control cultures were transferred 400 times
in broth without lecithin but under anaerobic
conditions, no loss in virulence was noted.
Effect of carotene and riboflavin. Vasil'eva
(220, 221) investigated the effect of carotene
and riboflavin on S. typhi. He observed that
the presence of 50 mg carotene in 100 ml of
the substrate suppressed the growth of the
organism. Low concentrations (10-s to 10 -1° rag/
100 ml) had a stimulatory effect and the opti-
mum dose (1.5 × 10 -1° rag/100 ml) produced
a 75% increase in cell numbers after 24 hr.
Riboflavin at concentrations of 4 × 10-5 to
2 × 10-~1 rag/100 ml suppressed the growth
of S. typhi, whereas stimulation of growth was
noted when the level was reduced to 5 × 10-12
rag/100 ml. Maximum stimulation was observed
with 2.5 × 10-is rag/100 ml, at which concen-
tration an 87% increase in growth was noted
after 5 hr, a 1,140% increase after 24 hr, and
a 2,642% after 48 hr of incubation.
Effect of spices and essential oils. Dold and
K n a p p (57) tested the inhibitory properties
of different spices by sprinkling them on plates
previously inoculated with S. typhi. Maximum
inhibition of the test organism was associated
with garlic, moderate inhibition with onion,
and slight inhibition with cinnamon, radish,
horseradish, and nutmeg. No inhibition was
observed with celery, caraway, fennel, cori-
ander, parsley, thyme, marjoram, chives, pap-
rika, and pepper.
Experiments to determine the effect of es-
sential oils on growth of S. typhi were con-
ducted by Kellner and Kober (99). They ap-
plied the test substance to filter paper discs
which were inserted in petri dishes inoculated
with the test organism and incubated in an
inverted position for 24 hours. Growth of
S. typhi was inhibited by oils of hyacinth,
chenopodinm, cassia, camphor, cinnamon, cara-
way, synthetic rose, carnation, coriander, syn-
thetic wintergreen, hops, spike, thyme, and
thymol. The effect of spices and essential oils
on microorganisms is discussed more completely
in a review by Marth (116).
Effect of sulfhydryl compounds. Thomas and
Cook (208) demonstrated that the germicidal
action of basic phenylmercuric nitrate on S.
typhi could be neutralized by the sulfhydryl-
containing compounds eysteine, homocysteine,
and glutathione but not by cystine and me-
thionine.
Effect of pressure. Cells of S. paratyphi C
 SALMONELLAE
were exposed to a pressure of 2,800 kg per cm~
for 5 rain by ¥ i g n a i s et al. (224). This treat-
ment was accompanied by cessation of multi-
plication. Reduction of the pressure to at-
mospheric was accompanied by resumption of
growth after a lag period. Respiration gen-
erally followed changes in cell multiplication,
ceasing when a pressure of 3,700 kg/cm ~ was
attained and being noticeably affected at a
pressure of 3,300 kg/cm :.
Occurrence of Salmonellae in Raw Milk
Occurrence of salmonellae in raw milk has
been verified repeatedly by investigators who
studied eertain outbreaks of salmonellosis in
humans. Some of these incidents will be de-
scribed in the sections dealing with sources of
salmonellae in raw milk. A comparison of two
surveys on food-borne illnesses in England
and Wales shows that the ineidence of salmo-
nellosis attributable to raw milk has not
changed significantly during the interval be-
tween 1951 and 1965. Cockburn and Simpson
(49) found that four of 235 and four of 274
food-poisoning incidents in 1951 and 1952 were
caused by salmonellae. I n 1965, according to
Vernon (223), the number of outbreaks had
increased to 4,091 and of these six resulted
from consumption of raw milk contaminated
with S. typhimuxium.
Information on the concentration of salmo-
nellae in raw milk supplies seems to be lacking
in the literature and only a few surveys have
been made on the kind of salmonellae present.
Yarela and Olarte (219), in 1952, examined
Mexican certified milks and recovered 11 differ-
ent salmonellae from 25 out of 520 samples.
Organisms identified included: Salmonella es-
sen (occurred six times); S. paratyphi (oc-
curred five times); S. derby (occurred three
times); S. typhi~ Salmonella bredeny, and S.
newport (each occurred twice), and Salmonella
abony, Salmonella muenchen) Salmonella bovis-
mordificans, and S. senftenberg, (each oc-
curred once). Ritchie and Clayton (159) ex-
amined 762 milks from England, Scotland, and
Ireland for Salmonella dublin and were able
to recover the organism from one sample. 2k
nmre recent (1966) survey to determine the
incidence of sahnonellae in Northern Ireland
milk supplies was conducted by Murray (138).
l i e was unable to recover these organisms from
bulk-collected samples, but did find S. dublin
in the milk from two individual producers.
Raw milk is most frequently, although not
exclusively, contaminated by cows. Other
sources include human carriers, water supplies,
AND SALMONELLOSIS 295
and equipment. Outbreaks of salmo~ellosis at-
tributable to raw milk contaminated from the
various sources will be described below.
Infected cows and calves as sources of con-
tamination. Salmonellae infections in dairy
cattle have been observed and reported by
numerous investigators. Some of the reports
have resulted from surveys conducted on cat-
tle, whereas others had their origin in out-
breaks of human illness attributable to con-
sumption of raw milk. Reports on surveys
will be considered first and details of food-
borne illnesses associated with raw milk con-
taminated by the cow will be given later.
Surveys. Field (64) conducted a survey in
Wales during 1946 and 1947 and found evidence
of salmonellosis on 70 farms. Calves were
affected on three of the farms and cattle from
one to 14 years of age on the other 67 farms.
The infective organism on 64 farms was S.
typhimurium. Eight outbreaks of bovine sahno-
nellosis were investigated by McFarlane and
Rennie (121). Three of the outbreaks occurred
in adult cattle and five in calves. Organisms
isolated from the diseased cattle include: S.
typhimurium, Salmonella orientalis, Salmonella
newington~ S. anatum, S. muenchen, and Sal-
monella london.
Murdock and Gordon (137) examined fecal
samples from apparently healthy cows in an
attempt to learn about the incidence of S.
dublin. Results of tests on 272 fecal samples
revealed the presence of S. dublin in 7.4% of
the specimens. A similar study on 1,250 cows
was carried out by Smith and Buxton (189),
who found S. dublin in fecal samples from six
cows. Post-mm~em examinations on 17 cows
culled because of chronic mastitis were con-
ducted by Zagaevskii (240), who recovered S.
dublir~ from the udders of five of the animals.
Nystr5m et al. (142), working in Norway,
noted S. dublin infections in 69 dairy herds
tested during 1962 and 1963.
A rather extensive survey on dairy cattle
in Germany was carried out by Rasch (152)
during 1948 to 1956. l i e examined 1,427 daia'y
herds and found that 325 cows in 277 herds
were carriers of salmonellae. Guinee et al.
(81), working in the Netherlands, conducted
a recent survey on the incidence of sahnonellae
in certain tissues obtained from cows and
calves. Salmonellae were recovered from the
mesenterie lymph nodes of two out of 600 nor-
real cows and 11 out of 265 normal calves.
A somewhat more detailed study was made on
1,504 normal calves at the time of slaughter.
Tests on the mesenteric and portal lymph nodes,
gall bladder, and feces revealed the presence of
J . DAIRY SOIEIgC~, VOL. 52, NO. 3
296 ~IARTH
salmonellae in one or more samples taken
from each of 216 calves. When an even more
intensive study was made on 416 calves (sam-
ples were taken from musculature, diaphragm,
spleen, liver, gall bladder, portal lymph nodes,
feces, and all mesenteric nodes), salmonellae
were found in one or more samples from each
of 63 calves. Salmonellae isolated from the
calves belonged to 32 different serotypes, with
S. typhimurium appearing in 61.4% and S.
dublin in 8.5% of the infected animals. The
musculature and organs of infected calves
rarely contained salmonellae, but the lymph
nodes, gall bladder, and intestinal contents
were frequently infected. According to Roth-
enbacher (163), S: typhimurium infections
were responsible for u large number of deaths
among dairy calves.
Raw milk-borne illnesses. The literature
reports numerous outbreaks of human illness
attributed to raw milk contaminated with sal-
monellae by the cow. Numerous serotypes of
Salmonella have been noted in these outbreaks.
