mastivac i: staphylococcus aureus vaccine

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ISRAEL JOURNAL OF
VETERINARY MEDICINE
MASTIVAC I: STAPHYLOCOCCUS AUREUS
VACCINE - PREVENTION OF NEW UDDER
INFECTION AND THERAPEUTIC EFFECT ON
COWS CHRONICALLY INFECTED WITH S.
AUREUS UNDER FIELD CONDITIONS
Vol.
2003
59
(4) Leitner G. 1,* Krifucks O.1 , Glickman A.1, Vaadia Y.3, Friedman S.4,
Ezra E.5, Saran A.1, and Trainin Z.2
1. National Mastitis Reference Center
2. Immunology Dept, Kimron Veterinary Institute, PO Box 12, Bet
Dagan 50250, Israel
3. Clinical veterinarian
4. Mastitis Laboratory, National Udder Health Service, Caesarea, Israel
5. Israel Cattle Breeders Association, Caesarea, Israel
* Corresponding author: Gabriel Leitner. National Mastitis Reference
Center, Kimron Veterinary Institute, PO Box 12, 50250 Bet Dagan, Israel
Abstract
Previously we described the development of a new Staphylococcus aureus
vaccine denoted MASTIVAC I. The present study describes an attempt to
reduce S. aureus udder infection by MASTIVAC I vaccination in both
infected and uninfected animals in a herd of 267 multiparous IsraeliHolstein cows, of which 22.1% were chronically infected with S. aureus.
Four groups were comprised: 1) 137 S. aureus infection-free cows and
heifers that were exposed to vaccination; 2) 135 S. aureus infection-free
cows and heifers that were not vaccinated and served as a control group; 3)
29 S. aureus-infected cows that were exposed to vaccination; and 4) 30 S.
aureus-infected cows that were exposed to a placebo. During the 348 d of
the experiment only 2/137 (1.46%) immunized cows in group 1 were found
to be infected with S. aureus, compared with 14/135 (10.4%) cows in group
2, this difference in the new infection rate (NIR) was found significant (P <
0.05). Twenty out of 29 cows (69%) in group 3 remained in the herd,
compared with only 14 out of 30 cows (46.7 %) in group 4. Of the
remaining cows, six of 20 cows (30%) in the immunized group (group 3)
were found to be infection-free, compared with one out of 14 cows (6.25%)
in the placebo group (group 4). However, this difference in the cure rate
(CR) was not significant. These results strengthen findings elicited in
previous experiments indicating that MASTIVACS I vaccine reduced the
number of S. aureus infected animals and exerted positive effects on both
the quantity and the quality of the milk production, by eliciting specific
protection as well as non-specific udder health improvement. Moreover, this
treatment helps to eliminate the infection from S. aureus-infected cows and
reduces their somatic cell count.
(Key words: mastitis, Staphylococcus aureus, vaccine)
Introduction
Staphylococcus aureus is still one of the major pathogenic agents causing
mastitis worldwide and therefore necessitates the use of antibiotics in dairy
herds. The increasing public concern with food safety, expressed in this
context in the desire to minimize antibiotic residues in milk, on the one hand,
and the need to reduce somatic cell counts on the other hand, strengthen our
determination to combat S. aureus mastitis by means of vaccination.
MASTIVAC I, is a new vaccine (Patent no. IL122829, PTC/IL 98/00627, AU
746285, USA09/582692) developed for this purpose. The vaccine is
composed of three field strains of S. aureus, which exhibit a broad spectrum
of antigenic and immunogenic properties (1). In controlled experiments, the
vaccine was found effective against challenge with a virulent field strain of S.
aureus (2); 70% of the S. aureus-inoculated quarters (11/17 quarters) in nine
Israeli-Holstein cows were free of infection compared with less than 10%
(2/21 quarters) in the 10 control cows. A large-scale vaccination field trial
extended over two consecutive years and involved 452 (vaccinated and
control) Israeli-Holstein heifers in seven dairy farms. The vaccinated cows in
first and second lactation had SCC that were over 40% lower than those of
the control cows, and they yielded 0.5 kg/d more milk (3) .
