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American Journal of Infectious Diseases
Year: 2009  |  Volume: 5  |  Issue: 1  |  Page No.: 11 - 16

Serological Evaluation of Brucella abortus S99 Lipopolysaccharide Extracted by an Optimized Method

Ali Sharifat Salmani, Seyed Davar Siadat, Mohammad Reza Fallahian, Hojat Ahmadi, Dariush Norouzian, Parichehr Yaghmai and Mohammad Reza Aghasadeghi    

Abstract: Problem statement: Brucellosis is a globally found infectious disease and there is no licensed vaccine against human brucellosis. The present study carried-out to evaluate the potency of our modified extracted lipopolysaccharide (LPS) of B. abortus to elicit specific anti-Brucella antibodies in animal model (Rabbit) as a part of a candidate vaccine for brucellosis. Lipopolysaccharide is one of the main virulence factors and the most immunogenic structure of smooth strains of Brucella. Approach: Lipopolysaccharide of B. abortus S99 (S-LPS) initially extracted through an optimized method as described previously. After biochemical and pyrogenicity evaluations of the extracted S-LPS humoral immune response against the extracted LPS analyzed in animal model through serological assays such as Rose Bengal assay, Rapid agglutination (Rapid Wright) test and Standard agglutination test (SAT or Wright test) to demonstrate the specific elicited antibodies against the injected LPS. In addition, the interaction of LPS and anti-LPS antibodies was demonstrated by Agarose Gel Immunodiffusion (AGID) assay. Results: Higher doses of B. abortus S99 LPS caused less or equal body temperature increase in comparison to E. coli LPS doses. Sera of immunized animals had been reported positive by RBT because of B. abortus LPS immunogenicity which we extracted through our optimized method. The highest titer of anti-Brucella antibodies detected two weeks after the third immunization (assayed by rapid slide agglutination and standard agglutination tests). Anti-Brucella antibodies of immunized animals reacted more specifically with the LPS of B. abortus in comparison with E. coli LPS and precipitation lines between B. abortus LPS and immune sera appeared after 30 min while detected after three hours for E. coli LPS. Conclusions/Recommendations: The properties of B. abortus S99 LPS concluded from the present study results, suggest the possible use of this component as a carrier or a part of a sub-unit or conjugated vaccine for human brucellosis.

Fig. 1 shows, higher doses of B. abortus S99 LPS causes less or equal body temperature increase in comparison to E. coli LPS doses. According to this graph, 10-1 mg mL-1 of B. abortus S99 LPS caused 6°C rise of body temperature following the injection while 10-5 mg mL-1 of E. coli LPS increased equal body temperature.

RBT: As the Table 1 shows, sera of immunized animals have been reported positive by RBT while the negative control serum (sample taken before immunization) did not react with rose bengal antigen (agglutination was not observed).

Rapid slide agglutination test: Titer of anti-Brucella antibodies in the sera of immunized animals, taken 2 weeks after the first, second and third immunization reported to be 160, 320 and 640, respectively while no agglutination has been observed in the negative control (Table 2).


Fig. 1:

Rabbit pyrogen test. Increased body temperatures (from the baseline) of animal models injected with B. abortus S99 LPS and E. coli LPS at the doses indicated. Ba-LPS: B. abortus LPS, Ec-LPS: E. coli LPS


Table 1: Results of RBT

Table 2: Results of rapid slide agglutination test

Table 3: Results of tube agglutination test

Fig. 2:

The Increase of anti-B. abortus antibody titer after booster injections (assayed by standard agglutination test)

Standard agglutination test: As the Table 3 shows, no agglutination ring appeared at the bottom of the negative control tube after 24 h. The highest titer of anti-Brucella antibody has been shown in the sample that was taken 2 weeks after the third immunization (1280) while the titer of samples taken 2 weeks after the first and second immunization reported to be 320 and 640, respectively (Fig. 2).

AGID test: The precipitation reaction between the pooled serum of immunized animals and the extracted LPS of B. abortus S99 has been observed after 30 min but this reaction did not occur between standard LPS of E. coli and the pooled serum of immunized animals in the same period of time. Finally after three h a thin line between E. coli LPS and the pooled serum appeared, indicating some structural similarities between the LPS of B. abortus S99 and E. coli LPS, while it was thick and sharp between B. abortus S99 LPS and the pooled serum because of the specificity of synthesized antibodies against the LPS of B. abortus S99 (Fig. 3).


