Abstract: A prospective longitudinal study was performed of brucellosis’ monthly incidence during a period of six years, in a bovine dairy farm revaccinated with RB51 and endemic presentation of brucellosis, monthly serologies were done to the total amount of cows (average of 340). Prevalence at the beginning of the study was 15.3%. All negative animals were vaccinated with RB51 reduced doses and revaccinated. Card Test (CT) was executed and the positive sera were tested by rivanol. Sampling on day 150 showed incidence of 21%. During the first 360 days of the study, the cows that were positive to serology were not eliminated. During the period between 390 to 960 days, positive animals were eliminated, obtaining with this type of handling, incidences below 2%. Between days 990 to 1830 we returned to the criteria of not eliminating positives and therefore monthly incidences were 2%. From day 1830 on, all seropositive cows were eliminated with incidences during day 2160 and 2190, last day of our study, of 0.5 and 0.25%, respectively. The conclusion that we arrived at is that to have an efficient control of brucellosis, a vaccination program appropriate for epidemiological conditions of brucellosis in the region is required.
Introduction
Brucellosis is an infectious and contagious disease that affects humans as well as domestic animal species. It causes economic loss due to abortions, lengthening of the period between calvings, loss of genetic lines, infertility and sterility, in bovines, milk production is reduced by up to 30% (Acha and Zyfres, 1986).
Brucella abortus is an intracellular pathogen that survives inside macrophages in order to avoid the hostile environment. During the course of infection the bacteria remains within the cells and therefore these are the ones responsible for the destruction of the pathogen or be themselves destroyed so that other mechanisms can gain access to the Brucella and eliminate it, for example antibody mediated means (Golding et al., 2001). In Mexico, beginning in 1998, RB51 vaccine was used in control programs within the official campaign (Castell, 1998).
B. abortus RB51 is an attenuated rough strain stable vaccine, derived from the virulent strain 2308. This vaccine strain is incapable of producing the polysaccharide O chain (Cheville, 2000; Vemulapalli et al., 2000). The lack of this chain allows us to use this vaccine without the production of antibodies that may interfere with the serological diagnosis of this disease. Due to this fact, any positive reaction to official tests of the animals is diagnosed as infected (Uzal et al., 2000).
Nevertheless, this statement is valid under the circumstances found in countries with low prevalence of the disease; such is the case of the United States, where brucellosis is practically eradicated and incidence is very low. In contrast, in Mexico, the disease is endemic and the circumstances are very different. It has been observed that bovines without infection, either vaccinated or revaccinated with strain RB51, come out positive to serological testing several months post-immunization, which causes diagnosis confusion (Díaz-Aparicio et al., 2004; Leal-Hernández et al., 2005).
Serological samplings in affected populations facilitate taking decisions for the culling of animals and therefore a reliable laboratory should be accessible, in order to have opportune results with the implicit acceptance of the results. Once the results are available, it is necessary to identify positive animals in a permanent manner in order to facilitate segregation or elimination of the animals from the farm (Luna and Mejía, 1998). The objective of this study was the determination of monthly incidences during a six-year period in a bovine dairy herd infected with brucellosis and revaccinated with RB51.
Materials and Methods
Animals
Serological follow-up was performed of a bovine dairy herd with cows of
Holstein breed, located in the Industrial Agricultural and Livestock Complex
of Tizayuca, Hidalgo, Mexico. In this dairy basin, brucellosis has an endemic
status and this dairy had B. abortus biotype 1 infection demonstrated
by isolation.
Tizayuca, Hidalgo, Mexico, dairy basin has around 25,000 Holstein breed cows, specialized in milk production and distributed in 126 dairies; throughout the basin there is 20% brucellosis prevalence.
The dairy herd that was studied is within the dairy basin, its handling system is intensive, the entrance to the dairy has sanitary trenches for vehicles as well as persons, four pens with individual stalls, feed bunks and drinkers, feed is integrated by a mixer car and the animals are milked three times per day. Seropositive animals to brucellosis are identified and grouped in a specified pen where they are no longer inseminated and when their milk production is reduced or they have any health problem they are gradually eliminated from the dairy.
Vaccination
At the beginning of the study (day zero) the total amount of negative animals
were vaccinated with RB51 at a 2 mL reduced dose at a concentration of 3x109
cfu mL-1, application was subcutaneous in the left axillary zone.
Revaccination took place at day 90 and every 360 days during the six years that the study lasted, with the same reduced doses of RB51 and the same route detailed previously.
Samplings
Serological samplings were performed for all the animals every 30 days during
a 2190 days period (72 months), the monthly average of worked cows was 340.
Blood samples were taken from the ventral coccygeal vein.
Milk and vaginal samples were taken, from the 37 seropositive cows that were detected at experiment day 0.
