# Brucella abortus Strain RB51 Vaccine:  Immune Response after Calfhood Vaccination  and   Field Investigation in Italian Cattle Population

Authors: Manuela Tittarelli; Barbara Bonfini; Fabrizio De Massis; Armando Giovannini; Mauro Di Ventura; Donatella Nannini; Vincenzo Caporale
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Immune response toBrucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%–100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated  animals.


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## Body

## 1. Introduction

The
objective of brucellosis eradication was introduced in the Italian legislation
in year 1994
[1], thus forbidding the
vaccination of cattle against this disease. Brucella abortus strain 19 vaccine,
in smooth phase, induces the production of antibodies that are detectable with
the official tests and are not distinguishable from those found in infected
animals. A similar problem is present with the B. abortus strain 45/20
vaccine, even though this strain is in rough phase [2].
B. abortus strain RB51 vaccine (RB51), rough mutant of the virulent strain B.
abortus 2308, does not lead to the production of antibodies that can be
detected using the conventional serological tests [3, 4]. Given the possibility of an illegal use of RB51, this
characteristic may represent a serious interference in the national eradication
plan and may seriously harm the achievement of officially Brucellosis-free (OBF)
qualification for herds and territories, as well as the already gained
qualifications. Therefore, it has been considered necessary to provide
diagnostic tools able to guarantee an effective surveillance. Available
literature reports the possibility to detect specific RB51 antibodies with a complement
fixation test (CFT) using RB51 as antigen ([RB51-CFT]; [5, 6]). Nevertheless, the kinetics of the specific RB51
immune response in cattle after calfhood vaccination, thus the probability of
identifying an illegal vaccination carried out on those animals, have never been
investigated. Moreover, the RB51-CFT has never been evaluated on national
cattle population. Thus, the aims of the present study are to identify the
antibody kinetics of RB51-CFT in cattle vaccinated with RB51 at calfhood, to
evaluate the RB51-CFT on Italian cattle population, and to carry out a
preliminary study on the possibility that RB51 vaccine has been used in this
population.


## 2. Materials and Methods

### 2.1. Vaccine

The
RB51 vaccine was kindly provided by CZ Veterinaria (Pontevedra,
Spain), the European
distributor of the product, under license from the Colorado Serum Company (Denver, Colo,
USA). Once
reconstituted, the vaccine contained5×109 CFU/mL of B. abortus strain RB51.


### 2.2. Animals and Vaccination

Fifteen
Friesian calves, aged between four to six months and obtained from OBF herds,
were randomly selected and divided into two groups. One group (n=10) was
vaccinated subcutaneously with RB51 in accordance with the instructions of the
manufacturers (2 mL reconstituted solution, containing 10×109 colony-forming
units (CFUs)). The other group (n=5) was used as nonvaccinated con-trol and the
animals were inoculated subcutaneously with 2 mL sterile saline solution. All
the animals were stabled with adequate space and fed with a standard diet (hay
and nutritional supplements) for the entire duration of the experiment; all
stages were conducted with consideration for their welfare and all procedures
with animals were carried out in accordance to appropriate humane methods.


### 2.3. Serological Testing and Antigens

All animals were tested for anti-B. abortus and
anti-RB51 antibodies before vaccination, on the day of vaccination (day zero)
and then periodically until Day 300 postvaccination (pv). Anti-B. abortus antibodies
were verified using the Rose Bengal test (RBT) and CFT, both performed using
the B. abortus biovar 1 strain 99 as antigen (VLA Weybridge, UK) and
according to the methods described in the 5th edition of the OIE Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals [7]. Anti-RB51
antibodies were monitored with an RB51 antigen-specific CFT (RB51-CFT). The antigen was
prepared from the same RB51 strain used for vaccination, after growth in
Brucella agar added with 5% bovine foetal serum. The antigen used in the
RB51-CFT was crosstitrated with a positive serum
coming from a
heifer vaccinated with RB51, as described in literature [5, 6].