Fig. 3:

AGID test confirms the specificity of synthesized antibodies in the animal model for the extracted LPS of B. abortus S99. Also after 3 h a thin line observed between pooled serum of immunized animals and E. coli LPS. 1: LPS of E. coli, 2, 3, 4: LPS of B. abortus S99

DISCUSSION

Lipopolysaccharide (LPS) is the main expressed antigenic structure at the surface of smooth strains of Brucella abortus. In the present study we analyzed the efficacy of the extracted LPS of Brucella abortus S99 (extracted through our optimized method) [14] to induce the anti-Brucella antibodies in the animal model following the intramuscular injection of this antigen and two booster injections. Sera of all the immunized animals have been detected as positive samples, indicating the potency of this extracted LPS of Brucella abortus S99. The highest antibody titer has been detected 2 weeks after the second injection (demonstrated by tube and slide agglutination methods and has been reported to be 640 and 1280, respectively). Booster injections have efficiently increased the titer of elicited antibodies against Brucella and there was a considerable rise in the assayed titer of analyzed serum samples (Fig. 2). The results of Rose Bengal Test (RBT) has been in parallel with Serum agglutination assay results but as RBT is not a quantitative method it may be applied only to detect positive and negative samples and should be followed by agglutination assays to detect the titer of samples.

Currently Serum Agglutination Test (SAT) is the most commonly used and accepted assay to detect the titer of anti-Brucella antibodies [17]. Other serology methods such as rose bengal and agarose gel immunodiffusion tests may be applied to distinguish positive and negative serum samples (but not the titer). Since the tube agglutination assay is a time-consuming assay and the process of this method takes at least 24 h, slide (rapid) agglutination assay may be applied to detect the titer of anti-Brucella antibodies in the serum of patients and also in the animal models. In the present study, tube agglutination assay results were in parallel with the slide (rapid) agglutination results but there were one titer difference between these two methods results.

In the present study the specificity of synthesized antibodies in the sera of immunized animals for B. abortus S99 LPS assayed by agarose gel-Immunodiffusion method in comparison to E. coli LPS. As it is mentioned in the Results, pooled serum of immunize animals react rapidly with B. abortus S99 LPS during the first 30 min of the test and a sharp precipitation line observed between the wells containing B. abortus S99 LPS and the well containing pooled serum of immunized animals, while no precipitation occurred between the pooled serum and E. coli LPS during the same time. Interestingly a thin line of precipitation between pooled serum and E. coli LPS observed after 3 h. Since the animals have been injected with B. abortus S99 LPS (not E. coli LPS) it could be concluded that this relative reaction may be a sign of some structural similarities between B. abortus S99 LPS and E. coli LPS. It is also previously reported that the main structural differences between Brucella and E. coli LPS is in the O chain of these two kind of LPS and other parts of this structure is almost the same in Brucella and E. coli. These structural similarities may lead to a poor cross-reaction between the antibodies elicited against B. abortus S99 LPS with E. coli LPS and it seems to be the main reason of reactivity of pooled serum of immunized animals with E. coli LPS in this study.

AGID test has been previously applied to detect positive serum samples of infected dogs and cattle with rough strains of B. canis and B. melitensis 16 M, respectively and reported to be a sensitive, rapid and reliable method[18]. In the present study, we showed that this method would be efficiently used for smooth strains of Brucella such as B. abortus S99.

Rabbit pyrogen test is one of the Invivo assays to evaluate the pyrogenicity of biologic compounds such as LPS and other bacterial antigens. In the present study we evaluated the pyrogenicity of our extracted LPS of B. abortus S99 in comparison with E. coli LPS. As the Fig. 2 shows injection of low doses of E. coli LPS cause higher increase of body temperature in comparison to B. abortus LPS which confirms that E. coli LPS is more toxic and pyrogenic than B. abortus S99 LPS. As the graph shows, 10-1 mg mL-1 of B. abortus S99 LPS and 10-5 mg mL-1 of E. coli LPS have equally increased the body temperature following the injection. These data confirm previous study and the fact that Brucella abortus LPS is 10,000 fold less pyrogenic than E. coli LPS [15]. Nowadays decreasing the pyrogenicity of immunogenic compounds with microbial origins is one of the strategies to make them applicable for Invivo trials and immunization. Unfortunately, the process of decreasing the pyrogenicity usually leads to less immunogenicity, too. According to this fact, the application of B. abortus S99 LPS for Invivo and immunization aims would be advantageous because of its low pyrogenicity and high potency to induce antibodies against Brucella.

CONCLUSION

This modified extracted LPS of B. abortus S99 has efficiently promoted the synthesis of high levels of anti-Brucella antibodies. Furthermore, elicited antibodies reacted specifically with the extracted LPS which has been intramuscularly administrated to animal models (demonstrated by AGID).

Potency of LPS to induce high titers of specific antibodies against Brucella in parallel with low pyrogenicity of this cellwall structure (defined by rabbi pyrogen test) suggests the possible application of this component as a part of a sub-unit or conjugated vaccine for human brucellosis.

ACKNOWLEDGEMENT

The researchers would like to thank all colleagues in the Department of Bacterial Vaccines and Antigens Production, Pasteur Institute of Iran, for their kind assistance and advices in the laboratory.

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