Evaluation of three different antigens in an indirect enzyme-linked immunoassay for the detection of antibodies against Brucella abortus SRB51 in vaccinated heifers

Robles, Carlos Alejandro; Nielsen, Klaus; Gall, D.; Willems, Priscila

doi:10.1016/j.vetimm.2008.09.007

Cited by 8 publications

(9 citation statements)

References 15 publications

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“…In our experiment, direct dialysis of phenol phase containing RLPS after evaporation of chloroform and petroleum ether as explained by Nielsen et al (2004) and Robles et al (2009) resulted in accumulation of RLPS in a small amount of phenol which remained inside the dialysis bag even after 5 days leading to poor yield in aqueous phase. Thus, we induced precipitation of RLPS in phenol phase by cold methanol reagent (Moreno et al, 1979) which facilitated separation from phenol and increased our yield.…”

Section: Discussionmentioning

confidence: 65%

“…However, due to the roughness of RB51 vaccine, these tests in which smooth antigens are used cannot be useful. Rough lipopolysaccharide of RB51 vaccine strain has been successfully utilised to develop serological test for detection of antibody responses to the vaccine strain (Nielsen et al, 2004;Robles et al, 2009). While hot phenol-water method has been used for the preparation of LPS from rough brucellae (Kreutzer et al, 1979;Kreutzer & Robertson, 1979;Moreno et al, 1979;1984;Kianmehr et al, 2015) including strain RB51 (Schurig et al, 1991), phenol-chloroform-petroleum ether method was reported as a specific method for preparation of rough Brucella LPS (Jones et al, 1973) and utilised by many other researchers for extraction and characterisation of LPS from rough Brucella mutants and strains (Freer et al, 1995;González et al, 2008;Fontana et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…While hot phenol-water method has been used for the preparation of LPS from rough brucellae (Kreutzer et al, 1979;Kreutzer & Robertson, 1979;Moreno et al, 1979;1984;Kianmehr et al, 2015) including strain RB51 (Schurig et al, 1991), phenol-chloroform-petroleum ether method was reported as a specific method for preparation of rough Brucella LPS (Jones et al, 1973) and utilised by many other researchers for extraction and characterisation of LPS from rough Brucella mutants and strains (Freer et al, 1995;González et al, 2008;Fontana et al, 2016). In addition, the RB51 RLPS used in development of ELISA assays for evaluation of post-vaccination serological responses to RB51 vaccine was prepared by the latter method (Nielsen et al, 2004;Robles et al, 2009). Therefore, we used phenol-chloroformpetroleum ether method for extraction of RLPS from B. abortus strain RB51 and since no information was available about characterisation of RB51 RLPS prepared by this method, immunochemical characterisation was done.…”

Section: Discussionmentioning

confidence: 99%

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Extraction and characterisation of Brucella abortus strain RB51 rough lipopolysaccharide

A.¹,

Farivar²,

Peymani³

et al. 2019

BJVM

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Brucellosis is an important zoonotic disease with considerable impacts on human and animal health. Brucella abortus strain RB51 vaccine is used for prevention of bovine brucellosis in Iran. Due to strain roughness, available serological tests cannot detect vaccinated animals. Detection of serological responses to the vaccine is important to monitor accurate vaccination implementation. Rough lipopolysaccharide (RLPS) of RB51 strain was extracted and characterised to develop serological tests for diagnosis of vaccinated animals. RLPS was extracted using phenol-chloroform-petroleum ether and evaluated by limulus amebocyte lysate (LAL) assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and agar gel immunodiffusion (AGID). According to our results, the extracted RLPS caused positive reaction in LAL assay. In SDS-PAGE, a band with a molecular weight around 14 kDa was identified after specific staining using silver nitrate. Double AGID of the RLPS with a hyperimmune serum resulted in a precipitation line formation. Our study showed that the method can be successfully used to extract RLPS from Brucella abortus strain RB51 as confirmed by LAL assay, PAGE and AGID.

