Abstract: In this study, the efficacy of vaccination with Brucella abortus RB51 strain as a measure for bovine brucellosis control was evaluated by a clinical assay in double purpose cattle that are naturally infected under tropical conditions. A herd with eight reactors to rivanol test with an initial serum reaction of 5% was selected. Confirmation of infected herd was carried out by isolation and identification of Brucella abortus from reactor animals, using bacteriological procedures. Also, the milk samples were analyzed by PCR technique whereby Brucella abortus infection was corroborated. Vaccinated and non-vaccinated groups were formed with 88 females each. Reactors were not eliminated nor segregated from the population. During 18 months of monitoring three new cases happened in the vaccinated group and therefore the initial serum reaction rate increased from 10 to 12.5%. The rate of vaccinated group remained at 0% due to 100% of protective efficacy that RB51 strain provided to the total vaccinated population (RR = 0; C.I. 95% 0-0). The conclusion is that under extensive double purpose livestock rearing conditions tropical climate, strain RB51 is a biological product efficacious for brucellosis control in infected herds with a prevalence of 6%.
INTRODUCTION
Vaccination is an indispensable practice in the control of bovine brucellosis and at the international level the RB51 and 19 strains of Brucella abortus (Halling and Boyle, 2002). Brucella abortus RB51 strain does not have the “O” polysaccharide and therefore, it does not induce the formation of antibodies against this bacterial lipopolysaccharide fraction. Thus, when animals are vaccinated with this strain it does not interfere with the routine diagnostic serology tests and thus it allows identification of vaccinated animals separating them from the infected ones; this does not happen with strain 19 (Moriyon et al., 2004). The use of strain RB51 was integrated into the eradication programs of the disease in Colombia, Costa Rica, Chile, United States of America, Mexico, Paraguay, Uruguay and Venezuela. Nevertheless, Argentina suspended its use due to a low protective response (Samartino et al., 2000; Garin et al., 2005). In view of the above, controversies on its efficacy allow us to suppose that there are important differences in test results provided by controlled experiments and how the vaccines really work in the field, exposed to different conditions and challenges (Aparicio et al., 2003; Samartino, 2005). Therefore, the objective of this study was the evaluation of the RB51 strain vaccine in herds that had cattle naturally infected with brucellosis under tropical conditions.
MATERIALS AND METHODS
Study Site
This research was carried out in the El Desengaño, community in Las
Choapas, Municipality of Veracruz, Mexico between August 2006 and February 2008.
During the first stage a transverse epidemiological study was carried out in
order to identify herds that were naturally infected with brucellosis.
Inclusion Criteria
Units dedicated to double purpose production in an extensive system in a tropical climate and without brucellosis vaccination were the ones taken into consideration. All animals six months old or more were sampled according to NOM-041-ZOO-1995 National Campaign against Brucellosis in Animals (SAGDR, 1996). The infected herd was defined as that one where there were animals, reactors to the buffered-tampon antigen test or Card Test (CT) with antigen at an 8% concentration and with at least one positive case to the precipitation action by Rivanol Test (RT).
Serology Diagnosis
Five milliliter blood sample was taken from the coccygeal vein with vacutainer
without anticoagulant. Samples were transported in refrigeration to the Microbiology
Laboratory of the Faculty of Veterinary Medicine and Animal Husbandry of the
University of Veracruz. The serum was placed in vials identified with the sample
number and preserved at -20°C until processing by the CENID Laboratory -
Animal Microbiology of INIFAP in Palo Alto, D.F., by CT and RT according to
NOM-041-ZOO-1995 National Campaign against Brucellosis in Animals. By RT any
agglutination value equal to or above 1:25 was considered as positive (SAGDR,
1996).
Clinical Assay
Based on the inclusion criteria, for the clinical assay a herd infected
with brucellosis was selected. To estimate the sample size and establish the
vaccinated and non-vaccinated groups the Win Episcope 2.0 program was used under
the modality of finding difference between proportions by estimating an expected
proportion of 6% of brucellosis positive animals in vaccinated population and
20% positive animals in the non-vaccinated population, with a level of confidence
of 95% and potency of 80%. Thus, the sample size was estimated at 88 animals
per group (Thrusfield et al., 2001). Vaccinated
and non-vaccinated groups were randomly selected and identified by ear tags.
From the time of vaccination, both groups were evaluated quarterly by serology
using CT and RT tests during 18 months in modalities of screening and confirmatory,
respectively.
Vaccination
All females that had negative results to CT and RT were vaccinated subcutaneously
once, in the middle third of the neck on the left side. Strain RB51 vaccine
was used in doses of 5x1010 Colony Forming Units (CFU) in females
6 to 12 months of age and in doses of 3x108 to 3x109 CFU
in animals older than 12 months including gestating females (SAGDR,
1996). Vaccination of animals was carried out in the month of August 2006;
at the time of experimental group establishment, 32 gestating females were integrated
into the vaccinated group and 36 gestating females in the non-vaccinated group.