The present paper describes an attempt to reduce S. aureus udder infection
by MASTIVAC I vaccination of both infected and uninfected animals in a herd
of 267 multiparous Israeli-Holstein cows, of which 22.1% were chronically
infected with S. aureus mastitis.
Materials and Methods
Animals
A total of 267 multiparous Israeli-Holstein cows from one herd, at various
times during milking or during pregnancy, and with various levels of udder
infection were included in this study. In addition, 70 heifers that were more
than 100 d in pregnancy and not less than 30 d before parturition were
included. S. aureus was isolated from 59 cows (22.1%). All cows and heifers
were bled for the detection of specific antibodies to S. aureus, and cows
whose udder S. aureus infections did not match their antibody levels were
tested again. Cows assigned to pairs according toS. aureus udder infection,
S. aureus-specific antibody, lactation number, time in milking, pregnancy
status, milking group in the herd, and SCC in the last 2 mo before the start of
the experiment. Heifers that matched in time of pregnancy and S. aureusspecific antibody level were assigned to pairs; they were considered to be free
of S. aureus infection. Pairs of S. aureus infection-free cows and heifers were
then assigned to two groups, and the S. aureus-infected ones were assigned
to another two groups. The various groups comprised: 1) 137 S. aureus
infection-free cows and heifers that were exposed to vaccination; 2) 135 S.
aureus infection-free cows and heifers that were not vaccinated and served as
a control group; 3) 29 S. aureus-infected cows that were exposed to
vaccination; and 4) 30 S. aureus-infected cows that were exposed to a
placebo. Cows and heifers in groups 1 and 3 were immunized with
MASTIVAC I (as described in Vaccine and Vaccination) and re-immunized
(boosted) 49 d later. Cows in group 4 were administered placebo according to
the same schedule as the immunizations. All animals were bled at 49, 92 and
348 d after the first immunization, for antibody determination. Milk was
sampled individually for bacteriological examination at drying-off, after
parturition, upon occurrence of clinical manifestation (of mastitis), or upon
observation of an increase in SCC. The milk of all lactating cows was sampled
at 244 d after the first immunization. Milk yield and composition, including
SCC, were recorded monthly (Israeli Cattle Breeders Association, Caesarea).
Cows were milked three times daily in two different milking parlors: a
herringbone and a robot parlor (during the experiment all cows were
transferred to the robot parlor). Food was offered in mangers located in freestall barns. During the study some cows were removed from the herd for
management reasons. At the end of the experiment the numbers of cows
remaining in groups 1, 2, 3 and 4 were 104, 104, 20 and 14, respectively.
Vaccine
and
Vaccination
MASTIVAC I (VIREO, Biological Laboratories, POB. 300, Or-Akiva 306000,
Israel) S. aureus vaccine comprises derivatives of three field strains of S.
aureus - exosecretion of VLVL8407, and bacterial fragments of ZO3984 and
BS449 - in the ratio of 1:1:1, with the addition (ratio of 1:1) of the 206 ISA
(SEPPIC, d’Orsay, Paris, France) adjuvant. The vaccine has been fully
described in patents, # IL122829, PTC/IL 98/00627, AU 746285,
USA09/582692, and in a previous manuscript (1). Placebo included PBS with
adjuvant in the same ratio. Dose for immunization was 2ml, administered
subcutaneously
under
the
tail
root.
Bacteriological
Examinations
Quarter foremilk samples were taken aseptically according to the International
Dairy Federation (IDF) procedures (4) and were submitted to the laboratory
within 1 h. Bacteriological analysis was performed according to accepted
standards (5). A 0.01-ml aliquot from each milk sample was spread over
blood-agar plates (Bacto-Agar; Difco Laboratory, USA) containing 5% sheep
red blood cells. All plates were incubated at 37oC and examined for growth at
18 and 42 h. Colonies suspected to be staphylococci were tested for
coagulase production {(tube test)Anilab, Rehovot, Israel} and strains were
identified with the ID 32 Staph Kit (Bio Merieux S.A., 69280 Marcy-l’Etoile,
France).