### 2.4. Field Investigation

The
field investigation covered the 2001–2004 period and was divided in two steps.
For the 2001–2003 period, the investigation was carried out on sera coming from
the National Serum Bank (NSB) placed in the Istituto Zooprofilattico
Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZS A&M). The NSB
collects and stores sera coming from cattle used as sentinel in the framework
of the National Bluetongue Surveillance System (NBSS). Sentinels are bled
regularly with variable frequency, depending on the season and bluetongue
infection rate in the area concerned [8]. The herds to be
tested were randomly selected from the sentinel herds that were present in the
12 regions in which the NBSS was in force during the period considered. Sera of
all animals present in the sampled herds were tested with RB51-CFT (22,416 sera
analysed from 1,948 herds, Table 1). For year 2004, sera collected by Local
Veterinary Services in the framework of the National Brucellosis Eradication
Plan (NBEP) were analysed by the local Istituti Zooprofilattici Sperimentali
(IZS), Italian National Veterinary Laboratories. Sera were sampled according to
a systematic sampling method, with a sampling interval (intended as the number
of samples in between the sample selected and the next to be selected)
calculated for each region on the basis of the number of samples tested for the
NBEP in the year 2002. The number of serum samples expected from each region
was set to 300, but not all the regions were able to reach the objective. All
sera were analysed with RB51-CFT (4,559 sera analysed from 928 herds, Table 2).
Moreover, NSB serum, coming from Piedmont region (2,489 sera), NBEP sera from Veneto region (300 sera), Friuli-Venezia
Giulia region (185 sera), and the autonomous provinces of Trento
and Bolzano
(300 sera), were used to calculate the threshold of RB51-CFT (3,274 sera
analysed in total).Table 1
Number of sera from the National Serum Bank (NSB, collected during the
2001–2003 period) and tested with RB51-CFT, on regional basis.
Region of originNo. of sera testedNo. of herds testedAbruzzo2,123177Apulia1,836207Basilicata1,957222Calabria1,064111Campania1,062117Liguria2,552193Marche2,825238Molise2,584217Piedmont2,489139Sardinia1,282110Sicily1,446114Umbria1,196103Total22,4161,948Table 2
Number of sera from the National Brucellosis Eradication Program (NBEP,
collected during year 2004) and tested with RB51-CFT, on regional basis.
Region of originNo. of sera testedNo. of herds testedApulia30261Basilicata30059Calabria26654Campania30059Emilia-Romagna30060Friuli-Venezia Giulia18538Liguria25155Lombardy30060Marche31579Piedmont30061Sardinia30062Sicily30057Trentino-Alto Adige30061Umbria24046Valle d'Aosta30056Veneto30060Total4,559928


### 2.5. Statistical Analysis

In
animals under experiment, and for each sampling day, mean value, 25th
percentile, 75th percentile, minimum value and maximum value of titres
resulting from the RB51-CFT were recorded. Sensitivity and specificity values
were estimated and compared using a Bayesian approach [9].
Bayesian
inference is an application of the Bayes theorem [10] that allows the
investigator to integrate any previous knowledge (expressed as a prior
probability distribution), with the likelihood of obtaining a certain result if
the animal is infected or if the animal is healthy (likelihood functions), with
the results obtained by the application of the tests to a given population
(collected data). The likelihood functions depend on the sensitivity and
specificity of the test(s) employed and on the uncertainty of their values. The
final results are probability distribution of the number of infected animals
correctly identified as infected (sensitivity) or of the number of healthy
animals correctly identified as healthy (specificity) in the sample or in the
population (posterior probability). Probabilities of the various possible
sensitivity values were estimated using a binomial likelihood function and an
uninformed Uniform(0,1) prior distribution. As existing knowledge on the sensitivity
or specificity of tests was considered to be virtually nil, an uninformed
Uniform(0,1) prior distribution was used. The Uniform(0,1) distribution states
that prior to the collection of data, all true probability values are
considered possible within the range defined for the number of true positives
(sensitivity calculation) or true negatives (specificity calculation). The
RB51-CFT results were expressed as the percentage of positive animals on
tested; the upper and lower 95% Credibility Intervals (CI) were calculated
using a beta probability distribution [11].


## 2.1. Vaccine

The
RB51 vaccine was kindly provided by CZ Veterinaria (Pontevedra,
Spain), the European
distributor of the product, under license from the Colorado Serum Company (Denver, Colo,
USA). Once
reconstituted, the vaccine contained5×109 CFU/mL of B. abortus strain RB51.