Included are S. dublin (7, 48, 51, 79, 85, 11],
119, 203, 206, 216), S. thompson (236), S.
heidelberg (53, 101), S. newport (35), S. en-
teritidis (28), and S. typhimurium (3, 241,
157). The reports available for the prepara-
tion of this paper suggest that S. dublin was
recovered with greater frequency than were
other sahnonellae when raw milk contaminated
with these organisms was associated with hu-
man illness. Goats also have been reported as
able to contract S. dublin infections (73).
Many authors repo~ that cows were shedding
sahnonellae in their milk and some suggest these
organisms may cause symptomless cases of
mastitis. There is not complete agreement,
however, and some authors feel that contam-
ination with fecal material from infected cows
may be an important means by which the orga-
nisms enter milk. Scott and Minett (179)
conducted some experiments in an attempt to
better understand the behavior of salmonellae
in cows. They drenched a cow with S. typhi
at concentrations far greater than would be
ingested if the animal consumed contaminated
water. No illness resulted from this treatment
nor could the organism be recovered from feces
or milk. Two calves received a similar treat-
ment; one contracted the disease, whereas the
other calf shed the organism in its feces for
48 hr. The authors also infused S. typhi cells
directly into the udder and found that: a) ten
quarters ceased to excrete S. typhi in ten days,
b) two quarters of one cow excreted the orga-
nism for 27 days, and c) two quarters of one
cow excreted the organism for 85 days.
J . DAIRY SCIENCE YOL. 52, 1~0.
Cows may become infected (or contaminated)
with salmonellae from a variety of sources,
including feed and water. The role of water
will be considered later, ttutchinson (92), in
1964, described an outbreak of human illness
which may have had its origin in a feed ma-
terial. Examination of feces from 20 persons
who consumed raw milk from a common source
revealed S. heidelberg. Upon further investiga-
tion the organism was recovered from milk
samples and also from the feces of several
cows suffering from enteritis. The cows were
on the farm that furnished the raw milk in
question. F a r m personnel were also found
to excrete S. heidelberg. The original source
of infection is believed to have been barley,
which was fed to cows and which contained
the pathogen. The barley probably became in-
fected from rats, since their fecal material was
evident in the grain.
Calves on a dairy farm can serve as sources
of salmonellae which contaminate milk. Two
such incidents have been described in recent
literature. Dobrier (55), in 1963, reported on
an outbreak of hmnan illness caused by S. en-
teritidis present in raw milk. Inspection of
the farm revealed that calves were suffering
from an illness caused by this organism. Since
hygienic practices on the farm were substan-
dard, it was believed that the organisms from
the calves entered the milk and, hence, caused
illness in the consumers.
An outbreak of gastroenteritis in which dis-
eased calves may have been important oc-
curred in the United States in January and
February of 1966 and was described by Holter-
man and Mather (90). Eleven persons in five
families were affected and all of them had
consumed milk produced on a single farm.
The farmer responsible for the milk was not in
the business of selling raw milk but gave his
surplus to relatives and friends. Although the
exact route of contamination remained un-
known, several possibilities were apparent to
the authors. They found that the farmer was
busy throughout the winter making trips to
a beef-cattle feeding lot several miles from his
farm. Apparently, 16 calves were in the feeding
lot and three died early in J a n u a r y from diar-
rheal disease. Fecal samples were taken from
six of the calves five weeks after recovering
from the disease and S. typhimurium was re-
covered from one sample. The farmer admitted
that he often worked late in the evening with
the calves while they were ill and, after return-
ing home, had neglected to wash his hands
prior to milking. Milk was collected in open
buckets and then poured into large wide-
 SALMONELLAE AND SALMONELLOSIS
nlouthed jars. I n addition to possible contami-
nation from the hands, manure and other barn-
yard materials could have easily fallen into the
bucket during the milking process. The milk
was cooled slowly after collection; more than
2 hr were required to bring the temperature
to 40 F, thus allowing considerable time for
bacterial growth.
Water as a source of contaminatiou. Water
can contribute salmonellae directly if it is
added to milk, as well as indirectly if it con-
tanfinates the cow and she then adds the orga-
nisms to milk. Wallace and Mackenzie (228)
conducted epidemiological studies on a milk-
borne outbreak of paratyphoid fever. Their
investigations suggested that a small stream
was contaminated with salmonellae by an in-
efficient cesspool. DailT cattle waded in the
stream, became contaminated, and carried the
organisms on their flanks. When the cows were
milked, sahnonellae entered the milk and then
proliferated, since cooling facilities were in-
adequate.
Run-off water may serve to contaminate
streams in a fashion similar to the cesspool
incident just described. Miner et al. (129)
examined such water collected from two feed-
lots near the Kansas State University campus
and recovered Salmonella infantis.
A n incident of enzootic salmonellosis in a
herd of cattle, that was caused by an infected
stream which furnished drinking water, is
described by Schaal (173). He indicates fur-
ther that fencing the stream and removal of
carrier animals served to control the disease
in this herd.
An unusual outbreak of gastroenteritis which
involved both raw milk and water occurred in
Yakima County, Washington, in 1967 and is
reported by l~rancis and Allard (68). At least
40 persons were involved and S. typhimurium
was recovered from nearly all of them. More
than 50% of those initially infected consumed
raw milk supplied by a single dairy farm.
Tests on samples taken at the farm showed
S. typhimurium to be present in the milk from
three cows, in the feces from one calf, and in
water from the stream which supplied the cattle.
Extensive sampling of the stream revealed it
to be contaminated for 15 to 18 miles. A fur-
ther seaxch was made and a gunny sack con-
taining a dead calf was found in the water just
above the highest point at which S. typhimu-
rium was recovered. The pathogen was found
in a stool sample taken from the calf. I t
was postulated that the calf died of the S.
typhimurium infection, was thrown into the
297
stream, and served to contaminate the water.
The dairy herd downstream then consumed the
water, became infected, and produced contami-
nated milk.
An outbreak of typhoid fever in St. Louis
caused by raw milk contaminated by water
fl'om a cistern was described by Meyer et al.
(127), and an outbreak of paxatyphoid fever
resulting from raw milk contaminated with
water supplied by a hydraulic ram was re-
ported by P a r r y (145).
Water also can serve to contaminate equip-
ment which, in turn, can add sahnonellae to
milk. I n at least one instance S. dublin was re-
covered from a washed milking machine cluster
(138).
Some years ago it was common practice
in Egypt to dilute buffalo milk with water
and urine. Mihaeloff (128) examined 200 sam-
ples of milk treated in this manner and found
that 50% contained S. typhi.
Addition of sewage to pastures has been
found to give rise to salmonellae in raw milk.
Bederke and Lunt (26) reported that S. para-
typhi entered milk from this source and Poppe
(148) made a similar observation with regard
to Salmonella manchester.
Humans as a source of contamination. Ty-
phoid fever transmitted by raw milk appears
to be the most common form of salmonellosis
which can be traced to human contamination.
More often than not a carrier is responsible
for infecting the milk. Such incidents have
been reported by Flammer and Fleisch (65),
W a t t (230), Landau (102), Merrilles (125),
Snfith (187), Steele (198), the Baltimore City
I-Iealth Department (23), and Bekenn and Ed-
wards (27). Other salmoneIIae reported as
having been added to milk by faxmers who
were ill include S. typhimurium (8) and S.
paratyphi (77).
Occurrence in milk of antibodies against sal-
monellae. Porterfield (149) exposed dry and
lactating cows to high doses of inactivated S.
pullorum cells, in attempts to develop antibodies
in the milk against the organism. He found
that intramammary infusion during the dry
period resulted in a higher antibody content
in colostrum than in blood. Intramuscular
and subcutaneous injections of the organism
were accompanied by lower antibody contents
in both colostrum and blood. Intramammary
infusions during lactation were associated with
the appearance of antibodies in 2 hr, which
persisted for 246 to 288 days.
While Porterfield was working with S. pul-
lorum, similar experiments with S. enteritidis
were being conducted by Sarantsev (169). Ite
,.T. DAIRY SOlEI'qOE VOL 52, NO'. 3
298 ~A~TH
concluded that milk from cows hyperimmunized
with S. enterit{dis possessed therapeutic and
prophylactic properties against paratyphoid
infections in calves.
