ENHANCED RESPONSES IN ANTIGEN PRIMED DOGS AFTER THE ADMINISTRATION OF A COMPLEX MICROBIAL IMMUNOSTIMULATING COMPOUND

Florinel BRUDASCA, Carmen Dana SANDRU, Constantin CERBU,
Aurel VASIU, Marina SPINU

University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Abstract. The research aimed to investigate the efficacy of antiparvoviral monovalent vaccine for puppies
treated with a complex poly-bacterial compound. The experiment was carried out on 17 Labrador Retriever
puppies, from 41 to 63 days of age. The two groups (experimental and control) were subjected to: a) a polybacterial
preparation and antiparvoviral vaccine (n = 12) and b) to antiparvoviral vaccine (n = 5), respectively.
One ml of a poly-bacterial preparation was administered on days 0, 2, 4, 6, while the antiparvoviral vaccine
Duramune KF11 was injected on day 8 of the experiment. Phagocytic activity and total lysozyme concentrations
were measured on days 0, 8 and 22 of the experiment. The global immunostimulating effects of the poly-bacterial
preparation on the innate immune system of vaccinated puppies increased when compared to those of the
control variant.
Key words: Puppies; Vaccine; Poly-bacterial preparation; Immune indicators; Phagocytosis; Lysozyme.

INTRODUCTION

Immune modulating products enhance the immune responses to antigen priming as well as the
protective capacity of the organism against diseases and stress in both farmed and companion animal
species. The most proficient vaccines fail to induce immunity against microbes in case of an
immunologically week host. Immunostimulating compounds not only increase the immune response,
but also facilitate the interaction of the immune effectors in expressing an appropriate reaction (Roitt,
I. 1991; Sigal, L.H., Ron, Y. 1994; Stites, P.D., Terr, A.I. 1991).
The canine parvovirus infection is one of the most important diseases in puppies brought up in
kennels, sometimes in spite of the previous vaccinations of the mother or the involved individuals.
Optimizing the immunization protocols represents an aim in order to eradicate the disease and maintain
healthy the kennel’s sanitary status.
The investigations were carried out to establish the possible positive influence of using a polybacterial
immunostimulating compound on the results of antiparvoviral vaccination, by monitoring humoral
and cell-mediated innate immune functions.
MATERIALS AND METHODS
The poly-bacterial preparation tested in this experiment, commercialized for its immune modulating
capacity, consisted of a mixture of 13 Gram-positive and Gram-negative suspended rods and cocci,
inactivated by heat, partially lysed by bile (1%), diluted in saline (0.875%). The standard concentration
of this mixture was of 48 million inactivated germs/ml of the product. The active composition consisted
of: Staphylococcus aureus 6 million/ml, Streptococcus pyogenes 3 million/ml, Streptococcus viridans
3 million/ml, Streptococcus faecalis 11 million/ml, Streptococcus pneumoniae 6 million/ml, Escherichia
coli 10 million/ml, Klebsiella pneumoniae 6 million/ml, Neisseria catarrhalis 3 million/ml, preserver
phenol (max 0.25%). It was included into the pharmaceutical group of immune modulators.
Experimental design. Labrador Retriever puppies, aged of 41 days (n=17) were divided in two
groups as follows:
a) group I, poly-bacterial preparation treated, vaccinated, n = 12;
b) group II, untreated, vaccinated, n = 5.
The animals were fed and watered ad libitum, under the same maintenance conditions. The
investigations were carried out till the animals reached the age of 63 days.
The experimental protocol included for group I, unlike for group II, intramuscular poly-bacterial
preparation injections (1 ml) on days 0, 2, 4, 6 after the beginning of the trial (aged of 41, 43, 45 and 47
days). Both groups were subjected to administration of a modified live antiparvoviral vaccine, according
to the instructions, on day 8 of the experiment (49 days of age) and blood samplings on days 0, 8 and
22 (41, 49 and 63 days of age). Blood was taken from the brachial vein, 0.5 ml for heparin (50 IU/ml)
to monitor the phagocytosis and 1.5 ml for serum, on special clotting gel, to monitor lysozyme activity.
The experimental protocol was drawn in table 1.
Phagocytic activity. The carbon particle clearance test was used to monitor the in vitro nonspecific
cell mediated activity, reducing the blood amounts to minimum aliquots necessary to perform
the test (micromethod) (Ghergariu, S. et al. 2000; Rapuntean, Gh. et al. 1994; Dumitru, C. et al. 1996).
For that purpose, 0.5 ml aliquots of heparinized blood were co-incubated at 370C with 1.5 ml of a
supernatant of India ink, in sterile tubes. At 0, 30 and 60 minutes of incubation, 150 ml of the mixture
were transferred to 3 ml of saline. Finally, all the tubes containing saline + blood + India ink were
centrifuged 10 minutes at 1500 rpm.

Supernatant optical densities, rendering 0,20 and 40 minutes of incubation for each sample, were
red in 96 well plates, using a multichannel spectrophotometer (SUMAL PE2, Karl Zeiss, Jena), at 535
nm wavelength (d=0.5 cm). Phagocytosis was estimated as the reciprocal of the regression slope of
the optical densities that were read (Vasiu, C. et al. 1995). Phagocytic activity index was calculated as
the difference between the natural logarithms for the 0-20·min and 20-40·min intervals.
The agar gel diffusion test against Micrococcus lysodeicticus was used to assess the lytic capacity
of serum lysozyme in the puppies during the experiment (Ghergariu, S. et al. 2000; Rapuntean, Gh. et
al. 1994). A standard curve built with pure lysozyme (Sigma Aldrich) was used to quantify the amounts
of lysozyme in the puppy sera in micrograms/ml.
Mean values, standard deviations and statistical significance of the differences were obtained using
the Excel program.