## 2.2. Animals and Vaccination

Fifteen
Friesian calves, aged between four to six months and obtained from OBF herds,
were randomly selected and divided into two groups. One group (n=10) was
vaccinated subcutaneously with RB51 in accordance with the instructions of the
manufacturers (2 mL reconstituted solution, containing 10×109 colony-forming
units (CFUs)). The other group (n=5) was used as nonvaccinated con-trol and the
animals were inoculated subcutaneously with 2 mL sterile saline solution. All
the animals were stabled with adequate space and fed with a standard diet (hay
and nutritional supplements) for the entire duration of the experiment; all
stages were conducted with consideration for their welfare and all procedures
with animals were carried out in accordance to appropriate humane methods.


## 2.3. Serological Testing and Antigens

All animals were tested for anti-B. abortus and
anti-RB51 antibodies before vaccination, on the day of vaccination (day zero)
and then periodically until Day 300 postvaccination (pv). Anti-B. abortus antibodies
were verified using the Rose Bengal test (RBT) and CFT, both performed using
the B. abortus biovar 1 strain 99 as antigen (VLA Weybridge, UK) and
according to the methods described in the 5th edition of the OIE Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals [7]. Anti-RB51
antibodies were monitored with an RB51 antigen-specific CFT (RB51-CFT). The antigen was
prepared from the same RB51 strain used for vaccination, after growth in
Brucella agar added with 5% bovine foetal serum. The antigen used in the
RB51-CFT was crosstitrated with a positive serum
coming from a
heifer vaccinated with RB51, as described in literature [5, 6].


## 2.4. Field Investigation

The
field investigation covered the 2001–2004 period and was divided in two steps.
For the 2001–2003 period, the investigation was carried out on sera coming from
the National Serum Bank (NSB) placed in the Istituto Zooprofilattico
Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZS A&M). The NSB
collects and stores sera coming from cattle used as sentinel in the framework
of the National Bluetongue Surveillance System (NBSS). Sentinels are bled
regularly with variable frequency, depending on the season and bluetongue
infection rate in the area concerned [8]. The herds to be
tested were randomly selected from the sentinel herds that were present in the
12 regions in which the NBSS was in force during the period considered. Sera of
all animals present in the sampled herds were tested with RB51-CFT (22,416 sera
analysed from 1,948 herds, Table 1). For year 2004, sera collected by Local
Veterinary Services in the framework of the National Brucellosis Eradication
Plan (NBEP) were analysed by the local Istituti Zooprofilattici Sperimentali
(IZS), Italian National Veterinary Laboratories. Sera were sampled according to
a systematic sampling method, with a sampling interval (intended as the number
of samples in between the sample selected and the next to be selected)
calculated for each region on the basis of the number of samples tested for the
NBEP in the year 2002. The number of serum samples expected from each region
was set to 300, but not all the regions were able to reach the objective. All
sera were analysed with RB51-CFT (4,559 sera analysed from 928 herds, Table 2).
Moreover, NSB serum, coming from Piedmont region (2,489 sera), NBEP sera from Veneto region (300 sera), Friuli-Venezia
Giulia region (185 sera), and the autonomous provinces of Trento
and Bolzano
(300 sera), were used to calculate the threshold of RB51-CFT (3,274 sera
analysed in total).Table 1
Number of sera from the National Serum Bank (NSB, collected during the
2001–2003 period) and tested with RB51-CFT, on regional basis.
Region of originNo. of sera testedNo. of herds testedAbruzzo2,123177Apulia1,836207Basilicata1,957222Calabria1,064111Campania1,062117Liguria2,552193Marche2,825238Molise2,584217Piedmont2,489139Sardinia1,282110Sicily1,446114Umbria1,196103Total22,4161,948Table 2
Number of sera from the National Brucellosis Eradication Program (NBEP,
collected during year 2004) and tested with RB51-CFT, on regional basis.
Region of originNo. of sera testedNo. of herds testedApulia30261Basilicata30059Calabria26654Campania30059Emilia-Romagna30060Friuli-Venezia Giulia18538Liguria25155Lombardy30060Marche31579Piedmont30061Sardinia30062Sicily30057Trentino-Alto Adige30061Umbria24046Valle d'Aosta30056Veneto30060Total4,559928