Senft and Porter (182), working with S.
pu~lorum, studied antibody formation in goats.
They noted that a significant antibody response
occurred after the third treatment, when the
antigen was infused into the mammary gland at
four-day intervals. A second goat developed
antibodies after the second infusion which, in
this instance, was given at ten-day intervals.
Both goats produced abnormal milk for 36
hr following the first two or three infusions.
Experiments by Dvorak (58) and Lassila et al.
(105) indicated that agglutinins were noted in
milk within 12 to 24 hr after inactivated cells
of S. pu~lorum~ S. gallinarum, or S. unatum
were infused into the udder. The agglutinins
produced as a response to S. pullorum were
found by Greene et al. (78) able to resist nor-
mal pasteurization of milk.
Occurrence of Salmonellae in Milk Products
Pasteurized ~nilk. Although the heat treat-
ment given milk during pasteurization is ade-
quate to destroy salmonellae, pasteurized milk
can become contaminated and, hence, has been
associated with a number of sahnonellosis out-
breaks.
RiddetI (156) reported an outbreak of food
poisoning involving 300 cases, caused by con-
sumption of raw and pasteurized milk con-
laminated with S. typhimurium. I t was postu-
lated that raw milk infected pasteurized milk
at the bottling machine. The contaminated raw
milk was produced on a farm where several
cattle had been infected with S. typhimurium
and where the farmer was suffering from diar-
rhea at the time of the outbreak.
A n incident in which 34 persons became
infected with S. dublin f r o m raw and pas-
teurized milk was described by NystrSm et al.
(142). Investigations at the dairy which sup-
plied the contaminated milk reveMed that pas-
teurized milk was pumped through unclean
pipelines used previously for raw milk.
Fifty-five cases of gastroenteritis caused by
S. paratyphi occurred in two Welsh counties.
According to Thomas et al. (209) nearly all
patients regularly consumed pasteurized milk
processed by a single plant. No evidence of
inadequate heating could be obtained, but oc-
casional samples of water taken from the jets
of bottle-washing machines gave positive pre-
sumptive coliform test results and a residual
chlorine content of 0.3 to 1.0 p p m or lower.
River water, which after filtration and ehtorina-
J . DAIRY SCIENCE VOL. 52, NO. 3
lion, served as the water supply for the plant
was found to be heavily contaminated with S.
paratyphi.. I t was believed that occasional
infection of milk bottles through use of inade-
quately treated river water caused this out-
break.
Werner and ZSckler (232) described an out-
break of paratyphoid fever which involved 635
persons and resulted from consumption of
contaminated pasteurized milk. A woman suf-
fering h'om paratyphoid fever was living on
the dairy premises and it was thought that the
infection was transferred from her to pas-
teurized milk by workers in the dairy.
A somewhat similar situation was reported
by Ruys (165). She indicated that an outbreak
of typhoid fever involving 175 primary and
282 secondary cases resulted from consump-
tion of pasteurized milk. The milk was con-
taminated after pasteurization by a carrier
working at the plant where it was processed.
The behavior of S. typhimurium in refriger-
ated pasteurized milk was ascertained by
W u n d t and Schnittenhelm (238). These investi-
gators inoculated the milk, after pasteuriza-
tion, with S. typhimurium and then held it at
7, 4, 2, and --10 C. A f t e r six days the number
of viable salmonellae had declined by 68, 58,
80, and 95%, at the respective temperatures
indicated above.
Mathur (118) recorded an outbreak of gastro-
enteritis caused by Sahnonella weltevreden in
boiled milk. The milk, boiled at the farm, was
transferred to another container which, ap-
parently, was responsible for its contamination.
Fermented milks. The literature on the be-
havior of salmonellae in cultured milks presents
a Confusing picture. Wilson and Tanner (234)
reviewed the literature covering the period from
1886 to 2944 and found more than 25 reports
which suggested r a p i d (within three to four
days or less) death of salmonellae in a variety
of cultured milks. S. typhi was most frequently
used in these tests, although a few other sal-
monellae were examined. Most often the test
organism was introduced into already sour
milk and the duration of viability (frequently
at room temperature or above) was determined.
I n contrast to the above, more than ten reports
were cited which suggested that salmonellae
survived for extended periods (often several
weeks or longer) in cultured milks.
I n some of their own experiments Wilson and
Tanner (234) noted that growth in milk of
S. typhi, S. paratyphi, and S. schottmuelleri
was inhibited by p H values of 4.5 to 4.8, 4.3 to
4.8, and 4.8 to 4.9, respectively. They observed
that the three organisms, in some instances,
 SALMONELLAE AND SALMONELLOSIS
survived for more than 63 days in buttermilks
containing u p to 0.73% titratable acid. Survi-
val in acidophilus milks (maximum acidity
1.3%) ranged from one to 58 days.
Probably more tests have been conducted
on the sm-cival of salmonellae in yogurt tban
on their behavior in other cultured milks.
Schmidt and I-Iannemann (176) compared the
bactericidal effects of biogurt, a special yo-
gurt, and milk fermented by Lactobacillus
casei. They concluded that the three cultured
milks acted similarly against Salmonella spp.
and that the pathogens were viable in all prod-
ucts after 15 days at 4 C. I n contrast to these
observations, Todorov (213) reported that S.
typhimurium and S. enteritidis remained viable
in yogurt for periods ranging from a few
hours to a few days, depending on the activ-
ity of the lactic acid bacteria and on storage
conditions. I n another report Todorov (212)
suggested that Lactobacillus bulgaricus and
Streptococcus thermophilus produced proteina-
ceous substances which together with lactic acid
were responsible for inhibiting salmonellae in
yogurt. Giirsel and Fisek (82) had concluded
earlier that inhibitory activity of yogurt was
associated with the lactic acid present.
The behavior of S. typhi in kefir was studied
by Habaj and Michalewicz (83). When milk
was inoculated with S. typhi before the starter
culture was added, the pathogen survived for
eight days in low-acid kefir made from the
mixture and for 72 hr in a high-acid product.
Kazberyuk (98) inoculated mare's milk with
S. paratyphi and an hour later added a kumiss
culture at the rate of 25% of the total milk
used. Six hours later he was unable to recover
sufficient viable S. typhi cells to induce illness
in mice. A fermented milk product known as
curds is made from buffalo's milk in the south-
ern province of Ceylon. The fermentation is
probably similar to that encountered in kumiss
and kefir, since yeasts, S. lactis, and a lacto-
bacillus are involved. Nicholls et al. (140)
added S. typhi to the whey from curds and
were unable to recover viable cells after 3 hr.
P a n j a and Ghosh (144) found that the p H
values of 27 samples of dahi (an I n d i a n fer-
mented milk) ranged from 4.2 to 4.7. Upon
adding S. typhi to these samples, they found
the pathogen destroyed in all 27 products after
60 rain, in 20 samples after 30 rain, and in 16
fermented milks after 15 rain. Solutions of
hydrochloric, acetic, and citric acids at p i t 4.4
were less bactericidal than either a solution of
lactic acid at p i t 4.4 or whey from dahi. More
recent investigations on dam were completed
by Tiwari and Singh (211). They added S.
299
paratyphi to milk and stored the fermented
product at 3 to 5 C and 22 to 25 C. Destruc-
tion of S. paratyphi was accomplished in 144
hr at the former and in 72 hr at the latter
temperature.
The behavior of salmonellae in mazun (a
sour milk preparation resembling acidophilus
milk) was investigated by Kazaryan (97). He
observed that simultaneous cultivation of the
lactic acid bacteria and salmonellae (S. typhi
and paratyphi) did not hinder growth of the
latter. Additional studies with white nlice
showed that the virulence of S. typhi and S.
paratyphi was unchanged after 18 to 24 hr
in an acidic milk product. I n later studies
Polonskaya et al. (147) prepared sterile fil-
trates from cultures of Lactobacillus acidophilus
and found them able to inhibit growth of Sal-
monella sp.
Abd-E1-Malek and Demerdask (1) added S.
typhi and S. paratyphi to sterile milk, which
was then made into Zabady (another fer-
mented milk product). They noted that the
salmonellae survived the manufacturing process
which involved incubation at 43 to 45 C and
then remained viable for up to 51 hr in the
finished product held at 4 to 5 C or 25 C.