RESULTS AND DISCUSSIONS

Phagocytic function is one of the most important mechanisms within the “first line of defense”
against microbes, being performed by neutrophils as small phagocytes, and also by monocytes, as
macrophages (Stites, P.D. and Terr, A.I. 1991). Their activation is being triggered by the presence of
particulate antigens, which are subsequently engulfed and destroyed. The oxidative burst plays an
important role in this process (Sigal, L.H., Ron, Y. 1994).
The nervous system, due to the presence of specific receptors on the cells of the immune system,
can respond to the secretion of neurohormones. Thus, the nervous system exerts a modulatory action
upon the immune response. This could happen by a direct hypothalamic or cortico-hypothalamic
stimulation, where bacteria or bacterial products could intervene, by neural and humoral pathways and
also by hypothalamus-pituitary-adrenal axis. The immune response triggering and enhancement by the
nervous system are essential, since it could favorably influence the outcome of infectious diseases
(Baciu, I. 1992; Baciu, I. et al. 2003).
Age is an important factor influencing the non-specific immunity. In an experiment aimed at monitoring
the neutrophil level in puppies on the first day of their life, it was indicated that it was about three times
higher than the one of lymphocytes. There was a change in the proportion of these leukocyte populations,
with a decrease of neutrophils and increase of lymphocytes, the latter becoming dominant as numbers.
By the end of the first month of life, neutrophils level could be compared to the one in adults. The
phagocytic activity of neutrophils for the same period was somewhat, but non-significantly higher, in
puppies. The experiment indicated that the puppies with very low levels of maternal antibodies developed
by the age of two weeks a specific immune response to a parvovirus antigen. This development was
continuous, differing from the one of the adults till at least 3 months of age (Toman, M. et al. 2002).
Attempts to stimulate the phagocytosis along with the antibody response and cell mediated adaptive
immunity were done. The use of glucan jointly with anti-parvovirus vaccination led to significantly
increased phagocytic ability, metabolic and chemotactic activity of polymorphonuclear leukocytes and
also improved the blastogenic response of lymphocytes. Similarly, the antibodies against canine parvovirus
infection were statistically significantly increased by day 28 after vaccine and glucan administration,
as compared to control group (Haladová, E. et al. 2011).
Bacteria were also used to stimulate the immune system. A diet supplemented with 5 × 108 colony
forming units (cfu)/d of probiotic Enterococcus faecium (SF68) from weaning to 1 y of age, led to
higher fecal IgA and distemper vaccine–specific circulating IgG and IgA. The percentage of mature
B cells was also increased in puppies fed with the probiotic when compared to the control group
(Benyacoub, J. et al. 2003).
Other treatments, such as chemotherapy, could change the phagocytic function, impeding the immune
response against bacteria, in the case of chronic disease. Thus, induction chemotherapy diminished the
percentage of neutrophils capable of oxidative burst in dogs with lymphoma, but phagocytosis improved
over time (LeBlanc, A.K. et al. 2015).
Phagocytic activity was presented for the stimulated group in figure 1 and for the control group in
figure 2. Average phagocytic indices were presented for the two periods, from 0 to 20 and from 20 to
40 min for each sampling.

As shown, the phagocytic activity is in the positive field, with an increase towards the end of the
experiment in the stimulated group, as opposed to the control group, where the majority of the indices
are negative. The enhanced phagocytic activity of the poly-bacterial preparation was obvious. The
comparison for the first reading period, third sampling, between the two groups indicated a high
statistically significant difference (p<0.001). The differences for the first and second sampling, second
period (20 to 40 min) were also statistically supported (p<0.05-0.01). Although the puppies at birth can
respond to external stimuli, by augmenting the non-specific cell mediated activity, the poly-bacterial
preparation could be of help in increasing the post-vaccinal immunity and lowering the caseload in
parvovirus infection (Haladová, E. et al. 2011).
The lysozyme (N-acetyl-muramyl-hydrolase) is involved in the lysis of the bacterial wall especially
in Gram positive cocci (Sigal, L.H., Ron, Y. 1994). Alterations of lysozyme concentrations were found
during the development of infectious diseases (Babíčková, K. et al. 2009). Figure 3 presents the mean
values of the lysozyme concentrations in the experimental groups. The value obtained at the first
sampling was the highest and decreased towards the second sampling in the immune stimulated group,
with no influence on the health status of animals. There was a second increase at the third sampling.
Compared to the experimental group, the increase in lysozyme concentrations was gradual by the end
of the experiment, but the levels were lower than in the control group. The differences in the dynamics
of lysozyme concentrations were not supported statistically, except for the first sampling. There were
no significant differences at the end of the experiment between the groups.

One of the purposes of poly-bacterial preparation treatment preceding vaccination in puppies was
to increase the basic non-specific immune response, in order to enhance protection against parvovirus
infection. Most probably, the enzyme levels decreased since it was sensitive to the manipulation/
vaccination stresses, and these exerted a more pronounced influence than the antigen priming. The
initial increased value could be the result of a different microbism of the environment, with a heavier
load in the experimental group. Since the poly-bacterial preparation treatment did not significantly
influence this parameter, probably there was a different pathway and other mechanisms were involved
in ensuring the health of animals.

CONCLUSIONS

1. Phagocytosis was positively influenced by the poly-bacterial preparation treatment and antigen
priming, suggesting an increase in the effectiveness of cell mediated non-specific immunity and a
possible use of the procedure to improve vaccinal status of the puppies.
2. Lysozyme concentrations were negatively influenced by the combined poly-bacterial preparation
and vaccination procedure, indicating supplementary pathways in the increased resistance against
parvovirus infection.

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Data prezentării articolului: 23.04.2017
Data acceptării articolului: 17.05.2017