Serology Studies
To identify animals that are seropositive to Brucella, all animals
were tested by Card Test (CT) and positive results were confirmed by rivanol
(Alton et al., 1988). By rivanol test, sera were considered positive
when they presented an agglutination reaction to any of the dilutions from 1:25
to 1:400.
Bacteriology Studies
Milk and vaginal exudate samples were inoculated in duplicate in TSA and
Farrell (1974), incubated during 10 days with 10% CO2 at 37°C.
Conventional methodology was used to identify isolated strains (Alton et
al., 1988).
Epidemiological Studies
A prospective longitudinal study was performed estimating monthly brucellosis
incidence and prevalence during a period of six years, according to the presence
of B. abortus antibodies detected by CT serological and rivanol tests
(Anonymous, 1978).
Results
Brucellosis seroprevalence in the dairy when the study began was 15.3% (37/241). Incidence by samplings on days 90, 120 and 150 was: 34.5% (56/162), 19.3% (30/155) and 20.9% (30/143), respectively (Table 1).
Bacteriology studies performed on vaginal exudates and milk samples gave as a result isolation and identification of four strains of B. abortus, biotype 1 with smooth phenotype.
During the first 360 days seropositive cows of the study were not eliminated
from the dairy, nevertheless they were placed in a pen specific for them.
| Table 1a: | Monthly prevalence and incidence in bovine herd infected for brucellosis, where cattle was revaccinated with RB51 |
| Table 1b: | Continued |
| Fig. 1: | Monthly prevalence and incidence in dairy cow herd infected with brucellosis, where cattle was revaccinated with RB51 |
During the interval between 360 and 960 days animals began slowly to be eliminated, taking into consideration the rivanol antibody serology titers, the animals with 1:100, 1:200 and 1:400 titers were slowly eliminated; nevertheless, cows with 1:25 and 1:50 were left in the designated pen and only if during the next sampling titers went up they were eliminated. This handling resulted in incidences below 2% during this period.
But, from day 990 to 1830, the non-elimination of positive cows was implemented again and monthly incidences were around 2%.
Beginning day 1830, all seropositive cows were eliminated from the dairy and incidences that were observed were 0.5% (2 of 401) on day 2160 and 0.25% (1 of 401) at day 2190. The study was ended on this day (Fig. 1).
Discussion
Vaccination strain RB51, has been used in Mexico, since 1998, very successfully and it slowly has been replacing strain S19. Discussions began since 2005, on how well strain RB51 worked, so strain S19 started gaining ground again. At this time both vaccines are being used in bovines indistinctly.
These discussions on RB51 vaccines occur mainly in endemic zones of our country, where after vaccination, cows are found positive to card test and rivanol tests with low titers. The reaction is transitory since it becomes negative after two or three months and this is attributed to the immune response facing exposure to a field strain of Brucella that is favorably resolved by the immune system. This should speak highly of RB51 vaccination, but it is not so because the vaccines are sold under the indication that no post-vaccination reactions occur and if there are positive animals to card tests, these undoubtedly are infected animals. But this claim happens only in zones with null or low prevalence and it is not the case in endemic areas, where animals are constantly challenged.
This response has been experimentally documented by Stevens and Olsen (1996) who demonstrated that RB51 immunized cows; later infected with B. abortus strain 2308, produce small amounts of IgG but not IgM. Also Cloeckaert et al. (2002) observed that strain RB51 produces low levels of an antigen that is similar to M; nevertheless, subsequent exposures to Brucella has some animals responding to chain O and are detected by conventional tests.
In controlled experiments, animals vaccinated with RB51 strain developed titers against Brucella after challenge with virulent 2308 strain and they have constant or decreasing titers until calving (Cheville et al., 1993; Olsen, 2000; Bagnat and Maneti, 2002).
Under field conditions, with endemic presence of brucellosis, our research group has found that when animals are naturally challenged, there is a recurrent serological response that is transitory (Leal-Hernandez et al., 2005; Herrera, 2002; Cantú et al., 2005). This response is derived from the primary immune response against B. abortus due to vaccination with RB51 and later challenge due to the extensive dissemination of field strains in endemic zones. Cows then develop a secondary response when they come into contact with these field strains. Antibody presence demonstrates that the animals have had contact with field strains, but their immune response was capable of controlling the infection (Díaz-Aparicio et al., 2004).
These data contrast with what Lord et al. (1998) found, since under these conditions of high prevalence (39%), bovines were vaccinated and revaccinated with RB51, at 5x109 cfu doses and later revaccinated with S19, finding that all vaccinated animals with RB51 resulted negative to conventional serological tests notwithstanding the natural challenge by field strains. These field results obtained with RB51 vaccination (Lord et al., 1998), are difficult to analyze and compare with those made under controlled conditions (Cheville et al., 1993; Olsen, 2000; Bagnat and Maneti, 2002). Under a more controlled scheme, Moriyón et al. (2004) observed that with this type of experiments, protection conferred by RB51 is useful under a moderate challenge, but when challenge is increased, RB51 is insufficient to prevent infection.
