Seroprevalence of brucellosis rate by RBPT in goat flocks of Xaltepec municipality Perote, Veracruz, Mexico.
Abstract
The aim of this study was to determine the seroprevalence of Brucella spp. in a goat flock and the seroconversion of three groups of animals vaccinated with Rev-1 (Brucella melitensis), RB51, and RB51-SOD (Brucella abortus) to estimate the level of protection conferred on susceptible females. Seventy-two animals were used by group. Goats were older than 3 months, seronegative to brucellosis, not vaccinated previously, and kept within positive flocks. Vaccinated animals received 2 mL of product subcutaneously in the neck region. The first block was injected with Rev-1; the second received RB51, and the third group was injected with RB51-SOD. Follow-up sampling was performed at 30, 60, 90, and 365 days postvaccination. The general prevalence of brucellosis for the three groups was 1.2% (95%CI:0.5–2.7). The seroconversion rate by day 30 after vaccination was 77.7% (95%CI:61.9–88.2) for goats vaccinated with Rev-1. At 365 days post vaccination, the percentage of seropositive goats declined to 13.8% (95%CI:6.0–28.6). At day 365 after vaccination, 2.7% (95%CI:0.4–14.1) and 5.5% (95%CI:1.5–18.1) of animals vaccinated with RB51 and RB51-SOD, respectively, became positive. Results show that the seroconversion induced by Brucella abortus RB51 and RB51-SOD vaccines is lower than that by Brucella melitensis Rev-1.
Keywords
- Brucella
- vaccine
- seroprevalence
- seroconversion
- goat
1. Introduction
The brucellosis is a highly contagious disease and one of the zoonoses worldwide; most importantly, it is caused by bacteria of the genus
In small ruminants, the brucellosis is caused by
In underdeveloped countries, vaccination is the main tool used in the control of this disease [12, 13], in particular as a preventive measure in small ruminants, and is considered necessary given the economic and medical consequences of having brucellosis in animals and people infected [14]. The main indicator of brucellosis reduction in animals is a concomitant reduction of human cases [13, 15]. In endemic areas, intensive vaccination with
In Mexico, the vaccine RB51 was approved since 1998 as the official one for use in cattle females. The strain has been evaluated in both goat and sheep under controlled conditions with good protection against the experimental challenge with
Nowadays, the homologous overexpression to induce a greater and more effective immune response for the improvement of protective immunity of the vaccines has been developed. This can be achieved by introducing a plasmid within the RB51 strain with the gene that encodes the antigen expressed, along with appropriate promoters. In mouse (
2. Material and methods
2.1 Study design
A phase III field trial was performed from September to December 2016 in order to determine the seroprevalence and seroconversion of goat flocks positive to brucellosis in the Xaltepec community municipality of Perote, Veracruz, Mexico, and to evaluate the protection conferred by vaccines with Rev. 1
2.2 Experimental design
The experiment was performed in two stages. In the first one, 546 animals from 14 herds with similar management, grazing, feeding, and confinement conditions were used to determine the prevalence of goat brucellosis in Xaltepec. In the second stage, groups required for vaccine evaluation were integrated by randomly selecting animals negative to serological tests meeting the inclusion criteria. Positive animals remained in the herds under field conditions in order to function as a challenge for healthy and vaccinated animals.
Sample size was calculated using Win Episcope Version 2.0 for simple random sampling, considering the 0.52% prevalence in goats reported in Veracruz by Román-Ramírez et al. of [12], a confidence interval of 95%, and an error margin of 5%. Since each animal had an identification number on its metallic earring, females were randomly assigned to each group and subgroup. For each group, the minimal calculated sample was 72 animals; each group was integrated by a vaccinated subgroup (36) and a not vaccinated or control subgroup (36). Studied groups were integrated by goats older than 3 months, seronegative to brucellosis, and not vaccinated previously and kept within positive flocks. Animals were randomly split into three groups and kept together 8 months in the flock to maintain exposure to
2.3 Vaccination of animals
Animals in each vaccinated group received 2 mL of vaccine subcutaneously applied in the neck region. The first group was injected with Rev. 1 (
2.4 Sample collection
Follow-up sampling was performed at 30, 60, 90, and 365 days post vaccination by blood sampling collected from the jugular vein in vacutainer tubes without anticoagulant (BD Vacutate, Oxford, UK). Each tube was identified with the number in the animal earring. Tubes containing blood samples were placed in a tilt position about 2 hours at room temperature allowing the separation of serum from the blood package. Later, tubes were placed into coolers at 4°C and transported to the laboratory and then were centrifuged at 1000 ×
2.5 Serological testing
Serum samples were analyzed by series using the following tests: 3% RBPT as screening and simple radial immunodifusion test (SRD) as confirmatory [5, 22].