Meyer (126) studied the fate of S. paratyphi
added to raw milk which was then allowed to
sore" naturally. I n some samples the pathogen
could not be recovered after 24 hr, whereas
others contained an almost pure culture of
S. paratyphi at this time. Tests on additional
soul" milk samples demonstrated that the para-
typhoid organisms remained viable for over
one year.
Cultured buttermilk is the most popular fer-
mented milk in the United States and is often
made with the aid of Streptococcus lactis. Cer-
tain strains of S. lactis produce the antibiotic
nisin; therefore, its effect on salmonellae might
be questioned. Marth (116) has reviewed the
literature dealing with nisin and does not list
a report which deals with its action on these
organisms. Inhibitory substances are also pro-
duced by Leuconostoe citrovorum, one of the
flavor- and aroma-producing bacteria used in
the lnanufacture of cultured buttermilk. Again,
according to Marth (116), the literature pro-
vides no data on how these agents affected sal-
monellae. Recently, Vedamuthu et al. (222)
observed that S. lactis var. diacetilactis, an-
other organism able to produce flavor and
aroma substances, inhibited S. typhi on agar
plate cultures.
Dried milk products. Contamination of in-
stant nonfat dried milk with salmonellae made
headlines in the United States during 1966
J . DAIRY SCIENCE VOL. 52, 1~O. 3
300
and 1967. This, however, was not the first
time these organisms were reported as present
in dried milks. The British Ministry of Health
(130) reported that 12 cases of sahnonellosis
in 1950 were associated with the consumption
of dried milk contaminated with S. typhimu-
rium. I n 1954, according to Cockburn and Ver-
non (50), another outbreak of salmonellosis
resulting from S. typhimurium was caused by
contaminated dried milk. Similar outbreaks
in Bulgaria during 1945-1950 were recorded
by Iordanov et al. (93).
The recent United States experience with
sahnonellae in dried milk began in January,
1966, when the Division of Epidemiology of
the Michigan State Department of Public
Health investigated two cases of gastroenteritis
resulting from S. new-brunswick (10, 11). The
cases occurred in males less than six months
old, residing in different areas of the state;
each had received a formula made from instant
nonfat dry milk. Following this outbreak, it
was noted that S. new-brunswick had been
reported only rarely in the United States be-
tween 1947 and 1964 (0.02% of all isolations).
Between April, ]965, and January, 1966, there
were 29 reported isolations of this serotype
(as compared to 13 from 1947 to 1964) from
humans, a situation which suggested a com-
mon source of infection.
As a consequence, state health departments
that reported isolation of this organism were
asked to submit epidemiologie information
about the cases. Of the 29 persons from whom
S. new-brunswick was isolated, 25 were avail-
able for further studies. All of these persons
had symptoms characteristic of salmonellosis,
but the cases were distributed throughout the
United States. Although dietary histories were
impossible to obtain, it was determined that
dried milk was the only item consumed with
greater frequency than was expected. Twenty
of the 25 persons had ingested this food within
30 days of their illness.
A f t e r dried milk was implicated, bacteriolog-
ical examinations were made on hundreds of
shelf samples of many brands of dried milk.
The same rare serotype, S. new-brunswick, was
subsequently isolated from many samples of
instant nonfat dry milk produced by a single
plant in the midwestern United States. The
organism was also isolated within the plant and
from other milk products on the premises.
Schroeder (178) has detailed conditions in the
plant where the contaminated powder was pro-
duced. The plant handled about 11 million lb
of milk per year, most of which was instantized.
Raw whole milk was obtained from about 250
J. DAIRY SOIENCE ~OL. 52, NO. 3
~ART~
farms and raw skimmilk came from four dairies.
During processing, the skimmilk passed through
a plate heater, where it was supposed to at-
tain a temperature of 72.2 C and be held at
that temperature for 10 to 15 see. Thermo-
static controls, a flow diversion valve) and time
controls were lacking and, hence, there was
no assurance of adequate pasteurization. No
heat treatments later in the process were suf-
ficient to destroy salmonellae, if they were
present. A f t e r heating, milk was concentrated
in a series of three vacuum pan evaporators
at temperatures which may have favored growth
of microorganisms present. The concentrated
milk was s p r a y dried and, with the aid of
a pneumatic system, transported to a sifting
apparatus where coarse aggregates were re-
moved. Dried milk was then subjected to the
instantizing or agglomerating process in which
moisture in the form of steam is added to the
powder. The moistened powder was dried and
larger particles obtained as a result of this
treatment.
Although much of the equipment used to
handle the milk was easily cleaned, inspection
of the plant revealed: a) a baffle plate inside
the dryer and a trough and screw used to
remove the powder had opened seams and
crevices, b) the instantizing system had several
welded joints with rough surfaces, numerous
crevices and open seams, together with a series
of rods and dividers in the after-dryer, all of
which permitted accumulation of caked mate-
rial, c) the s p r a y dryer was dry cleaned (with
brushes) daily and wet cleaned monthly, d) the
agglomerator was wet cleaned weekly but prod-
uct deposits accmnulated in much less than a
week, and e) air intakes for the dryer and
after-dryer were located in the same room with
sifting and bagging operations, where the at-
mosphere contained a high level of dried milk
solids.
Schroeder's (178) study on the occurrence of
salmonellae in dried milk led him to the fol-
lowing conclusions: a) Since a large number
of producers supply a processing plant, it is
difficult to identify the original source of infec-
tion, but it is important to guard against such
contamination, b) temperature requirements
for pasteurization should be followed carefully,
c) equipment used to dry and to instantize
milk is very difficult to clean and, hence, once
salmonellae are introduced into a plant great
efforts are required to eradicate them, and
d) dried milk is distributed extensively at the
wholesale level and, hence, contaminated prod-
uct can received widespread distribution.
The product from this plant was recalled
 SALMONELLAE AND SALMONELLOSIS
from the market in April, 1966, and a careful
cleanup and remodeling of the plant instituted.
Additional recalls of' instant dry milk pro-
duced at other locations occurred during 1966
and 1967.
A large-scale testing program was instituted
by the U S D A during 1966. During the initial
phase of the program, A p r i l through August,
1966, 2,741 samples from 156 plants in 23
states were tested. Thirty-four samples of
nonfat dried milk and 27 environmental sam-
ples were found to contain sahnonellae. Dried
milks contaminated with sahnonellae were re-
covered from plants in Idaho, Iowa, Minne-
sota, South Dakota, Washington, and Wiscon-
sin. Additionally, environmental samples from
plants in the following states were found to
contain salmonellae: Iowa, Minnesota, Mis-
souri, North Dakota, Oklahoma, South Da-
kota, Vermont, Washington, and Wisconsin (10,
11).
Salmonellae recovered from the dried prod-
ucts include: S. montevideo~ S. oranienbnrg,
S. heidelberg, S. tennessee, S. senftenberg, Sal-
monella alachua~ S. cubana) and Salmonella
orion (10, 11, 15, 16). Pickett and Agate
(146) also isolated a lactose-fermenting strain
of S. newington from instant nonfat milk pow-
der after the product had given rise to nine
cases of salmonellosis.
Tests on environmental samples from dried
milk plants revealed the presence of the fol-
lowing serotypes: Salmonella sehwarzengrund,
S. tennessee, S. newport, S. cubana, S. typhi-
~turium, S. anatum~ Salmonella worthington,
Salmonella kentucky~ S. infantis, S. oranien-
burg, S. heidelburg, and S. orion (10, 11).
Meister (11) reported results obtained in the
USDA survey during the first six months of
1967. Product and environmentM samples were
taken from approximately 200 dry milk sam-
ples in 19 states. Results indicated that 34
(1%) of 3,315 product samples and 121 (8.2%)
of 1,475 environmental samples were contam-
inated with salmonellae.
Final survey data (14, 15, 16) showed that
0.2% of all products tested contained salmo-
nellae and these were manufactured in 13% of
the plants.
According to Severs et al. (184), the New-
foundland Public Health Laboratories reported
20 isolations of S. newport from stool speci-
mens examined during F e b r u a r y 1 to March
20, 1968. The 20 patients, who came from 17
different households in the St. John's district,
gave a history of diarrhea and vomiting; five
preschool children and two adults required hos-
pitalization. At least 12 other persons in the
301
families of confirmed cases of S. newport in-
fection gave a history of diarrhea and vomiting.
