Antibacterial properties of saliva: Role in maternal periparturient grooming and in licking wounds

Abstract

Canine saliva was tested for its bactericidal effects against pathogens relevant to the presumed hygienic functions of maternal grooming of the mammary and anogenital areas and licking of wounds. Both female and male saliva were bactericidal against Escherichia coli and Streptococcus canis but only slightly, and nonsignificantly, bactericidal against coagulase positive staphylococcus and Pseudomonas aeruginosa. E. coli is the cause of highly fatal coliform enteritis of neonatal mammals and E. coli and S. canis are the main pathogens implicated in neonatal septicemia of dogs. The bactericidal effects of saliva would facilitate the hygienic function of maternal licking of the mammary and anogenital areas in protecting newborns from these diseases. E. coli and S. canis along with coagulase positive staphylococcus and P. aeruginosa are among the common wound contaminants of dogs. Wound licking, and the application of saliva, would thus reduce wound contamination by E. coli and S. canis. The resistance of staphylococcus to bactericidal effects of saliva may be a factor in the high frequency (46 percent) with which coagulase positive staphylococcus was isolated from wounds compared with much lower frequency (9-17 percent) with which E. coli and S. canis were isolated.

Full text

Physiology & Behavior. Vol. 48, pp. 383-386. © Pergamon Press plc, 1990. Printed in the U.S.A. 0031-9384/90 $3.00 + .00
Antibacterial Properties of Saliva:
Role in Maternal Periparturient
Grooming and in Licking Wounds
BENJAMIN L. HART AND KAREN L. POWELL
Department of Physiological Sciences, School of Veterinary Medicine
University of California, Davis, CA 95616
Received 27 March 1990
HART, B. L. AND K. L. POWELL. Antibacterial properties of saliva: Role in maternal periparturient grooming and in licking
wounds. PHYSIOL BEHAV 48(3) 383-386, 1990. --Canine saliva was tested for its bactericidal effects against pathogens relevant to
the presumed hygienic functions of maternal grooming of the mammary and anogenital areas and licking of wounds. Both female and
male saliva were bactericidal against Escherichia coli and Streptococcus canis but only slightly, and nonsignificantly, bactericidal
against coagulase positive staphylococcus and Pseudomonas aeruginosa. E. coli is the cause of highly fatal coliform enteritis of
neonatal mammals and E. coli and S. canis are the main pathogens implicated in neonatal septicemia of dogs. The bactericidal effects
of saliva would facilitate the hygienic function of maternal licking of the mammary and anogenital areas in protecting newborns from
these diseases. E. coli and S. canis along with coagulase positive staphylococcus and P, aeruginosa are among the common wound
contaminants of dogs. Wound licking, and the application of saliva, would thus reduce wound contamination by E. coli and S. canis.
The resistance of staphylococcus to bactericidal effects of saliva may be a factor in the high frequency (46 percent) with which
coagulase positive staphylococcus was isolated from wounds compared with much lower frequency (9-17 percent) with which E. coli
and S. canis were isolated.
Saliva Maternal behavior Bacteria E. coli Dogs Grooming
SALIVA is a very complex fluid subserving a number of important
functions (20). One of the early known properties of human saliva
was an antibacterial effect which was attributed to the lysozyme
content (8, 10, 18, 37, 38). Subsequent to these earlier studies,
saliva has been found to contain a variety of other antibacterial
substances including lactoferrin, leukocytes, lactoperoxidase, an-
tibodies and cationic proteins (5, 20, 21). The more recent studies
on the bactericidal effects of various constituents of human saliva
have been aimed at understanding the role of saliva in inhibiting
the action of bacteria which cause oral pathology (1, 21, 35, 36).
Among the other substances in human saliva are the histidine-rich
peptides which have direct antifungal properties (29) and sub-
stances that interfere with the ability of bacteria to adhere or attach
to soft tissues (21).
In animals, particularly rodents and carnivores, one can point
to functions other than maintenance of oral hygiene for the
putative antibacterial properties of saliva. One of these functions is
postcopulatory genital grooming. In a previous study, we found
that saliva of male and female rats had bactericidal effects against
two genital pathogens, PasteureUa pneumotropica and Myco-
plasma pulmonis (14). This bactericidal effect of rat saliva
presumably enhances the physical washing effect of genital
grooming in preventing the transmission of genital pathogens from
females to males during copulation.