Males were not vaccinated and animals seropositive to RT were not segregated
or eliminated from the herd.
Statistical Analysis
To determine seroprevalence rates, Relative Risk (RR) and Confidence Intervals
(CI) of 95% were estimated according to Thrusfield (2005).
Statistical significance of observed frequencies in vaccinated and non-vaccinated
groups was estimated by Chi-square and significant differences were considered
when p<0.05 (Daniel, 1999).
Vaccination Efficacy
It was estimated by the formula (Orenstein et al.,
1985):
Where:
| VE | = | Vaccination efficacy |
| CDR | = | Diseased animal rate within the control group |
| VDR | = | Diseased animal rate within the vaccinated group |
Bacteria Isolation
Bacteria isolation was considered an inclusion criteria necessary to confirm
infection of the herd by Brucella abortus and thus, be able to evaluate
efficacy of strain RB51 in the presence of field strains; therefore, in each
monitoring, milk samples were collected in sterile Falcon type tubes and bacteria
isolation was carried out following procedure by Alton et
al. (1988) of all animals reactors to RT. Samples were maintained in
refrigeration from the time of collection until processed. From the milk fat,
duplicate primary seeding was carried out in Farrell selective media and incubated
in aerobiosis and micro-aerobiosis environments; media were incubated at least
during one month at 37°C and checked for colony development every other
day. Isolations suggestive of Brucella sp. by colony morphology were
seeded again in Trypticasein Soy Agar (TSA) until pure cultures were obtained
and identified by biochemistry tests. Also, samples were sent refrigerated to
the Microbiology Department of the National School of Biological Sciences of
the National Polytechnic Institute, for confirmation diagnosis by Polymerase
Chain Reaction (PCR) (Matar et al., 1996; Hamdy
and Amin, 2002).
RESULTS
Serological Diagnosis
The transverse selection study allowed the identification of a brucellosis
infected herd by a serum reaction rate to RT of 5% (8/176) with agglutination
reactions between 1:25 and 1:400 that were considered positive in non-vaccinated
animals as established in NOM-041-ZOO-1995 National Campaign against Brucellosis
in Animals (SAGDR, 1996).
Clinical Assay
In Table 1, we are found the new cases of animals that
were reactors to RT during each one of the quarterly post vaccination follow-up
monitoring.
In the first year of monitoring three new infections cases happened in the non-vaccinated animals (Table 1). During all the research the rate of serum reaction of the non-vaccinated group (CDR) increased from the initial 10 to 12.5% in 18 months, while in the vaccinated group (VDR) it was 0%. Dissemination of the disease in animals of the non-vaccinated group propitiated an increase of the initial serum reaction rate of 5% to an accumulated serum reaction rate of 6% in the herd during the study period (Fig. 1).
When estimating frequencies observed in the vaccinated and non-vaccinated groups a year and half after vaccination, significant differences were found between them (p<0.05); nevertheless no association was found between groups and serum conversion (RR = 0; C.I. 95% 0-0) and this indicates that strain RB51 vaccine protected the total amount of susceptible population. It must be underlined that none of the 32 gestating females of the vaccinated group aborted as a consequence of the biological product application nor did they develop the disease.
Vaccination Efficacy
Since, no serum positive or reactor animal was detected in the vaccinated
group during the 18 months of the research, the strain RB51 vaccine of Brucella
abortus had a protective efficacy of 100%.
Bacteria Isolation
In cultures carried out in micro-aerobiosis of the 26 milk samples, Brucella
abortus colonies showed up in 38% of the cases (10/26); for this, 10 reactor
animals were monitored and the quarterly isolations that were obtained came
from 60% of these (6/10) as can be seen in Table 2.
| Table 1: | New cases seropositive to rivanol test identified during quarterly serology monitoring in the non-vaccinated group |
| Fig. 1: | Serum reaction rates in the vaccinated and non-vaccinated groups and the herd during quarterly follow-up monitoring |
| Table 2: | Isolation of Brucella abortus from milk of animals reactors to rivanol test during the study |
| Num.: Identification, I: Isolation, +: Positive, –: Negative | |
Isolations that were obtained were confirmed as Brucella abortus through PCR studies carried out from the collected milk by the use of primers that amplify membrane protein OMP´s 31 kDa.
DISCUSSION
Serology Diagnosis
Rivanol Test (RT) test has a relative sensitivity between 86 to 97% and
therefore it is not recommended for eradication program final stages (Dájer-Abimerhi
et al., 1998); nevertheless, due to its high specificity (100%) it
identifies IgG antibodies derived from a strong antigenic stimulus and therefore
its presence implies an active infection, or chronic infection making it useful
as a confirming test in control programs or early stages of eradication campaigns
(Dajer-Abimerhi et al., 1995; Diaz
et al., 2001). The eight positive cases to RT that were identified
during the transverse study were females older than three years old, situation
that coincides with what Nicoletti (2005) mentions, referring
to the fact that the infection affects bovines of all ages but persists more
frequently in sexually developed animals.