All the families had purchased and consumed
a particular brand of dried skimmilk. Opened
3-1b boxes of product, all with the same batch
number, were obtained from three of the homes
and were found to contain S. newport. Dried
milk from eight unopened 3-1b boxes with the
same batch numbers as those taken from homes
of infected persons were obtained in St. John's
supermarkets and also found to contain S. new-
port. Additional details on this outbreak of
salmonellosis associated with dried milk were
not available to the author when this p a p e r
was prepared.
Ice cream and related products. Ice cream
and related frozen desserts can become con-
taminated with salmonellae before freezing.
Freezing does not destroy all of the organisms
and, hence, the contaminated frozen desserts
when consumed can give rise to salmonellosis.
Relatively few problems apparently have been
encountered with these products in the United
States, but the foreign literature suggests that
ice cream has been associated with a number of
salmonellosis outbreaks.
During 1923 to 1941, according to Savage
(171), 40 outbreaks of paratyphoid fever oc-
curred in Great Britain. Two of the outbreaks
were attributed specifically to ice cream, al-
though a variety of dairy produots was as-
sociated with 25 of the other outbreaks. Cock-
burn and Simpson (49) noted that salmonellae
in ice cream were responsible for three disease
outbreaks in England and :Wales during 1951-
52.
During September 1, 1939, to May 31, 1940,
Santiago, Chile, experienced outbreaks o f ty-
phoid and paratyphoid fevers. Molina (132)
reported that of 356 cases investigated, 107
involved ice cream as the sole source of infec-
tion and in 44 others as an auxiliary source.
Fecal coliforms were also observed frequently
in the ice creams from plants which had pro-
duced the product infected with salmonellae.
The problem in Santiago persisted and Tellez
Aguirre (205) studied outbreaks which oc-
curred during September, 1940, to May, 1941.
H e noted that 140 of 472 cases were traced to
ice cream. During 1940, ten of 600 fecal sam-
pies from dairy workers and ice cream vendors
contained typhoid or paratyphoid bacteria.
Again, many of the ice creams contained fecal
coliforms.
Moore (134) described an outbreak of p a r a -
typhoid fever which involved 25 cases and was
associated with ice cream from a particular
delivery van. Examination of the ice cream
$. DAIRY SCIEI~CE VOL. 52, NO. 3
302 ~A~TH
and vendor failed to reveal any infecting orga-
nisms, but later investigation showed that the
vendor's wife was discharging S. paratyphi in
her feces. I t was thought that she was in-
directly responsible for contaminating the ice
cream. Another outbreak of salmonellosis in-
volving 505 cases was also attributable to an
ice cream vendor. According to Evans (63)
all involved persons had consumed ice cream
dispensed by the infected person who, upon
subsequent examination, was found to be an
active carrier of S. typhi. The largest number
of typhoid fever cases in this outbreak occurred
in the 104 to 15-year age group. F o u r deaths
were associated with the outbreak.
Other instances of sahnonellosis resulting from
consumption of contaminated ice cream were re-
ported by Leinbrock and Kirhoff (106) and
Biersehenk (31). The former authors noted
that ice cream contaminated with S. typhimu-
rium during manufacture caused illness in 53
persons in a sanatorium near Bonn. The other
author reported that 180 persons became ill
after eating unpasteurized ice cream infected
with S. infantis.
W u n d t and ¥oss (239) studied a recent out-
break of gastroenteritis caused by commercial
ice cream containing 10,000 to 100,000 cells of
S. typhimur~m and Salmonella bareilly per
milliliter. These authors also inoculated va-
nilla, strawberry, and lemon ice creams with
S. typhimurium and then subjected them to
heat treatments of 60 to 80 C. Ten to 15 min
of exposure at the lowest temperature destroyed
the organisms in all types of ice cream, but
from 20 to 60 rain of additional exposure( de-
pending on the type of container) was needed
before the desired temperature was attained.
During 1967, 14 outbreaks of salmonellosis
occurred in New York, New Jersey, Connecti-
cut, and Maine, and were attributable to a
kosher imitation ice cream (19). Although the
imitation ice cream did not contain dairy in-
gredients, it is the type of product which
could easily be manufactured in a dairy plant.
Each outbreak occurred one to three days after
a catered banquet at which kosher food was
served. Of approximately 3,300 persons who
attended the banquets, an estinmted 1,800
(54%) developed diarrhea, abdominal pain or
cramps, fever, chills, headache, nausea, and
vomiting. The median duration of the illness
was three days, and there were no deaths.
Examination of stool specimens from 12 per-
sons who represented six of the banquets re-
vealed S. typhimurium. Product ]eft over from
the banquets also yielded S. typhimurium.
Studies were then carried out at the plant
J. DAIRY 8C~,NCE VOL. 52, NO. 8
which manufactured the ice cream. All en-
vironmental samples from the plant and em-
ployee stool cultures were free of salmonellae.
Ingredients used in the product were also ex-
amined and all were free o£ salmonellae ex-
cept the frozen egg yolks, which yielded S.
typhimurium. The frozen egg yolks were un-
pasteurized and were produced at a New York
City egg-breaking plant. Environmental sam-
ples taken at the egg-breaking plant revealed
several serotypes, including S. typhimurium.
As a consequence of the incident just de-
scribed, action was taken by the New York
City Health Department which resulted in the
temporary suspension of operations by the
frozen dessert and egg-breaking plants. All
imitation ice cream manufacturers in the city
have been placed under the milk code, which
will require production of a certified pathogen-
free product. The incriminated egg-breaking
plant has agreed to pasteurize its raw egg
products. Joint action by the city health de-
partment, state officials, and U.S. Food and
Drug Administration officials resulted in re-
covery or destruction of several thousand serv-
ings of contaminated product.
Although egg yolk was the ingredient re-
sponsible for contamination of the frozen des-
sert just described, other materials can also
serve as vehicles for the introduction of salmo-
nellae into these products. An example is
recorded by Ahmed and Nagib (2). They sug-
gested that guar flour be utilized as a stabilizer
in ice cream, but found that the flour con-
tained S. typhi, if not adequately processed.
Other ingredients which might contaminate
dairy products are discussed later in this paper.
Butter. Salmonellae survive for extended pe-
riods if they occur in butter and the product
has been associated with some outbreaks of
salmonellosis. A survey made during 1945-1950
by Iordanov et al. (93) revealed the presence
of S. typhb~urium in some samples of Bul-
garian butter.
Outbreaks of salmon ellosis. Cambessedes and
Boyer (41) reported that in June, ]944, several
hundred cases of typhoid fever appeared in
Paris. The victims were all customers of cer-
tain stores and dairies and the only food from
these sources eaten by M1 affected was butter
obtained from one wholesale merchant. The
causative agent, apparently, was not recovered
from the butter.
A study made by Studney (201) on two
explosive outbreaks of paratyphoid fever in
Graz, Austria, during 1940 and 1945 revealed
that butter was the likely source o£ infection.
Investigations at the dairy which supplied the
 S A L M O N E L L A E AND S A L M O N E L L O S I S
area showed that each time an outbreak oc-
curred one worker handling dairy products
was suffering from paratyphoid fever. Al-
though the causative organism was not isolated
from milk, cream, or butter during the in-
vestigation, it was thought that pasteurized
cream was infected before it was made into
butter. Later experiments demonstrated that
paratyphoid organisms could be recovered from
butter made from cream contaminated arti-
ficially after pasteurization.
Survival in butter. Several investigators have
demonstrated that sahnonellae survive for ex-
tended periods in butter. Siegmund (185) ob-
served that Salnwnella spp. survived for an
average of 52.5 days in butter held at 22 C
and for 91 days when the product was stored
at 4 C. He also tested Shigella spp. and found
the survival of these organisms to be an aver-
age of 59.5 and 63 days at the respective tem-
peratures cited above.