## 2.5. Statistical Analysis

In
animals under experiment, and for each sampling day, mean value, 25th
percentile, 75th percentile, minimum value and maximum value of titres
resulting from the RB51-CFT were recorded. Sensitivity and specificity values
were estimated and compared using a Bayesian approach [9].
Bayesian
inference is an application of the Bayes theorem [10] that allows the
investigator to integrate any previous knowledge (expressed as a prior
probability distribution), with the likelihood of obtaining a certain result if
the animal is infected or if the animal is healthy (likelihood functions), with
the results obtained by the application of the tests to a given population
(collected data). The likelihood functions depend on the sensitivity and
specificity of the test(s) employed and on the uncertainty of their values. The
final results are probability distribution of the number of infected animals
correctly identified as infected (sensitivity) or of the number of healthy
animals correctly identified as healthy (specificity) in the sample or in the
population (posterior probability). Probabilities of the various possible
sensitivity values were estimated using a binomial likelihood function and an
uninformed Uniform(0,1) prior distribution. As existing knowledge on the sensitivity
or specificity of tests was considered to be virtually nil, an uninformed
Uniform(0,1) prior distribution was used. The Uniform(0,1) distribution states
that prior to the collection of data, all true probability values are
considered possible within the range defined for the number of true positives
(sensitivity calculation) or true negatives (specificity calculation). The
RB51-CFT results were expressed as the percentage of positive animals on
tested; the upper and lower 95% Credibility Intervals (CI) were calculated
using a beta probability distribution [11].


## 3. Results

### 3.1. Serological Testing on Experimentally Vaccinated Animals

Sera
from all animals (RB51-vaccinated and controls) gave negative results to RBT
and CFT prior to vaccination, on Day zero and during the entire study. All the
animals were also negative to RB51-CFT prior to vaccination and on Day zero.
After vaccination, vaccinated animals developed a serological response to
RB51-CFT. The results of RB51-CFT on vaccinated animals and on controls are
shown in Figures1 and 2, respectively. Using the threshold resulted from the
field investigation, the percentage of animals correctly identified as
vaccinated or unvaccinated by the RB51-CFT are shown in Figures 3
and 4,
respectively.Figure 1
Median, maximum value, minimum value, 25th
percentile, and 75th percentile of RB51-CFT results in vaccinated animals (n=10).Figure 2
Median, maximum value, minimum value, 25th
percentile, and 75th percentile of RB51-CFT results in control animals (n=5).Figure 3
Percent vaccinated animals correctly
identified by the RB51-CFT and 95% Credibility Intervals.Figure 4
Percent control animals correctly identified
by the RB51-CFT and 95% Credibility Intervals.


### 3.2. Field Investigation

The
RB51-CFT threshold has been identified in the 100% of fixation at 1 : 4 serum
dilution. This result corresponds to the 90th percentile of the distribution of
titres of all sera that have shown reactivity in the RB51-CFT and to the 99.9th
percentile of the distribution of titres of sera coming from Piedmont,
Veneto and Fiuli-Venezia Giulia regions, and
the autonomous provinces of Trento and Bolzano.
RB51-CFT results on NSB sera (2001–2003 period), expressed according to the
region of origin and as percent of animals resulted positive on tested, are
shown in Figure5. RB51-CFT results in NBEP sera (year 2004), expressed according to the region of
origin and animals resulted positive on tested, are shown in Figure 6. Given
that they have been used for the threshold calculation, NSB sera from Piedmont
region, as well as NBEP sera from Veneto and
Fiuli-Venezia Giulia regions and the autonomous provinces of Trento and Bolzano, have not been
considered in Figures 5 and  6, respectively.Figure 5
Percent of sera coming from the National
Serum Bank (NSB, collected during the 2001–2003 period) and resulted positive to RB51-CFT, on
regional basis.Figure 6
Percent of sera coming from the National
Brucellosis Eradication Plan (NBEP, collected during year 2004) and resulted positive to RB51-CFT, on
regional basis.