Milk
and
Somatic
Cell
Count
Milk and SCC were determined with the Fossomatic 360 instrument at the
Central Milk Control Laboratory of the Israeli Cattle Breeders’ Association.
Immunological
Assay
ELISA. The assay was performed as previously described (6), with VLVL8407
S. aureus as the antigen. Positive and negative serum standards were added
to each plate. The optical density of each tested sample was normalized by
linear
regression
in
order
to
calculate
antibody
levels.
Statistical
Analysis
Milk quantity and SCC data were statistically analyzed with SAS Software
Release 8.2, 2002 (7) (SAS Inst., Inc., Cary, NC). Dependent variables were
milk (kg/d), SCC and Log SCC. The independent variables - treatment,
lactation number, month, cow, and days in milk - were examined according to
the
model:
Yijklm = u + Ti + Hj + Lk + Sl + dimijklm + eijklm
in which Y - dependent variable; U - overall mean; T - treatment; i = 1, 2; H group j = 1,2 or 3,4.; L - lactation number, k = 1,2,3+; M - month in lactation, l
= 1,..12; dim - days in milk is a continue variable; e - error. New infection rate
(NIR) and cured rate (CR) were examined by chi-square analysis.
Results
Safety
During the entire period of the experiment, none of the cows showed any
abnormal symptoms of sensitivity to the vaccine except for local swelling at
the inoculation area that lasted for up to 10 d after vaccination. No difference
could be detected in calf survival rate between the immunized and the control
cows.
S. aureus new infection and recovery
All cows and heifers in groups 1 and 2 were free of S. aureus infection
before immunization. During the 348 d of the experiment only 2/137 (1.46%)
immunized cows in group 1 were found to be infected with S. aureus,
compared with 14/135 (10.4%) cows in group 2 (control) (Table 1). This
difference in the NIR was significant (P < 0.05). Cows in groups 3 and 4 were
all S. aureus infected before immunization. Shortly after immunization, cows
of both groups were removed from the herd, for management reasons, with
no new information having been obtained on their udder status. Therefore CR
was determined twice: the first time according to the number of infection-free
cows out of the total number at the beginning; and the second time according
to the number of infection-free cows out of those remaining at day 348 after
immunization. Considering that all the cows that were removed were S.
aureus positive, the difference was not significant. While with the remaining
20 cows in the immunized group (group 3), six (30%) were found to be
infection-free, compared with one out of 14 cows (6.25%) in the placebo
group (group 4)(Table 2). Moreover, 20 out of 29 cows (69%) in group 3
remained in the herd, compared with only 14 out of 30 cows (46.7 %) in group
4.
Table 1. New S. aureus infection rate of udders of cows and heifers initially
free of S. aureus infection which were immunized with MASTIVAC I (group 1)
or remained unvaccinated (group 2), during 348 d after immunization.
Table 2. Numbers of cows at the beginning (start) and 348 d after immunization
(end), and S. aureus-infection curing rate in udders of cows immunized with
MASTIVAC I (group 3) or with placebo (group 4), during 348 d after immunization.
Group
Number of cows
Cured of infection1(%)
End/Start
(Remaining cows)
Pr > F
3 (vaccinated) 20/29 (69.0 %) 30.0 (6/20)
NS
4 (placebo)
NS
14/30 (46.7 %) 6.25 (1/14)
1 Analyses of CR were done on the cows which remained 348 d after immunization.
Antibody response
Before immunization, no antibodies to S. aureus could be detected in the cows and
heifers of groups 1 and 2 (Table 3). At the first testing (49 d after immunization), over
90 % of the immunized animals (group 1) were positive, with a mean antibody level
of 0.81 ± 0.06. After the secondary immunization, antibody levels increased to 1.07 ±
0.07 at day 92 and then decreased to 0.36 ± 0.05 at day 348 after the first
immunization. At the last examination, S. aureus antibodies could be detected in 65%
of the animals of group 1. In group 2, at day 348, S. aureus antibodies were detected
in 17% of the cows, i.e., most of them (82.4%) exhibited a new S. aureus udder
infection. Before immunization, 76.6% (44/59) of the S. aureus-infected cows (groups
3 and 4), had detectable S. aureus-specific antibody levels, whereas in the remaining
15 of them 59 cows, antibody could not be detected. After immunization, all the cows
in the vaccinated group were found to be positive, with increased antibody levels. In
the placebo group, the individual antibody levels fluctuated but few of the originally
antibody-negative cows had become antibody-positive.