Moriyón et al. (2004) mentioned that the experiment by Lord et al. (1998) has certain defects in the selection of animals free from Brucella. Those animals belonged to infected herds and not free from brucellosis herds and were only selected based on negative serology. This method doesn’t take into consideration young animals that acquire latent infections that are not manifested in serology tests and this introduces a bias in the selection of vaccinated groups. Also it is important that we highlight that none of the animals vaccinated with RB51 became positive to the serology tests even though they were in contact with a field strain. Even though the field studies are less reliable, the results should be taken with caution when they openly contradict the results of controlled studies (Moriyón et al., 2004).
In the dairy basin where this study took place, brucellosis has an endemic status and high prevalence. Bovines that enter the basin are vaccinated and revaccinated with RB51 and besides each year they are revaccinated with a reduced dose of RB51. This procedure exists since the nineties but the vaccines that were used were S19 (Aparicio et al., 2003; Bustamante et al., 2000, González et al., 2006). Transitory post-vaccination responses are common in this dairy basin, therefore the non-official diagnosis criteria is that a cow is considered as having brucellosis only when it comes out positive to card tests and rivanol at titers 1:100 or higher, if they have lower titers to the rivanol test, it is repeated every month until the titers disappear or increase.
Revaccination is recommended by the Mexican Official Standard (1996) but on condition it is done only in endemic zones that have high prevalence using reduced doses and what is most important is that revaccination is done only one time. Repeated revaccination is not recommended since brucellosis vaccines are live and only one dose is capable of eliciting a solid response that is sufficient to protect the animal during all of its productive life (Diaz-Aparicio et al., 2004; Elzer et al., 1999; Nicoletti, 1990), that in Mexico is around 3.16 lactations (Valdespino, 1999; Vitela et al., 2004).
Revaccination is not necessary since RB51 vaccine induces a good cell type immune response, where Th1 and early gamma interferon production have a prominent role in protection (Elzer et al., 1998). Also during its intracellular transit RB51 is destroyed in the phagolysome and the resulting peptides are presented to the T cells through the main histocompatibility complex (Arellano et al., 2004). What has happened is that repeated RB51 revaccination has promoted the presentation of anergic animals (Leal, 2003).
Undoubtedly vaccination is an important tool to avoid the transmission of brucellosis amongst animals. Nevertheless, a control program in dairies with high prevalence should be integral to avoid the dissemination of the disease. What is ideal is that before beginning vaccination the infected animals be known by periodic serology controls, also, an epidemiological surveillance plan should be instrumented as well as a sanitary handling program.
When comparing incidences, between periods, when reactors were eliminated and when they were not, we can observe that there is a marked difference between both incidences and thus we conclude that this type of handling is essential to control brucellosis in an infected herd.
The increase of new cases during the first year of study (at 360 days) is attributed to seropositive animals that were not eliminated from the dairy. The number of positive animals that were found in the herd was considerable, since prevalence reached up to 51.9% (132/254) at day 270. Elimination of animals seropositive to brucellosis was performed according to their production and reproduction stage. We must underline that in Mexico the main causes for culling are urogenital problems, especially low production and infertility (Vitela et al., 2004).
Presence of seropositive animals represents an important risk factor and therefore control programs should be implemented that take into consideration the use of quarantines and continuous serological monitoring, in order to identify opportunely cows that are beginning with the disease, thus avoiding that these animals disseminate the disease to others at the time of calving or abortion, besides the continuous gradual dissemination.
During the incubation period of the disease it is very difficult to perform serological diagnosis. In fact, it is known that 20% of daughters of reactor dams may remain seronegative since birth until they are about to calve, after that, they become seropositive, notwithstanding they may have been vaccinated as calves, thus making it necessary to follow-up daughters of reactor dams during 24 months. It has also been recommended that all those cows that have aborted or have reproductive problems have serology tests in order to obtain a negative result before insemination, otherwise they should not be includes in the process (Luna and Mejía, 1998).
The most appropriate parameter to determine the degree and progress of infection in a herd is incidence, in this study we performed a monthly determination during six years finding that vaccination notably reduced brucellosis incidence and during those times when seropositive animals were eliminated, incidence reduction was reinforced, especially during the first three years.
The conclusion is that in order to have an efficient brucellosis control, besides a vaccination program that is appropriate to the epidemiological conditions of brucellosis in the region, continuous serological monitoring should be implemented to opportunely identify cows that are beginning with the disease in order to eliminate contagion sources, taking also into consideration the gradual elimination of positive animals.