## 3.1. Serological Testing on Experimentally Vaccinated Animals

Sera
from all animals (RB51-vaccinated and controls) gave negative results to RBT
and CFT prior to vaccination, on Day zero and during the entire study. All the
animals were also negative to RB51-CFT prior to vaccination and on Day zero.
After vaccination, vaccinated animals developed a serological response to
RB51-CFT. The results of RB51-CFT on vaccinated animals and on controls are
shown in Figures1 and 2, respectively. Using the threshold resulted from the
field investigation, the percentage of animals correctly identified as
vaccinated or unvaccinated by the RB51-CFT are shown in Figures 3
and 4,
respectively.Figure 1
Median, maximum value, minimum value, 25th
percentile, and 75th percentile of RB51-CFT results in vaccinated animals (n=10).Figure 2
Median, maximum value, minimum value, 25th
percentile, and 75th percentile of RB51-CFT results in control animals (n=5).Figure 3
Percent vaccinated animals correctly
identified by the RB51-CFT and 95% Credibility Intervals.Figure 4
Percent control animals correctly identified
by the RB51-CFT and 95% Credibility Intervals.


## 3.2. Field Investigation

The
RB51-CFT threshold has been identified in the 100% of fixation at 1 : 4 serum
dilution. This result corresponds to the 90th percentile of the distribution of
titres of all sera that have shown reactivity in the RB51-CFT and to the 99.9th
percentile of the distribution of titres of sera coming from Piedmont,
Veneto and Fiuli-Venezia Giulia regions, and
the autonomous provinces of Trento and Bolzano.
RB51-CFT results on NSB sera (2001–2003 period), expressed according to the
region of origin and as percent of animals resulted positive on tested, are
shown in Figure5. RB51-CFT results in NBEP sera (year 2004), expressed according to the region of
origin and animals resulted positive on tested, are shown in Figure 6. Given
that they have been used for the threshold calculation, NSB sera from Piedmont
region, as well as NBEP sera from Veneto and
Fiuli-Venezia Giulia regions and the autonomous provinces of Trento and Bolzano, have not been
considered in Figures 5 and  6, respectively.Figure 5
Percent of sera coming from the National
Serum Bank (NSB, collected during the 2001–2003 period) and resulted positive to RB51-CFT, on
regional basis.Figure 6
Percent of sera coming from the National
Brucellosis Eradication Plan (NBEP, collected during year 2004) and resulted positive to RB51-CFT, on
regional basis.