Butter was heated for 30 rain at 63 C on
three successive days ~nd then inoculated with
S. typhi and S. paratyphi in experiments by
Guerzoni (80). Subculturing of the butter at
intervals revealed that both organisms re-
mained viable for more than 55 days. Recent
tests by Zagaevskii (240, 241) revealed that
S. dublin and S. typhimurium survived for up
to 285 days in butter stored at 0 to 4 C. To-
dorov (213) made sweet- and ripened-cream
butters containing S. typhimu~'i~m and S. en-
teritidis. When the butters were held at room
temperature, salmonellae survived beyond the
shelf-life o£ the products; whereas, at 0 C
storage salmonellae persisted for 124 days in
sweet-cream butter and 55 days in the ripened-
cream spread. Survival of S. typhimuriu~n in
butter patties was found by Orlandella and
Barresi (143) to be about 76 days.
Although free fatty acids have a destructive
effect on S. typhi in ordinary media, especially
at a p H value of 5 or less, tests by Brisou (36)
demonstrated that this was not true in rancid
butter. He noted that the p H of rancid butter
was near 6.0 and suggested this may have ac-
counted for survival of the organism in short-
term storage experiments.
Smirnova (186) made butter creams contain-
ing 35-41% sugar and inoculated them with
S. typhirnurium. The organism was able to
grow in the product during conventional stor-
age. According to this investigator, 64% sugar
was needed in the aqueous phase before growth
of the organism was stopped with certainty.
Cheese. Outbreaks of sahnonellosis have
been associated with the consumption of dif-
ferent cheeses. The early literature is con-
303
eerned, primarily, with typhoid fever, whereas
more recent investigations suggest that sahno-
nellae other than S. typhi may sometimes be
associated with chesee-induced illness.
Cheddar cheese. Gauthier and Foley (72)
described an epidemic of typhoid fever in
Canada during the autumn of 1941. Forty cases
were involved and six deaths resulted. The
only food common to all the patients was Ched-
dar cheese, made locally from raw milk and
consumed when it was about ten days old. Al-
though the factory where the cheese was made
lacked adequate sanitation, the source of the
epidemic was found to be a known typhoid
carrier who, against orders from public health
authorities, milked cows whose milk was used
by the factory to produce Cheddar cheese.
Another outbreak of typhoid fever attrib-
utable to Cheddar cheese occurred in Quebec
during February, 1944, and was described by
Foley and Poisson (66). The original source
of infection was never traced, although later
it was found that the cheesemaker's wife had
an active case. Nevertheless, the authors be-
lieve she was not responsible for infecting the
cheese. Foley and Poisson (66) also recom-
mended use of a three-month ripening period
when Cheddar cheese is made from raw milk.
One hundred and eleven out of 507 cases of
typhoid fever in Alberta between 1936 and
1944 were caused by Cheddar cheese, according
to Menzies (123). Samples of cheese from the
last three outbreaks in 1944 were recovered and
tested for S. typhi. The organism was found
in 30-day-old cheese, but could not be recov-
ered from 48- and 63-day-old cheese. As a
consequence of this outbreak, Alberta halted
sale of cheese made from raw milk unless the
cheese was ripened for at least three months.
SurvivM of S. typhi in Cheddar cheese was
studied by Ranta and Dohnan (151) and
Campbell and Gibbard (42). The former au-
thors mixed S. typhi with Cheddar cheese and
found that the organism survived for one month
at 20 C. Inoculation of S. typhi on to the sur-
face of cheese was accompanied by a similar
survival at room temperature, a longer sur-
vival period at refrigerator temperature, and
penetration of the organism to a depth of 4 to
5 cnl into the cheese after 17 days.
Campbell and Gibbard (42) inoculated milk
with S. typhi and used it to make Cheddar
cheese. All cheeses were ripened for two weeks
at 14.4 to 15.6 C, after which one cheese from
each duplicate set was transferred to storage
at 4.4 to 5.6 C. A t the lower temperature,
seven out of ten cheeses contained viable S.
typhi cells for more than ten months, whereas
J. D A I ~ Y ~CIENCE VOL. 52, NO. 3
304 MARTH
at the higher temperature the organism gener-
ally disappeared after three months of ripen-
ing. Size of inoculum and acidity of the cheese
did not appear to affect the longevity of S.
typhi.
Recently Goepfert, Olson, and Marth (74,
75) investigated the behavior of S. typhimuri-
urn during the manufacture and ripening of
Cheddar cheese. Pastuerized milk was inocu-
lated with S. typhimurium when the lactic
starter culture was added. A slight increase in
number of salmonellae occurred during the
time between inoculation and cutting of the
curd, followed by a rapid increase during the
interval between cutting the curd and draining
the whey. After accounting for concentration
of cells through coagulation, an average of
three and a half generations of salmonellae de-
veloped during this period. Salting of the curd
was associated with a reduction in the growth
rate and ripening of the cheese was accom-
panied by a decline in the salmonellae popula-
tion. Survival of S. typhimurium exceeded 12
weeks at a ripening temperature of 12.8 C and
16 weeks at 7.2 C. Limited tests demonstrated
that acetate accumulating in ripening cheese
may contribute to the denlise of salmonellae.
Colby cheese. A typhoid fever epidemic
started in January, 1944, in the northern part
of Indiana and covered 18 to 20 counties (6,
155). Approximately 250 cases and 13 deaths
were recorded in this outbreak. Thomasson
(210) noted that the carrier was never traced
but illness was associated with consumption of
Colby cheese. The cheese, made by a single
dairy in the area, was produced from raw miIk
preheated to 32.2 to 37.8 C and was not allowed
to ripen before sale.
Wright (237) and Tucker et al. (215) record
an incident in which 384 cases of illness in
Kentucky were caused by consumption of 12-
to 14-day-old Colby cheese infected by S. ty-
phimurium. Investigation revealed that a dead
mouse had been removed from a 1,000-gal vat
of milk used to produce the cheese. Tests on
infected cheese demonstrated that S. typhimu-
rium survived for 302 days during storage at
6.1 to 8.9 C.
Mold ripened cheeses. Mocquot et al. (131)
made blue cheese from milk inoculated with 10 ~
to 100 Salmonellae sp. cells per milliliter and
observed the behavior of the organisms during
manufacture and ripening. The death rate of
Salmonella was related to the p H value of
24-hour-old cheese and increased with a decrease
in the pH. Survival and death of the organism
were similar in the inner and outer portions of
the cheese. The percentage survival of salmo-
g. DAII~Y SCIENCE ~OL. 52, NO. 8
nellae in six-day-old cheese was less than 0.01.
Camembert cheese was responsible for an
extensive outbreak of illness in Germauy, with
more than 6,000 cases involved. According to
Bonitz (32) the cheese was infected with S.
bareilly, and rennet was thought to be the
original source of the contaminant. After con-
ducting additional experiments, Bonitz (33)
changed his mind and postulated that infection
probably entered the cheese via the glue used
to fasten labels to individual cheeses.
Other cheeses. The occurrence of salmonellae
in a variety of other ripened cheeses has been
reported. Many of these cheeses are not com-
mon in the United States, but the information
may be useful, in that it further demonstrates
the gaps which exist in our knowledge about
the behavior of sahnonellae in cheese. I n some
instances investigators simply stated that sal-
monellae were recovered from cheese, without
specifying the type of cheese being studied.
A few examples will be given at the end of
this section.
Bruhn et al. (38) studied the survival of
sahnonellae in Samsoe cheese. This cheese has
a p H value of 5.15 to 5.20 after 24 hr and
contains 44 to 46% moisture. Samsoe cheese
was ripened at 16 to 20 C for five to six weeks,
after which it was held at 10 to 12 C for an
additional seven to ten weeks. The authors
noted that a 60-day period was necessary to
achieve a 10,000-fold reduction in number of
viable salmonellae. The death rate was less
rapid at 10 to 12 C storage than at 16 to 20 C.
I n May of 1944 an unusually high number
of typhoid fever cases were reported in four
counties of California. Investigation, accord-
ing to ttalverson (84), showed that the sources
of infection were Romano Dolce, Teleme, and
high-moisture Jack cheeses, all made from un-
pasteurized milk. Over 90% of the affected
persons had consumed one or several of the
cheeses just mentioned (4). This outbreak
provided the impetus for the state of California
to pass laws controlling the manufacture and
sale of cheese in that state.
Wahby and Roushdy (227) studied the sur-
vival of S. enteritidis, S. typhi, and S. para-
typhi B in Damietta cheese, an Egyptian dairy
product. When the cheese was held at 20 to
25 C, S. enteritidis survived for 17 days, S.
typhi for 12 days, and S. paratyphi B for 27
days.