Two other behavioral patterns involving licking, and where
antibacterial effects of bacteria may be important, are periparturi-
ent licking by females of their mammary and anogenital areas and
licking wounds. Licking of the mammary and anogenital areas just
prior to parturition is evident in rodents (30), cats (32) and dogs (4)
and the shift of grooming from other parts of the body to the
mammary and anogenital areas during late pregnancy has been
quantitatively documented for rats (31). Newborn mammals,
which are born with a sterile gut, do not have the intestinal
bacterial flora which is protective against opportunistic pathogens
(11). Newborns are, therefore, at risk to some of the more virulent
strains of Escherichia coli commonly found in feces (6, 27, 39) if
exposed to this microorganism prior to ingestion of colostrum, as
they would be during the birth process or in attaching to nipples
prior to suckling. Severe enteritis with high mortality, caused by
E. coli (colibacillosis), is reported for young rats (12,22) and dogs
and cats (11, 17, 39). The intestinal epithelium of neonates is
permeable to bacteria for the first 48-72 hours after birth (11), and
in neonatal dogs E. coli and Streptococcus canis can be involved
in septicemia (1 I, 17). If saliva were bactericidal against E. coli,
and other potential pathogens, periparturient licking of the mam-
mary and anogenital areas would be particularly adaptive since
these are the body areas of the mother which could be contami-
nated by fecal-borne bacteria and which the newborns' mouths
come in intimate contact with during birth and suckling.
Finally, a common observation among carnivores and rodents
that have sustained cutaneous injuries is licking their wounds
immediately after the injury and rather frequently during the
healing process. One rather obvious function is to physically
cleanse the wound of foreign material, tissue debris and bacterial
383

384 HART AND POWELL
contaminants through the use of the tongue. If the saliva of
animals has antibacterial agents that are effective against potential
wound-tissue pathogens, then the licking of wounds would be
particularly adaptive in reducing bacterial contamination.
The purpose of this study was to examine saliva for its
bactericidal effects against pathogens relevant to the hygienic
functions of maternal periparturient grooming and wound licking.
Dogs were chosen as saliva donors because a large quantity of
saliva could be obtained for testing against several pathogens.
Additionally, we had access to a repository of bacteria isolated
from dog wounds.
METHOD
Subjects
The saliva donors were 46 gonadally intact male dogs and 42
female dogs that were in anestrus or ovariohysterectomized at the
time of saliva collection. The donors were of mixed breeding,
weighing a mean of 21.4 kg.
Saliva Collection
The procedures used for collecting saliva for dogs were
modified from those previously utilized in collecting saliva from
rats (2, 14, 15, 24). The subjects were anesthetized with sodium
thiamylal. At the time of anesthetization subjects were injected
with pilocarpine (0.02 mg/kg, SC) for cholinergic stimulation of
saliva. Saliva was collected by means of a funnel suspended below
the oral cavity and, after 5-10 rain, 20-25 ml of saliva was
collected. Atropine (0.1 ml/kg SC) was given after the specified
amount of saliva was obtained to counteract the effect of the
pilocarpine. Immediately after each collection procedure the saliva
from 6-8 dogs of the same sex was pooled and frozen. At a later
date this saliva was thawed, centrifuged at 10,000 rpm for 15 min,
sterilized by passage through a 0.45 tx millipore filter, frozen at
-70°C
and then lyophilized and stored at 5°C until reconstituted
and used in the tests. Given that the saliva collected by use of
pilocarpine was dilute, the sterilized saliva used for the bacterio-
logical tests was reconstituted to only 5 percent of the original
volume.