## 4. Discussion

The results of the present study confirm the
possibility to detect specific anti-RB51 antibodies with the RB51-CFT
[5, 6, 12]. Moreover,
negative results to RBT and CFT confirm the impossibility to detect specific
RB51 antibodies with the conventional serological tests [3, 4], while the use of Strains 19 or 45/20 would elicit the
production of antibodies detectable with conventional serological tests
[2]. This characteristic of
RB51 might be very useful for countries having a brucellosis control program
based on vaccination. Nonetheless, the choice of vaccines to be used should
take into consideration also other factors, namely:(i)
RB51 efficiency
(compared with S19) and its innocuousness remain controversial(ii)
field experience
indicates that it can induce abortion in some cases if applied to pregnant
cattle and that there is excretion in milk in a relevant number of vaccinated
animals(iii)
RB51 could
infect humans and it is highly resistant to rifampicin, one of the antibiotics
of choice for treating human brucellosis. In addition, the diagnosis of the
infection produced by RB51 requires special tests not available in most
hospitals.
All these factors,
together with the frequency of immunosuppressed persons in the human population
should be taken into consideration in the choice of a vaccine to be used in
animal populations.Beside the availability of tests with known
performances, to investigate on the possible illegal use of RB51 vaccine, it is
also necessary to know the antibody kinetics in cattle vaccinated at calfhood.
After showing an increase at Day 6 pv, the antibody level recorded in the 10
vaccinated animals remains practically constant for two months, and then
progressively decreases (Figure1). In particular, considering the threshold
identified for the RB51-CFT with the field investigation carried out in the
present study, only one animal is still correctly identified out of the 10
experimentally vaccinated at 119 days pv (Figure 3). All experimentally
vaccinated animals remain negative from Day 162 pv to the end of the study (day
300 pv). The RB51-CFT shows 100% sensitivity (CI 76.2%–100%) only at days 13 and 14 pv. Control animals
show results to the RB51-CFT always inferior or equal to a reaction of 100%
fixation at serum dilution 1 : 4 (Figure 2). Results observed in experimentally
vaccinated animals confirm the threshold identified with the field
investigation and show that the possibility to use RB51-CFT for the
identification of cattle vaccinated at calfhood with RB51 is limited in time.
The absence of positive results in the five control animals gives an estimation
of the specificity of 100%, with a CI between 60.7% and 100% (Figure 4).
Nevertheless, given that this CI is somewhat wide, the RB51-CFT specificity is
better defined when the results of the field investigation are considered
(specificity 99.9%, CI 99.73%–99.96%). The field investigation reveals that
in some Italian regions (namely, Basilicata, Calabria, Campania, Liguria, Apulia, Sardinia,
Sicily, and Umbria) the number of positive results
(Figure 5) is higher than what would be expected on the basis of the RB51-CFT
specificity (as it has been standardized). As a matter of fact, the expected
percentage of false-positive results to the RB51-CFT would be 0.1%, with a
lower CI of 0.04%. Moreover, in the case of Liguria,
Sicily, and Sardinia regions, the lower CI of the percent positive animals is higher than the upper CI
of the percent positive animals that would be expected on the basis of the test
specificity. Therefore, it is unlikely that the frequency of positive results
observed in these latter regions would be only due to the effect of case. The
results shown in Figure 5 (sera from the 2001–2003 period) are confirmed for
six regions (namely, Basilicata, Calabria, Campania, Apulia, Sicily, and
Sardinia) by the results observed on samples collected during 2004 (Figure 6).
These regions, in year 2004 also, show a percentage of positive animals higher
than expected on the basis of test specificity. The positive results observed
on sera collected during the 2001–2003 period from Umbria and Liguria
regions (Figure 5) are not confirmed on sera collected in year 2004, where no
positive result was recorded. Moreover, for some regions (namely, Calabria, Campania, Apulia, Sicily, and Sardinia) the lower CI of the percentage of positive
animals is higher than the percentage expected on the basis of the upper CI of
test specificity (Figure 6). Although a possible illegal use of RB51 vaccine
could be explained for some of these regions (Calabria, Campania, Apulia,
Sicily) by the high brucellosis prevalence, by the difficulties encountered in
the eradication process, and by the frequent reinfections of cleaned herds (as
detected during the NBEP, Italian Ministry of Health, personal communication),
an illegal use of this vaccine remains difficult to explain in Sardinia region,
which reached the status of OBF region in April 2003 [13]. Nevertheless, Sardinia has intensive animal trade with other Italian regions that in several occasions
was responsible of the reintroduction of Brucellosis in the island. In the case
of Sardinia, RB51-vaccinated animals could
have been imported from other Italian regions and this could explain the
presence of a positivity rate significantly higher than that expected on the
basis of the test specificity.Given the nature of the disease
(vaccination cannot avoid the infection of the animal and the related carrier
state), the passage from a brucellosis control program to a brucellosis
eradication program would necessarily imply to forbid the use of any kind of
vaccination. Once vaccination is forbidden, an illegal use of RB51 in cattle
can be expected in areas with a relatively high prevalence because farmers
would try to decrease the number of abortions (and the other economic losses
associated with brucellosis, such as the drop in fertility and/or in milk
production) by using a tool that cannot be identified by the Veterinary
Services with the routine brucellosis diagnostic procedures. Lacks in the
Official Veterinary Services inspection activities, together with the absence
of an effective surveillance system, are conditions that encourage the illegal vaccination
practices in those areas. The uncontrolled spread of the vaccinal strain would
have effects on the epidemiology of the field strain also, and this cannot be
properly assessed (and therefore controlled) without a reliable diagnostic tool
to identify animals vaccinated with RB51.The
results of the present study suggest that in case of RB51-CFT-positive results
observed in OBF areas (as the ones observed in Sardinia region) and, in any
case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT
alone is not sufficient but it would be appropriate to design a more
sophisticated diagnostic protocol. The use of a protocol which would include an
epidemiological investigation, the use of RB51-CFT, and the use of the skin
test with RB51 as antigen [12] could give important
elements to the final correct interpretation of RB51-CFT results and
identification of RB51-vaccinated animals.



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*Source: 584624-2008-04-09.xml*