RBPT was used as a screening test on the serum samples collected for the presence of
SRD was used as a confirmatory test, and the antigen was used at a concentration of 1 mg/mL on agarose gel prepared with a glycine buffer solution and native hapten obtained from
2.6 Analyses of data
Seroconversion produced during the observation period was calculated. Differences between groups and the significance of association were calculated by chi square (
3. Results
The results of initial seroprevalence of brucellosis in goat flocks at Xaltepec are shown in Tables 1 and 2. The seroprevalence in the three groups determined by the 3% RBPT as presumptive test resulted in 22.1, 26.1, and 16.0% (95%CI: 16.5–28.9, 19.9–33.2, and 11.1–22.3, respectively).
Strain | Sample size | RBPT | ||
---|---|---|---|---|
Positive | Seroprevalence (%) | 95%CI | ||
Rev. 1 | 185 | 41 | 22.1 | 16.5–28.9 |
RB51 | 180 | 47 | 26.1 | 19.9–33.2 |
RB51-SOD | 181 | 29 | 16.0 | 11.1–22.3 |
Strain | Sample size | SRD | ||
---|---|---|---|---|
Positive | Seroprevalence (%) | 95%CI | ||
Rev. 1 | 41 | 1 | 0.5 | 0.3–3.4 |
RB51 | 47 | 2 | 1.1 | 0.1–4.3 |
RB51-SOD | 29 | 4 | 2.2 | 0.7–5.9 |
The serum positive goats were confirmed with SRD, and the prevalence reduced to 0.5, 1.1, and 2.2% (95%CI: 0.3–3.4, 0.1–4.3, and 0.7–5.9, respectively). Thus, a general prevalence of 1.2% (95%CI: 0.5–2.7) was observed.
Tables 3
Group/subgroup | N | Time after vaccination (days) | |||
---|---|---|---|---|---|
30 | |||||
Positive | Seroconversion rate (%) | 95%CI | |||
Rev 1 | Vaccinated | 36 | 28 a | 77.7 | 61.9–88.2 |
Control | 36 | 4 b | 11.1 | 4.4–25.3 | |
RB51 | Vaccinated | 36 | 1 a | 2.7 | 0.4–14.1 |
Control | 36 | 0 a | 0.0 | 0.0–0.09 | |
RB51-SOD | Vaccinated | 36 | 2 a | 5.5 | 1.5–18.1 |
Control | 36 | 0 a | 0.0 | 0.0–0.09 |
Group/subgroup | N | Time after vaccination (days) | |||
---|---|---|---|---|---|
60 | |||||
Positive | Seroconversion rate (%) | 95%CI | |||
Rev 1 | Vaccinated | 36 | 27 a | 72.2 | 56.0–84.1 |
Control | 36 | 4 b | 11.1 | 4.4–25.3 | |
RB51 | Vaccinated | 36 | 1 a | 2.7 | 0.4–14.1 |
Control | 36 | 0 a | 0.0 | 0.0–0.09 | |
RB51-SOD | Vaccinated | 36 | 2 a | 5.5 | 1.5–18.1 |
Control | 36 | 0 a | 0.0 | 0.0–0.09 |
Group/subgroup | N | Time after vaccination (days) | |||
---|---|---|---|---|---|
90 | |||||
Positive | Seroconversion rate (%) | 95%CI | |||
Rev 1 | Vaccinated | 36 | 23 a | 63.8 | 47.5–77.5 |
Control | 36 | 4 b | 11.1 | 4.4–25.3 | |
RB51 | Vaccinated | 36 | 1 a | 2.7 | 0.4–14.1 |
Control | 36 | 0 a | 0.0 | 0.0–0.09 | |
RB51-SOD | Vaccinated | 36 | 2 a | 5.5 | 1.5–18.1 |
Control | 36 | 0 a | 0.0 | 0.0–0.09 |
Group/subgroup | N | Time after vaccination (days) | |||
---|---|---|---|---|---|
365 | |||||
Positive | Seroconversion rate (%) | 95%CI | |||
Rev 1 | Vaccinated | 36 | 5 a | 13.8 | 6.0–28.6 |
Control | 36 | 9 a | 25.0 | 13.7–41.0 | |
RB51 | Vaccinated | 36 | 4 a | 11.1 | 4.4–25.3 |
Control | 36 | 9 a | 25.0 | 13.7–41.0 | |
RB51-SOD | Vaccinated | 36 | 0 a | 0.0 | 0.0–0.09 |
Control | 36 | 7 a | 19.4 | 9.7–35.0 |
Tables 7
Group/subgroup | Time after vaccination (days) | |||
---|---|---|---|---|
30 | ||||
Positive | Prevalence rate (%) | 95%CI | ||
Rev 1 | Vaccinated | 1/28 | 2.7 | 0.49–14.1 |
Control | 0/4 | 0.0 | 0.0–0.49 | |
RB51 | Vaccinated | 0/1 | 0.0 | 0.0–0.79 |
Control | 0/0 | w.d.* | w.d.* | |
RB51-SOD | Vaccinated | 0/2 | 0.0 | 0.0–0.66 |
Control | 0/0 | w.d.* | w.d.* |
Group/subgroup | Time after vaccination (days) | |||
---|---|---|---|---|
60 | ||||
Positive | Prevalence rate (%) | 95%CI | ||
Rev 1 | Vaccinated | 1/27 | 2.7 | 0.49–14.1 |
Control | 0/4 | 0.0 | 0.0–0.49 | |
RB51 | Vaccinated | 0/1 | 0.0 | 0.0–0.79 |
Control | 0/0 | w.d.* | w.d.* | |
RB51-SOD | Vaccinated | 0/2 | 0.0 | 0.0–0.66 |
Control | 0/0 | w.d.* | w.d.* |
Group/subgroup | Time after vaccination (days) | |||
---|---|---|---|---|
90 | ||||
Positive | Prevalence rate (%) | 95%CI | ||
Rev 1 | Vaccinated | 1/23 | 2.7 | 0.49–14.1 |
Control | 0/4 | 0.0 | 0.0–0.49 | |
RB51 | Vaccinated | 0/1 | 0.0 | 0.0–0.79 |