Selection of Bacteria
One pathogen chosen for testing was E. coli because of its role
in coliform enteritis of newborn animals. Another organism
chosen was the 13-hemolytic S. canis because in neonatal puppies,
B-hemolytic streptococcus, along with E. coli, is a predominant
organism involved in septicemia (11,17). To address the question
of whether saliva is bactericidal for some common wound con-
taminants, we conducted a survey of bacterial isolates from dog
wounds as cataloged in a bacterial repository. The repository
consisted of stored and cataloged bacteria which had been isolated
and identified over a period of years from open wounds of 87 dogs
(Table 1). The two most frequently isolated aerobic wound
contaminants, coagulase positive staphylococcus (either Staphylo-
coccus intermedius or S. aureus) and Pseudomonas aeruginosa,
were chosen as the other test bacteria. The choice of E. coli, S.
canis, coagulase positive staphylococcus and P. aeruginosa re-
sulted in 2 Gram positive and 2 Gram negative bacteria (E. coli, P.
aeruginosa representing Gram negative and staphylococcus, S.
canis representing Gram positive). The particular test bacterium
chosen was one of the wound isolates for each of the different
species of bacteria.
Testing for Bactericidal Effects of Saliva
Each of the organisms selected for study was inoculated into
TABLE
1
PERCENT OF AEROBIC BACTERIA CULTURED FROM CANINE WOUNDS
Organism Percent of
Genus Species Wounds
Acinetobacter calcoaceticus 2.3
Actinomyces viscosus 3.5
Aeromonas species 1.2
Aeromonas hydrophilia 1.2
Bacillus species 2.3
Corynebacterium species 4.6
Eikenella corrodens 1.2
Enterobacter species 1.2
Enterobacter aerogenes 2.3
Enterobacter agglomerans 1.2
Enterobacter cloacae
4.6
Enterobacter sakazakii 1.2
Escherichia coli 17.2
Klebsiella pneumoniae
10.3
Microeoccus species 1.2
Moraxella species 1.2
Mycobacterium phlei 1.2
Pasteurella species 3.5
Pasteurella multocida
10.3
Pasteurella pneumotropica 1.2
Proteus species 2.3
Proteus mirabilis 3.5
Pseudomonas aeruginosa 18. 4
Serratia marcescens 1.2
Staphylococcus coagul, pos. species
46.0
Staphylococcus species 4.7
Streptococcus species 1.2
Streptococcus canis 9.2
Streptococcus faecalis 2.3
Streptococcus faecium 2.3
Streptococcus agglactiae 5.8
Streptococcus viridans 1.2
Streptococcus zymogenes 1.2
brain-heart infusion broth and incubated for 18-24 hr to achieve
maximum growth in the broth solution. Aliquots of this stock
broth were then frozen for future tests. The procedure for
quantitatively estimating the bactericidal effect of saliva was
similar to that used previously (14). This involved serial dilution
of the stock nutrient broth of each organism from 10 - 1 to 10-9 in
brain-heart infusion broth. At this time 0.1 ml aliquots of each
dilution were uniformly applied to washed bovine blood agar
plates to determine organism concentrations in the stock culture.
Colonies were counted after 18-24 hr of incubation. At the same
time, aliquots of 0.1 ml were taken from each of the bacterial
serial dilutions and 0.06 ml of 5 percent reconstituted saliva in
sterile water was added. The mixture was incubated at 37°C for
18-24
hours. An equal amount of sterile isotonic saline was added
to 0.1 ml aliquots of each bacterial serial dilution to serve as
control. Following incubation with saliva, 0.01 ml from each
serial dilution tube was uniformly applied to washed bovine blood
agar plates, and the plates incubated for 18-24 hr. The number of
bacteria killed was estimated by comparing number of colonies
with the number expected at that dilution from counts on stock
cultures. A plate with less than 10 colonies was counted as having
no growth. Two replications for each of 5 trials with each species
of bacteria were conducted separately for male and for female
saliva.

ANTIBACTERIAL EFFECTS OF SALIVA 385
100
.~
60
z~
g~ 40
~
2o
0
Psaudomonas Escharichia Staphylococcus
Str#ptococcus
aeruginosa coil cans
I I t
I r~z,;~l I
i I ~_////~i i
z I ~//Az i I P'/J~
i I ~'///At i I
~///~
Saline Male Female Saline Male Female Saline Male Female Saline Male Female
FIG. 1. Estimated mean number of bacteria killed ( ___ SEM) by 0.6 ml of
5 percent reconstituted saliva. Saline control killed no bacteria of any of
the bacterial species.