Somatic
cell
counts
and
milk
yields
The SCCs and milk yields (Israeli Herd Book data) for the 348 d after
immunization were analyzed (Table 4). Immunized cows and heifers in group 1 had
marginally lower value of SCC than the controls (group 2): 310 ± 19 x 103 cells/ml
compared with 324 ± 21 x 103 cells/ml. There was also a minimal, not significant
difference between their milk yields, with the immunized cows and heifers in group 1
producing 0.12 kg/d more milk than those in group 2. The immunized cows in group
3 had significantly (P < 0.001) lower SCCs than the placebo-treated animals in group
4: 905 ± 88 × 103 and 1335 ± 119 × 103 cells/ml, respectively. The animals in group
4 showed an advantage in milk yield over those in group 3: 0.12 kg/d (Table 4)
however
the
difference
was
not
significant.
Table 3. Antibody levels; means and standard errors, of S. aureus infection-free cows
and heifers, which were immunized with MASTIVAC I (group 1) or remained
untreated (group 2), and of S. aureus-infected cows which were treated with
MASTIVAC I (group 3) or with placebo (group 4), at 0, 49, 92 and 348 d after first
immunization.
Group
1
S. aureus infection
Antibody levels 1 (Days after first immunization)
Start
End 1
0
49
92
348
-
-
0.04±0.01
0.81±0.06
1.07±0.07
0.36±0.05
+ (2)
0
0.75±0.01
0.67±0.03
NT
(Vaccinated) 2
-
-
0.03±0.01
0
0
0.10±0.01
(Control)
-
+ (14)
0
0.25±0.25
0.56±0.24
1.03±0.19
3
+
- (6)
0.28±0.01
0.99±0.21
1.48±0.41
1.26±0.71
+
0.94±0.17
1.95±0.15
2.48±0.25
1.17±0.13
(Vaccinated) +
4
+
- (1)
0
0
0
0
(Placebo)
+
+
1.36±0.22
1.61±0.20
2.08±0.28
1.69±0.13
1 End = 348 d after first immunization
NT - not tested.
Table 4. Means and standard errors of SCC and daily milk yield (kg) during 348 d
after immunization of S. aureus infection-free cows and heifers which were
immunized with MASTIVAC I (group 1) or remained untreated (group 2), and of S.
aureus-infected cows which were treated with MASTIVAC I (group 3) or with
placebo (group 4).
Group
SCC x 1000 (ml/milk)
Milk (kg/d)
1 (Vaccinated)
310±19
31.11±0.28
2 (Control)
324±21
30.99±0.27
3 (Vaccinated)
905±88
26.91±0.71
4 (Placebo)
1335±119
27.03±0.59
Pr > F (1; 2)
NS
NS
Pr > F (3; 4)
0.001
NS
NS - non-significant.
Discussion
The present study aimed to elucidate whether MASTIVAC I vaccination of both
infected and uninfected animals in a herd of 267 multiparous Israeli-Holstein cows, of
which 22.1% were chronically infected with S. aureus mastitis is effective.