Forty samples of Kareish cheese (an Arabian
product) were examined chemically and bac-
teriologically by Moutsy and Nasr (136). They
observed that the cheeses contained from 2.33
to 11.38% salt, 0.75 to 2.7% titratable acidity,
 SALMONELLAE AND S A L M O N E L L O S I S
68 million to 6.3 billion bacteria per gram,
and 1,000 to 100,000,000 coliforms per gram.
One sample yielded S. typhimurium.
The behavior of S. typhimurium and S. en-
teritidis in Kachkaval cheese (a hard cheese)
was studied by Todorov (214). He added five
million to 350 million salmonellae per milliliter
of milk, which was then made into Kachkaval
cheese. Heating of the curd in a water bath
at 71 to 72 C for 70 to 90 see did not destroy
the salmonellae. Their survival in the cheese
ranged from four to 20 days, depending on
the initial level of contamination.
Vizir (226) studied the behavior of S. breslau,
a contaminant of Brynza cheese, in different
media fortified with salt. At 12 to 16 C the
organism remained alive in milk with 5, 10,
and 15% salt and in broth with 5 to 10% salt
during the 45 days of the experiment. Lactic
acid at a concentration of 0.9% or higher de-
stroyed the organisms in five to ten days, re-
gardless of temperature. Zagaevskii (241) noted
that S. typhimurium and S. dublin remained
viable in Brynza cheese for up to 22 months.
An outbreak of typhoid fever attributable
to a cheese made in a Norwegian home was re-
ported by ttemmes (87). A man ill with ty-
phoid fever was a part of the household when
the cheese in question was made. Studies on
the aged cheese revealed that viable S. typhi
were present after 40 but not 55 days.
Cheese produced in a factory in southern
Alberta, Canada, was responsible for an out-
break of typhoid fever in 1VIarch, 1944 (5).
Thirty-five cases and one death were reported.
The source of the infection was traced to one
cheese faeto~ 7 and it was believed that the
factory's water supply was contaminated.
A total of 5,387 food-poisoning incidents
were recorded in England and Wales during
1961. The vehicle of infection was identified
in 160 of the outbreaks. Four outbreaks were
caused by cheese, and salmonellae were re-
sponsible in one of these four incidents (9).
Rivas et al. (160) examined 524 samples of
different cheeses in Mexico between 1957 and
1962. Salmonellae were recovered from five
samples and the following serotypes were noted :
S. typhimurium, S. enteritidis, Salmonella car-
ran, and S. kentucky.
Quarg. Quarg is a soft, unripened cheese
product common in Europe and in other areas.
I t can be made by coagulating milk through
the use of lactic acid producing microorga-
nisms or rennet. The product is probably quite
similar to dry Cottage cheese curd. D'Yako-
nova (59) noted that an outbreak of food-borne
illness attributable to quarg occurred in the
305
Saratov district of Russia in 1960. Evidence
obtained suggested that the quarg was pre-
pared frmn milk produced by a cow infected
with S. typhimurium.
A n earlier epidemic attributable to quarg
occurred in South Baden, Germany, in 1940.
Merkle (124) reported that S. typhimurium
was recovered from the feces of 68 adults and
four infants. Two of the infants died later.
Quarg produced by a large dairy which sup-
plied much of the Baden area was found to be
the source of infection.
Survival of salmonellae in quarg has been
studied by a number of investigators. Todorov
(213) found that S. typhimurium and S. en-
teritidis survived for up to 20 days in rennet
quarg and for more than 35 days in whey
quarg. Siegmund (185) noted that salmonellae
survived an average of 16.5 days in quarg at
22 C and an average of 64.7 days when the
quarg was stored at 4 C. Rather extended sur-
vival periods were reported by Zagaevskii (240,
24•). He reported that S. typhimurium and S.
dublin survived up to 22 months in quarg at
0 to 4 C and that S. dublin remained viable
for four and three-fourths years in dried quarg
stored at 0 to 4 C.
Cream and Cottage cheeses. Reports on the
occurrence of salmonellae in cream cheese are
very limited. Savage (171) reported one out
of 40 outbreaks of paratyphoid fever in Great
Britain during 1923 to 1941 resulted from con-
sumption of contaminated cream cheese.
Studies on the behavior of salmonellae in
Cottage cheese are also quite limited. Re-
cently, Basarab et al. (24) reported an out-
break of salmonellosis which involved 12 cases
and resulted from consumption of Cottage
cheese contaminated with S. typhlmurium. The
pathogen was traced to the cheesemaker, and
he was responsible for infecting the product.
Lyons and Mallmann (114) introduced S.
typhi into Cottage cheese and held the re-
sultant mixture at 37, 24, and 10 C. Within
48 hr samples stored at 37 and 24 C had
dropped to a p i t value of 3.8 and viable cells
of S. typhi could not be recovered. Holding
of the cheese at 10 C was accompanied by a
survival period of 96 hr.
Recently, McDonough et al. (120) studied
the behavior of salmonellae during the manu-
facture of Cottage cheese by the short-set
method. A mixture of salmonellae was added
to the cheese milk approximately 1 hr prior
to the manufacture of cheese. Although a
rather high initial inoculum (200,000 to 500,000
salmonellae per milliliter) was used, there was
no evidence of growth during the interval
J. DAIRY SCIENCE TC'OL. 52, NO. 3
306 MARTH
between inoculation and cooking of the curd.
A cooking temperature of 51.7 C was needed
to ensure destruction of salmonellae during
the cheesemaking operation. The same investi-
gators also added sahnonellae to Cottage cheese
creamed with a sweet cream dressing, a dress-
ing containing Leucon.ostoc s p , and a dressing
cultured with Ia'ctie acid bacteria. Salmonellae
persisted for more than 12 days, regardless
of the cream dressing used on the cheese when
the products were held at 4.4 C. No marked
decrease in numbers was noted during the
storage period.
Other Products
Milk shake. A small outbreak of typhoid
fever traced to a milk shake was described by
Marmion and Martin (115). The product con-
sisted of dried milk, flavoring, saccharine, and
water. I t was impossible to determine how the
milk shake became contaminated, but the au-
thors speculate that water or workers pre-
paring the drink might have been sources of
S. typhi. This outbreak consisted of 11 cases
of confirmed typhoid fever and eight additional
cases of diarrhea and vomiting among people
who had consumed the drink.
Eggnog. A fatal case of S. enteritidis infec-
tion in an 11-year-old male resulted from a
family outbreak in a rural community in Ore-
gon in August of 1967 and was reported by
I-Iolmes et al. (89). Seven members of the
family became ill with severe gastroenteritis
following a luncheon. The suspected meal con-
sisted of chocolate eggnog made with raw
eggs, pancakes, and fried eggs. Normally, the
family consumed eggs collected from nests on
the farm; however, the suspected meal included
eggs collected from the hayloft and around
the farm. These eggs were highly contaminated
with fecal material and dirt, and the shells
were not cleaned prior to breaking the eggs
during food preparation.
The deceased child became ill with abdominal
cramps five hours after consuming the suspect
meal. Symptoms of the central nervous sys-
tem developed and the boy was brought to the
doctor's office, where he was pronounced dead.
Stool cultures from all members of the fam-
ily (including the deceased boy) and a blood
culture from the deceased boy were found to
contain S. enteritidis. Shells of eggs used for
the eggnog, pancake batter, and the chocolate
drink all yielded S. e~teritldia.
Cream. Sandiford (168) reported the isola-
tion of S. typhi from a can of sterilized cream
imported into England. Further tests on 225
cans of the same batch revealed that 33% were
J. DAIRY SCIENCE ~OL. 52, N0, 3
not sterile. The sealing of the cans in the
affected batch was imperfect, and it was sug-
gested that they became contaminated from
cooling water after sterilization. F o r this par-
ticular batch, the cooling water came from a
shallow well and not from the regular supply.
According to Scassellati Sforzolini (172),
S. typhi does not survive too well in frozen
cream. He inoculated cream, held it at --25
C for 60 days, and found that the numbers of
S. typhi declined by 98% during the treatment.
An outbreak of gastroenteritis associated
with a cream dessert was described by Lein-
brock and Ritter (107). The chocolate dessert
was made from milk and cream and S. enteri-
tldis was responsible for illness in at least 23
patients. The source of the contaminant was
not reported by the authors.