In pilot studies phenylephrine (0.3 mg/kg, SC) was given to
anesthetized dogs for adrenergic stimulation of saliva. Saliva was
produced much more slowly and less dependably after the admin-
istration of this stimulant than with the cholinergic stimulant. In
duplicate trials, adrenergic saliva was compared with cholinergic
saliva against each of the four bacteria. Since no difference in
bacteria killed was noted between adrenergic and cholinergic
saliva, the present experiment analyzed only cholinergic saliva
from pilocarpine administration.
RESULTS
AND
DISCUSS/ON
In all instances, the saline control supported growth or survival
of all four species of bacteria to a level in which a calculated serial
dilution would have yielded 10 or fewer bacteria. In other words,
saline killed no bacteria (Fig. 1). In contrast, the saliva added to
broth dilutions of E. coli and S. canis was estimated to have killed
a mean of about 40,000-75,000 bacteria (Fig. 1). The number of
bacteria killed in replications ranged from 3,000 to 230,000 for E.
coli and from 4,000 to 70,000 for S. canis. Saliva appeared to be
only slightly, and nonsignificantly, bactericidal towards staphylo-
coccus and P. aeruginosa in that an estimated mean of 500-700
bacteria were killed by the same saliva treatment (Fig. 1). The
number of bacteria killed in replications ranged from 0 to 2,200 for
staphylococcus and from 0 to 2,300 for P. aeruginosa. A sign test
on the replications confirmed a difference between the saliva and
saline treatment for E. coil and S. canis for both male and female
saliva (p<0.05), but no difference between male and female
saliva.
The finding of bactericidal effects of saliva against E. coli and
S. canis is supportive of the concept that maternal periparturient
licking of the mammary and anogenital areas is adaptive in
protecting the newborn from excessive exposure to these potential
pathogens. As mentioned, some strains of E. coil found in feces
cause severe enteritis with high mortality in newborn rats, cats and
dogs, and E. coli and S. canis are the most frequent pathogens
involved in septicemia in neonatal dogs and cats. When a mother
licks the nipples she is able to physically clean and apply to the
nipples saliva which is bactericidal to E. coli, S. canis and
possibly other (untested) pathogens. It would appear that the most
critical time for a mother to lick the anogenital and mammary areas
is just prior to parturition and immediately after birth since that is
when the newborn gut is most vulnerable and newborns have not
received any protective colostrum. Interestingly, in rats it is in the
periparturient phase that anogenital and mammary area licking
peaks (31). Rat pups will not attach to nipples that have been
experimentally washed, but attachment can be induced when
mother saliva is applied to the nipples (3). The reluctance of
newborn rats, and possibly also carnivores (13), to attach to
nipples to which maternal saliva has not been applied may be
thought of as a fail-safe mechanism to assure that infants do not
place their mouths on contaminated nipples.
In terms of wound licking the potential for 0.06 ml of 5 percent
reconstituted saliva to kill 40,0(O-75,000 bacteria may be quite
beneficial especially if most of the bacteria have been physically
removed by licking. In wild carnivores resting in dens or nest
areas, in which the wounds would be subjected to E. coli
contamination, the bactericidal effect of saliva against this poten-
tial pathogen may be particularly important.
The bactericidal effect of saliva on staphylococcus and P.
aeruginosa is probably biologically insignificant and this may be
one reason that staphylococcus is cultured from wounds so
frequently (from 46 percent of wounds compared with 9 and 17
percent, respectively, for S. canis and E. coil in the present study).
The ineffectiveness of saliva on staphylococcus is also reflected in
the fact that it is the most common pathogen isolated from canine
skin lesions such as superficial or deep pyoderma (25). Staphylo-
coccus was found in the previous study on rat saliva to be resistant
to the same saliva treatment that was effective in killing P.
pneumotropica and M. pulmonis (14).
Constituents of saliva that would help the wound repair process
are epithelial growth factor and nerve growth factor which are
found in saliva of rodents (23,28). Epidermal growth factor is
found in human saliva although in lower concentrations than in
rodents (9,34). Presumably, the growth factors would also be
found in carnivore saliva. Evidence that these growth factors play
a role in wound healing comes from findings that removal of
salivary glands in mice retards wound healing (16), and topical
application of epithelial growth factor (26) and nerve growth factor
(I 9) to wounds of mice, in which the salivary glands have been
removed, facilitates the closure of wounds.