Vaccination with MASTIVAC I against S. aureus mastitis did not affect pregnancy,
and no difference was observed between vaccinated and control heifers, in the
numbers of healthy calves delivered (data not shown). These results are consistent
with those of our previous large-scale vaccination field trial, which extended over two
consecutive years and involved 452 (vaccinated and control) Israeli-Holstein heifers
in seven dairy farms (3). Moreover, no general cytotoxic or local tissue damage was
observed except for local swelling at the inoculation area that lasted for up to 10 d
following administration of the vaccine. Thus the vaccine appears to be safe for
vaccination of pregnant cows and heifers. The most conspicuous aspect of the
efficacy of MASTIVAC I was the significant protection against new S. aureus
infection of the cows and heifers that were free of S. aureus udder infection at the
time of vaccination (1.5% and 10.4% respectively). In the previous large-scale
vaccination field trial (3), the specific protection against S. aureus infection imparted
by the vaccine could not be analyzed, because of the small number of first-lactating
heifers and second-lactating cows infected with S. aureus. The direct involvement of
the specific antibodies in the prevention of udder infection was not demonstrated in
the present study, but it was shown in a previous one (8) that immunoglobulin of IgG,
especially IgG1, are transferred from the blood into the milk regardless the presence
or absence of the infecting agent in the quarters. IgG is the principal immunoglobulin
of the mammary gland immune system, responsible for promoting neutrophil
phagocytosis (9-11), therefore, high levels of antibodies in the blood stimulate the
transfer of antibodies to the milk, to combat new infections.
The number and the distribution of the somatic cells in the milk represent a major
indicator of udder health: the higher the cell count, the poorer the udder health. It was
shown previously (1) that the SCC in an uninfected mammary gland is lower than
50x103 cells/ml, and that the presence of most contaminating bacteria, including
coagulase-negative staphylococci (CNS), in an udder quarter (12) causes the SCC to
increase to 105-106 cells/ml. Moreover, contaminants in one quarter have no
influence on the SCCs in the other quarters of the same cow; therefore, the total SCC
in the milk indicates the health status of the udder as whole. In the present study, the
overall SCCs during the entire period of the experiment (348 d), regardless of the
species of bacterial infection, were lower in the vaccinated cows and heifers than in
the control animals, though the difference was small. The milk yield was marginally
greater in vaccinated cows than in the control animals, and, although the differences
in SCC and in milk production were not significant in the present study; they were
consistent with the results of our previous large-scale vaccination field trial (3). This
large-scale trial showed that, despite the differing local conditions among the seven
herds studied, the immunized cows had significantly better udder health than the
control cows, as indicated by significantly lower SCCs and higher milk yields (0.5
kg/d) in each herd when considered separately as well as for the total of 452 animals
tested.
One may ask whether immunization with MASTIVAC I vaccine during lactation,
of cows already infected with S. aureus and/or CNS, would increase their SCC,
because of the local immune reaction to the agent that was already present. However,
no increase in SCC was recorded in the 29 infected with S. aureus at the time of
immunization, which seems to indicate that the vaccine is safe in this respect, for
administration to cows during lactation. The high level of antibodies in most of the S.
aureus-infected cows before vaccination and the fact that these cows were mostly
among the older members of the herd suggest that in most cases the infection was
long-established. Some cows were removed from the herd because of management
decisions, regardless of their immunization status, with the result that, at the end of
the experiment, 69% of the 29 initially S. aureus-infected cows that were immunized
remained in the herd, compared with 46.7% of the 30 placebo cows. Moreover, six of
the 20 immunized cows, which remained to the end of the experiment, were found
free from S. aureus in comparison with only one out of the 14 remaining placebotreated cows. Also, since one of the parameters for allocating an animal to
immunization or placebo treatment was the mean SCC over the 2 mo before the
experiment began, the significant difference in SCC between the vaccinated and
placebo-treated infected cows that developed in the course of the experiment could be
attributed to the elimination of S. aureus from the vaccinated cows. These finding
indicate that immunization of already infected animals with MASTIVAC I vaccine
cured their udder infection, probably by circumventing the immune tolerance caused
by the chronic S. aureus infection. Thus, MASTIVAC I can be regarded as a
therapeutic as well as a preventive treatment.
In conclusion, MASTIVAC I vaccine reduced the number of animals that became
infected with S. aureus, and exerted positive effects on both the quantity and the
quality of the milk production, by eliciting specific protection as well as non-specific
udder health improvement. Moreover, this treatment helps to eliminate the infection
from
already S.
aureus-infected
cows
and
reduces
their
SCC.
LINKS TO OTHER ARTICLES IN THIS ISSUE
References
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