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Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Extraction and characterisation of Brucella abortus strain RB51 rough lipopolysaccharide

A.¹,

Farivar²,

Peymani³

et al. 2019

BJVM

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show abstract

“…Recently, with the gradual use of RB51 vaccine, it became necessary to develop a method capable of identifying animals vaccinated with RB51 vaccine (Robles et al, 2003). This include a dot-blot assay using killed irradiated RB51 bacteria as an antigen , iELISAs using a 5% optical density heat-killed whole RB51 organisms as an antigen (Edmonds et al, 1999), and a crude rough LPS preparation from RB51 (Uzal et al, 2000), Dot-Blot ELISA (Fosgate et al, 2003;Diptee et al, 2007), an immunoblot analysis using sonicated cell lysates from RB51 (Edmonds et al, 1999), a complement fixation test, using RB51 cultured cells in calcium-magnesium-veronal buffer (Diptee et al, 2007;Galiero, 2009;Caporale et al, 2010) and an agar gel immunodifusion test using hot saline extract antigen from Brucella ovis (Robles et al, 2009). Unfortunately all these methods were unpractical and time consuming.…”

Section: Discussionmentioning

confidence: 99%

“…To overcome the problem of serological interference, RB51, a mutant vaccinal rough strain that is devoid of the LPS O-side chain was developed (Poester et al, 2006). Consequently, cattle vaccinated with RB51 do not seroconvert on conventional brucellosis serologic tests (Robles et al, 2009). Therefore, this vaccine is more appropriate than B. abortus S19 for control and eradication programs that rely on serologic testing and removal of positive animals.…”

Section: Introductionmentioning

confidence: 99%

The immunological response of RB51 vaccinated buffalo calves using brucella periplasmic proteins as ELISA antigen

El-Razik¹,

El-Hafez²,

Shell³

et al. 2011

Afr. J. Biotechnol.

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Immune status of RB51 vaccinated buffaloes was evaluated using tube agglutination test (TAT) and ELISA, using both periplasmic protein antigen (PPA) and lipopolysaccharide antigen (LPS). For this purpose, three groups of buffalo calves were used. The first one received S19 vaccine subcutaneously; the second was vaccinated once subcutaneously with RB51 vaccine. The third (control) group was injected similarly with sterile saline. Concerning the S19 vaccinated group, significant TAT titers were seen 1 week post vaccination (WPV) till the maximum at the 2 nd WPV. After that it was gradually decreased till the 7 WPV, then sharply before it completely disappeared at the 13 WPV. On the other hand, the LPS-ELISA showed an antibody titer as early as one WPV reached its peak at 2 WPV and persisted steadily till the 6 th WPV and decreased slowly when it reached minimal level at the 16 WPV till the end of the experiment. While in RB51 vaccinated buffalo calves using the PPA-ELISA, the antibody titer began and reach the maximum as early as the first WPV, still steady till 2 WPV, fluctuating till the 6th WPV, then dropped sharply when it disappeared at 11WPV till the end of the experiment.

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Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine

Ramnanan¹,

Diptee

Asgarali

et al. 2012

Trop Anim Health Prod

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Thirty-two water buffalo (Bubalus bubalis) calves aged 6–10 months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose-response study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I-III received the recommended vaccine dose (RD) twice 4 weeks apart, RD twice 18 weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16 weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson's correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P > 0.05).

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Evaluation of three different antigens in an indirect enzyme-linked immunoassay for the detection of antibodies against Brucella abortus SRB51 in vaccinated heifers

Cited by 8 publications

References 15 publications

Extraction and characterisation of Brucella abortus strain RB51 rough lipopolysaccharide

Extraction and characterisation of Brucella abortus strain RB51 rough lipopolysaccharide

The immunological response of RB51 vaccinated buffalo calves using brucella periplasmic proteins as ELISA antigen

Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine

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