The predisposition of dogs to lick their skin is carried to an
extreme in the syndrome known as acral lick dermatitis in which
they persistently and excessively lick the carpal or metacarpal area
to the point where the epidermis is damaged (25). Typically, the
epidermis on the periphery of the ulcerated lesion is hyperplastic,
possibly revealing the growth-promoting effect of epithelial growth
factor (7).
As an interesting historical note, it has been reported that in the
Middle Ages people sometimes encouraged dogs to lick their
wounds (33), perhaps in recognition of the value of the licking in
reducing bacterial contamination and accelerating healing.
ACKNOWLEDGEMENTS
This work was supported in part by Grant BRS 2 S07 RR05457 from
the National Institutes of Health. Dwight Hirsh and Lori Hansen of the
Department of Veterinary Microbiology kindly provided guidance for the
microbiological procedures and access to the bacterial repository.
REFERENCES
1. Arnold, R. R.; Brower, M.; Gauthier, J. J. Bactericidal activity of Res. 35:326-327; 1956.
human lactoferrin: Sensitivity of a variety of microorganisms. Infect. 3. Blass, E. M.; Teicher, M. H. Suckling. Science 210:15-21; 1980.
Immun. 28:893-898; 1980. 4. Bleicher, N. Behavior of the bitch during parturition. J. Am. Vet.
2. Bemarde, M. A.; Fabian, F. W.; Rosen, S.; Hoppert, C. A.; Hunt, H. Med. Assoc. 140:1076-1082; 1962.
L. A method for the collection of large quantities of rat saliva. J. Dent. 5. Bowen, W. H. Defense mechanisms in the mouth and their possible

386 HART AND POWELL
role in the prevention of dental caries. A review. J. Oral Pathol.
3:266-278; 1974.
6. Broes, A.; Drolet, R.; Jacques, M.; Fairbrother, J. M.; Johnson, W.
M. Natural infection with an attaching and effacing E. coli in a
diarrhetic puppy. Can. J. Vet. Res. 52:280-282; 1988.
7. Carpenter, G.; Cohen, S. Epidermal growth factor. Annu. Rev.
Biochem. 48:193-216; 1979.
8. Chauncey, H. H.; Lisanti, V. F. Determination of the enzyme content
of whole saliva. J. Dent. Res. 32:642; 1953.
9. Dagogo-Jack, S.; Atldnson, S.; Kendall-Taylor, P. Homologous
radioimmunoassay for epidermal growth factor in human saliva. J.
Immunoassay 6:125-136; 1985.
10. Goldsworthy, N. E.; Florey, H. Some properties of mucus with
special reference to its antibacterial functions. Br. J. Exp. Pathol.
11:192-208; 1930.
11. Greene, C. E. Clinical microbiology and infectious diseases of the dog
and cat. Philadelphia: Lea & Febiger; 1984.
12. Harkness, J. E.; Wagner, J. E. The biology and medicine of rabbits
and rodents. Philadelphia: Lea & Febiger; 1989.
13. Hart, B. L. Behavior of domestic animals. New York: Freeman Press;
1985.
14. Hart, B. L.; Korinek, E.; Brennan, P. Postcopulatory genital groom-
ing in male rats: Prevention of sexually transmitted infections.
Physiol. Behav. 41:321-325; 1987.
15. Holloway, P. J.; Williams, R. A. D. A study of the oral secretions of
rats stimulated by pilocarpine. Arch. Oral Biol. 10:237-243; 1965.
16. Hudson, J. M.; Niall, M.; Evan, D.; Fowler, R. Effect of salivary
glands on wound contraction in mice. Nature 279:793-795; 1979.
17. Jones, R. L. Special considerations for appropriate antimicrobial
therapy in neonates. Vet. Clin. North Am. [Small Anim. Pract.]
17:577--602; 1987.
18. Kesteren, M.; Van Bibby, B. G.; Berry, G. P. Studies on the
antibacterial factors of human saliva. J. Bact. 43:573-583; 1942.
19. Li, A. K.; Koroly, M. J.; Schattenkerk, M. E.; Malt, R. A.; Young,
M. Nerve growth factor: Acceleration of the rate of wound healing.
Proc. Natl. Acad. Sci. USA 77:4379-4381; 1980.