Whey. Data presented by Helm and Wede-
mann (86) indicate that S. paratyphi B and
S. enteritidis were able to survive in acid and
rennet wheys for a sufficient time to constitute
a possible hazard in the unpasteurized product.
Recently, S. derby has been recovered from
dried rennet whey resulting from the manufac-
ture of Swiss cheese (21).
Lactalbumin. A recent report (17) has indi-
cated that a governmental agency recovered
S. cubana from a sample of lactalbumin.
Salmonellae in ingredients used in some dairy
products. Salmonellae can enter dailT products
from some ingredients used in their manufac-
ture. The final section of this review will con-
sider some of these sources.
Coconut. Schaffner et al. (174) have ob-
served that raw, unprocessed coconut supports
the growth of salmonellae as well as other en-
teric bacteria. Original contamination of the
coconut does not result from carriers or pol-
luted waters, but instead from contact with
soils containing salnmnellae, followed by dis-
persion via infected coconut milk. Since sal-
monellae were particularly resistant to desicca-
tion (see earlier discussion in this paper), the
authors resorted to a pasteurization treatment
to destroy these bacteria in coconut. They
found that heating raw coconut meat in a water
bath at 80 C for 8 to 10 rain effectively killed
sahnonellae, did not injure the product, and
provided a prophylactic method now widely
used by the coconut industry. Exposures of
1 rain at 100 C and 5 min at 90 C destroyed
sahnonellae but caused product damage. I t
was reported that more than 75% of the Sal-
monella cultures recovered in the United States
from imported coconut were S. senftenberg,
whereas 20% were S. cubana. Other salmonel-
 SALMONELLAE AND
lae recovered from coconut include Salmonella
lexington, Salmondla stanleyj and S. bazeilly.
Eggs. I t is well known that salmonellae occur
with some frequency in dried eggs and other
egg products. I n one study, Schneider (177)
recovered salmonellae from 3.2% of 901 sam-
ples of high-quality dried egg. Salmonellae
encountered in his work include S. pul~orum~
S. ora~n~enburg, S. tennessee, Salmonella oregon~
and S. montevideo. Other references could be
cited to verify this point, but it appears un-
necessary. Sahnonella can grow rapidly in
reconstituted dried eggs, hence such products
should be used immediately after rehydration.
According to Solowey and Calesnick (191), no
more than 4 hr should elapse between prep-
aration and use if the eggs are at a temperature
of 25 to 45 C.
Effective J u l y 1, 1966, egg products entering
interstate commerce had t o be pasteurized.
I t is of interest to observe that of 76 to 112
official samples of egg products tested by a
governmental agency during the first and second
quarters of 1966 (before pasteurization), 46
and 23%, respectively, contained salmonellae.
During the third and fourth quarters (after
pasteurization) this same agency tested 92 and
29 samples and recovered salmonellae from 12
and 31%, respectively (18). Although use of
pasteurization has reduced the frequency with
which salmonellae were observed in egg prod-
ucts, it apparently has not completely elim-
inated the problem.
Dye. Carmine dye is prepared from the
cochineal insect, Coccus cacti, which lives on
cactus plants in tropical areas. The insects
are imported into the United States, primarily
from Peru and the Canary Islands. The dye
is then produced in the United States (as well
as in England, France, and Germany). Group
G salmonellae have been recovered from ship-
ments of the insect and from the carmine dye
manufactured in the United States and En-
gland. Lang et al. (104) reported that in-
terest in this dye developed after an outbreak
of S. cabana infections occurred among patients
in a Massachusetts hospital who had ingested
the dye for gastrointestinal studies.
Although contamination of carmine dye with
S. cubana was initially discovered through use
of the dye as a clinical diagnostic aid, the bulk
of the dye is used to color foods, cosmetics,
and drugs. Tests by federal and state labora-
tories in the United States (13) revealed S.
cubana in the following items which contained
the dye: carmine stock solution, pink summer
coating, rainbow peach coating, rainbow yel-
low coating, kiddy pops, r a s p b e r r y creams,
SALMONELLOSIS 307
other candies of various types, p a p r i k a mix,
meat binder, meat preservative, and pepper-
mint ice. t t u m a n illness was not, however,
traced to any of these products.
Fruit products. Data by Rochaix and Jac-
queson (161) indicate that fresh grape
juice with p I I values of 2.6 to 3.2 showed
definite bactericidal properties against S. typhi
and S. paratyphi A and B. These bacteria,
when placed in grape juice, were destroyed in
15 rain to 3 hr. Ryberg and Catheart (166)
prepared lemon, orange, pineapple, strawberry,
and apricot fillings from water, sugar, fruit,
salt, cornstarch, and egg yolk. They inoculated
the fillings with S. enteritidis (approximately
50,000 to 200,000 per gram) and held them
at 37 C for 24 hr. Although none of the prod-
ucts were free of the Salmonella at this point,
there was a drastic reduction in numbers dur-
i n g the incubation period. Xo .products eDn-
tained more than 3,000 per gram (apricot and
orange) and some (lemon, pineapple, and straw-
berry) had less than 800 salmonellae per
gram. The p H values of all products ranged
~rom 2.92 to 4.38.
Sirup. Lynovskii (113) inoculated the ty-
phoid and paratyphoid bacilli into 21 samples
of artificially flavored sirups containing from
30 to 53% sugar. A f t e r 24 hr, growth of
these bacteria was observed in two samples.
Products of animal origin. During 1966 and
1967 (12, 17) governmental laboratories re-
ported sahnonellae in a variety of animal prod-
ucts, some used as drugs and others in the food
industry. Of particular concern to the dairy
industry is the recovery of salmonellae from
pepsin and from edible gelatin (S. senften-
berg). During 1968 (20), S. newington also
was recovered from unflavored, edible gelatin.
In Conclusion
This review, lengthy as it is, has not cov-
ered all aspects of the Salmonella problem.
I t has primarily addressed itself to the ques-
tion of sahnonellae as they affect the dairy
industry. The reader interested in other as-
pects of this problem is referred to the books
by Edwards and Ewing (60), Kauffmann (96),
Van Oye (218), Seidel and Muschter (181),
Soltys (192), Well and S a p h r a (231), and ~he
chapter by Morgan (135) in Bacterial and
Mycotie Infections of Man. Reviews by Bow-
met (34), Buxton (40), and Prost and Rie-
mann (150) may also prove helpful. Persons
interested in methods for isolation of salmo-
nellae will find the p a p e r by Galton and Bor-
ing (71) to be a good starting point.
Perhaps a word should be said about control
J. D~RY SOI]~NOE V O L 52, NO. 3
308
of sahnonellae. Control of these organisms is
not different f r o m that of most other bacteria.
Initial contamination in raw materials should
be low, the product should receive sufficient
heat (or other treatment) to destroy the salmo-
nellae, and the processed product should be
protected f r o m reeontamination until it reaches
the consumer. Although these general state-
ments a p p l y in all instances, the details of ac-
complishing the goal of Salmonella control will
v a r y f r o m p l a n t to plant and f r o m product
to product. I t is impossible to indicate all of
the procedures which should be followed, but
the reader can develop m a n y of them as he
considers the information given in this review.
Finally, some comments are in order about
information which is missing. Obviously, much
is known about salmonellae. Although we m a y
be f a m i l i a r with p o l a r and a n t i p o l a r mutants
in the t r y p t o p h a n operon of S. typhlmurium
(22), we do not know the frequency with which
they occur nor the level of salmonellae in our
raw milk supply. I t is evident f r o m this re-
view that we really are not sure about the
behavior of these organisms during the manu-
facture of numerous dairy products. H o w do
various conditions associated with operating
a dryer affect their survival in dried milk
products? W h a t are the relationships between
starter bacteria and salmonellae? H o w do the
salmonellae behave during the manufacture of
Swiss or Brick cheese ? W h y are there so many
viewpoints in the literature dealing with sur-
vival of salmonellae in cultured milks? H o w
frequently do salmonellae occur in dried whey,
lactalbumin, casein, and sodium caseinate ?
These are some of the unanswered questions.
The careful reader will have m a n y others by
the time he completes this paper.
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