Clinical Assay
In 18 months of research a total of three new infection cases were identified
in the non-vaccinated group, in this sense, Renteria et
al. (2003) and Nicoletti (2005) underline that
the degree of crowding and animal population density are factors that favor
the transmission of the disease since thus there is a higher probability that
susceptible animals be exposed to the infection; nevertheless, Magana-Monforte
et al. (2006) indicated that neither crowding nor population density
are factors that characterize the extensive cattle production system and therefore
since that does not influence the population, contact between diseased and susceptible
animals is not facilitated. This in turn reduces the risk of infection; this
together with a low disease incidence observed during the study could explain
the reduced number of new cases identified during the first year in the animals
of the non vaccinated group.
Immunization with strain RB51 reduces susceptibility to the infection by providing immune protection from three to four weeks after its application and slowly reduces the level of exposure to the infection since the number of infected animals with brucellosis does not increase in the herd (Casas, 2003); this explains why the rate of serum reactors in the vaccinated group (VDR) was 0% and of the non-vaccinated group (CDR) was increased from the initial 10% to 12.5% during the study period. Different from what Van Metre et al. (1999) found in his study, reporting the infection of a gestating vaccinated female, during this study no gestating female of the vaccinated group aborted and neither did it get infected with brucellosis. In their respective studies, Edmonds et al. (1999) and Olsen (2000) come to the conclusion that vaccination with RB51 strain does not cause reproductive problems or abortion when applied to sexually mature or gestating females; these statements, coincide with observations of this study, of gestating females of the vaccinated group. Vaccination of females even during the last third of gestation with strain RB51 with doses of 3x109 CFU as used in this study, is considered by Uza et al. (2000) as safe, since there is no diagnostic interference and it does not cause abortion.
Vaccination Efficacy
Leal et al. (2005) mentioned that in endemic
zones, strain RB51 protects up to 94% of the vaccinated herd when challenged
by field virulent strains; thus, the 100% protective efficacy provided by strain
RB51 to animals of the vaccinated group, is above the rate reported by these
authors. The obtained result of vaccine efficacy coincides with that which was
expressed by Lord et al. (1998) and Ramirez
et al. (2002), who indicated that this strain does not induce serum
conversion in vaccinated females and protects 100% of the susceptible cattle
when it is used to control brucellosis in low prevalence herds.
Bacteria Isolation
As it is seen in Table 2 consecutive, alternate or only
isolations were obtained from the milk samples that came from reactor animals
and this may be due among other factors to the presence of the bacteria in this
product. Osorio (2004) mentioned that the bacteriological
procedures is not always successful due to the intermittency in the elimination
of the bacteria in milk; together with that, Renteria et
al. (2005) indicated that a large amount of bacteria are needed in the
sample or that the sample must be collected when Brucella sp. is being
excreted.
Rodriguez et al. (2005) indicated that approximately 50% of infected cows eliminate Brucella spp. in milk during several weeks or months after aborting or calving; notwithstanding the above, in this study 60% of the infected monitored cows eliminated the bacteria during the six quarters, situation that favored the isolation of the same in animals in production as well as in the dry period, in which, as indicated by these researchers, elimination is reinforced.
Lopez et al. (1992) reported that in milk and vaginal secretions approximately 10 bacteria/gram are eliminated even in the cases where no symptoms are observed, as was the clinical status presented by all reactor animals during this study. Milk of affected animals facilitates contamination of the environment and favors the dissemination and transmission of the disease to the susceptible population, especially in those farms where hygiene is deficient and there is the custom of throwing the first spurt of milk on the floor prior to milking (despunte in Spanish) (Rodriguez et al., 2005); this is a procedure that is carried out in this production unit during milking.
According to what has been mentioned by Dajer-Abimerhi et al. (1998) and Diaz et al. (2001), isolation and identification of the bacteria obtained during 5 quarters of field monitoring together with the presence of new cases, allowed the confirmation of infected herd.
Moreno et al. (2002) and Renteria et al. (2003) consider that the reactor animals within the population are a risk factor that favors the transmission and permanence of the disease in the herd. The presence of a circulating field strain of Brucella abortus that comes from the reactor cattle and that has not been eliminated, propitiated that vaccinated and not-vaccinated animals had at all times a constant natural challenge, that allows the evaluation of the protective efficacy of strain RB51.
Results of this study allows us to conclude that under extensive double purpose cattle production system in tropical climate, strain RB51 is an efficacious biological product for the control of bovine brucellosis in naturally infected herds with a prevalence of 6%.
ACKNOWLEDGMENTS
This study is part of the requirements that the first author must cover to obtain the degree of Doctor in Agriculture and Livestock Sciences granted by the Autonomous University of Yucatan who carry out an economic support with a CONACYT scholarship holder. Research received support and financing from the Project Comparative study of strain RB51 and strain S19 efficacy in the prevention of brucellosis in herds with different sanitary conditions of the National Forestry, Agriculture and Livestock Research Institute (INIFAP) called for by SAGARPA-CONACYT 2004 Sector fund 23.