20. Mandel, I. D.; Ellison, S. A. Naturally occurring defense mechanisms
in saliva. In: Tanzer, J. M., ed. Animal models in cardiology: Proc.
symp. workshop in cardiology. Washington, DC: Information Re-
trieval; 1981:367-379.
21. Mandel, J. D. The functions of saliva. J. Dent. Res. 66:623-627;
1987.
22. Moltz, H. Of rats and infants and necrotizing enterocolitis. Perspect.
Biol. Med. 27:327-335; 1984.
23. Murphy, R. A.; Watson, A. Y.; Metz, J.; Forssmann, W. G. The
mouse submandibular gland. J. Histochem. Cytochem. 28:890-902;
1980.
24. Navia, J. M. Collection of salivary secretions in animals. In: Animal
models in dental research. Tuscaloosa, AL: University of Alabama
Press; 1977:234-237.
25. Nesbitt, G. H. Canine and feline dermatology. A systematic ap-
proach. Philadelphia: Lea & Febiger; 1983.
26. Niall, J. M.; O'Brien, B. M. The effect of epidermal growth factor on
wound healing in mice. J. Surg. Res. 33:164-169; 1982.
27. Olson, P.; Hedhammar, A.; Fads, A.; Krovacek, K.; Wadstrrm, T.
Enterotoxigenic Escherichia coli (ETEC) and Klebsiella pneumoniae
isolated from dogs with diarrhea. Vet. Microbiol. 40:577-589; 1985.
28. Petrides, P. E.; Brhlen, P.; Shively, J. E. Chemical characterization
of the two forms of epidermal growth factor in murine saliva.
Biochem. Biophys. Res. Commun. 125:218-228; 1984.
29. Pollock, J. J.; Denepitiya, L.; Mackay, B. J.; Iacono, V, J. Fungi-
static and fungicidal activity of the human parotid salivary histidine-
rich polypeptides on Candida albicans. Infect. Immun. 44:702-707;
1984.
30. Rosenblatt, J. S.; Lehrman, D. S. Maternal behavior of the laboratory
rat. In: Rheingold, H. L., ed. Maternal behavior in mammals. New
York: John Wiley and Sons; 1963:8-57.
31. Roth, L.; Rosenblatt, J. S. Changes in self-licking during pregnancy
in the rat. J. Comp. Physiol. Psychol. 63:397--400; 1967.
32. Schneirla, T. C.; Rosenblatt, J. S.; Tobach, E. Maternal behavior of
the cat. In: Rheingold, H. S., ed. Maternal behavior of mammals.
New York: John Wiley and Sons; 1963:122-168.
33. Serpell, J. In the company of animals. New York: Basil Blackwell;
1986.
34. Starkey, R. H.; Orth, D. W. Radioimmunoassay of human epidermal
growth factor (urogastrone). J. Clin. Endocrinol. Metab. 45:1144-
1153; 1977.
35. Tenovuo, J.; Mansson-Rahemtulla, B.; Pruitt, K. M.; Arnold, R.
Inhibition of dental plaque acid production by the salivary lactoper-
oxidase system. Infect. Immun. 34:208-219; 1981.
36. Tenovuo, J.; Moldoveanu, F.; Mestecky, J.; Pruitt, K. M.; Ra-
hemtulla, B. M. Interaction of specific and innate factors of immu-
nity: IgA enhances the antimicrobial effect of the lactoperoxidase
system against Streptococcus mutans. J. Immunol. 128:726-731;
1982.
37. Thompson, R. Lysozyme and its relation to the antibacterial properties
of various tissues and secretions. Arch. Pathol. 30:1096-1134; 1940.
38. Thompson, R. Certain antibacterial properties of saliva and tears not
due to lysozyme. J. Bacteriol. 41:77; 1941.
39. Wasteson, Y.; Olsvik, O.; Skanacke, E.; Bopp, C. A.; Fossum, K.
Heat-stable-enterotoxin-producing Escherichia coli strains isolated
from dogs. J. Clin. Microbiol. 26:2564-2566; 1988.
40. Zimmer, J. F.; Pollock, R. V. H. Esophageal, gastric and intestinal
disorders of young dogs and cats. Vet. Clin. North Am. [Small Anim.
Pract.] 17:641-661; 1987.