Control | 0/0 | w.d.* | w.d.* | |
RB51-SOD | Vaccinated | 0/2 | 0.0 | 0.0–0.66 |
Control | 0/0 | w.d.* | w.d.* |
Group/subgroup | Time after vaccination (days) | |||
---|---|---|---|---|
365 | ||||
Positive | Prevalence rate (%) | 95%CI | ||
Rev 1 | Vaccinated | 0/5 | 0.0 | 0.0–0.43 |
Control | 0/9 | 0.0 | 0.0–0.29 | |
RB51 | Vaccinated | 1/4 | 2.7 | 0.49–14.1 |
Control | 1/9 | 2.7 | 0.49–14.1 | |
RB51-SOD | Vaccinated | 0/0 | w.d.* | w.d.* |
Control | 1/7 | 2.7 | 0.49–14.1 |
4. Discussion
Goat herds in the present study had similar conditions of feeding, handling, and confinement. Each group was exposed to animals infected with
The permanent vaccination program for goat herds has been operating in the area since 1994 achieving the requirements for the control phase according to the Official Mexican Standard (NOM-041-ZOO-1995) National Campaign against brucellosis in animals. These findings may suggest that the vaccine used is not protecting all animals, the vaccine is not properly managed or injected, or vaccination is not timely applied, resulting in the possibility of maintaining infection in the animals. Furthermore, the animal may not develop the infection, but the immune response capability is then detected by the diagnostic screening test without being a truly infected animal. As a result, the recognized agglutination serological tests (RSBT) leads to diagnostic confusion determining infected animals to remain in the herds. Hence, it is necessary to evaluate the vaccine strain to be used in the brucellosis control programs, since the results shown in Table 1 demonstrate that more than 50% of the animals reacted to the screening test, but are not infected as shown by the SRD test (Tables 7–9), which possess a greater sensitivity. This situation determines the need to invest in confirmatory tests [25, 26, 27, 28, 29].
When vaccinated groups of goats were evaluated by the RSBT, animals vaccinated with Rev. 1 strain had a seroconversion rate of 77.7% (95%CI: 61.9–88.2), 72.2% (95%CI: 56.0–84.1), 63.8% (95%CI: 47.5–77.5), and 13.8% (95%CI: 6.0–28.6) at 30, 60, 90, and 365 days post vaccination, respectively (Tables 3
As observed in Tables 3
El Idrissi et al. compared the vaccine efficacy of Rev. 1 and RB51 strains in sheep. Considering seroconversion, they conclude that after vaccination, all sheep vaccinated with Rev. 1 were positive to RBPT and complement fixation test (CFT) at 2 weeks, reaching their maximum between 2 and 6 weeks [7]. Then the percentage decreased and was zero 14 weeks after challenge. Animals vaccinated with RB51 did not produce anti-O side-chain antibodies, as measured by RBPT and CFT. After exposure to challenge, anti-O side-chain antibodies, measured by RBPT, were detected in the serum of vaccinated animals and controls [19].
Out of the animals vaccinated with RB51-SOD strain, 2/36 were seroconverted, representing 5.5% (95%CI: 1.5–18.1) (Tables 3
The RB51-SOD strain was obtained from
5. Conclusion
When evaluating the Rev. 1, RB51, and RB51-SOD vaccine strains, seroconversion in animals vaccinated with Rev. 1 strain was higher than that shown by the strains RB51 and RB51-SOD by conventional serological tests in infected herds during the evaluated period. Therefore, vaccination with Rev. 1 originates the need to perform confirmatory tests causing an increase in diagnosis costs. According to results of the present study, the RB51-SOD vaccine represents an alternative for controlling one of the most important worldwide zoonosis in goats. However, further studies are required to evaluate the performance of immune response, vaccine safety, and efficacy at field level.
Acknowledgments
We thank the state committee for the promotion and protection of livestock of Veracruz and the product system goats of Veracruz. This work was supported by SEP-PRODEP research grant project [DSA/I103.5/I5/14220] “Support for the integration of thematic networks of academic collaboration.”
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