JOURNAL OF CLINICAL MEDICINE OF KAZAKHSTAN (E-ISSN 2313-1519)

Original Article DOI: 10.23950/1812-2892-JCMK-00802

Comparative study of in vitro

prepared Rose Bengal Plate Test
(RBPT) antigen with commercially
available antigens
Mujeeb ur Rhaman1, Amir Ullah2, Junaid Ali Shah3
1College of Life Sciences, Northwest University, Xi’an, Shaanxi province, China.
2Department of Laboratory Medicine, Southern Medical University, Guangzhou, China.
3College of Life Sciences, Jilin University, China Changchun city, jilin Province

Received: 2020-06-18. Abstract

Accepted: 2020-08-03 Background and aim: Brucellosis is one of the world most common
zoonotic diseases. The current study was aimed to prepare the Rose
Bengal Plate Test (RBPT) antigen for the diagnosis of brucellosis and to
determine its specificity and sensitivity.
Material and methods: The Rose Bengal plate test antigen
prepared from Brucella abortus (B. abortus) strain 99 was compared
with two commercial Rose Bengal Plate Test antigens and its specificity
and sensitivity are determined.
Results: The were Rose Bengal plate test and I-ELISA result show
that the in vitro antigen was superior to RBPT antigen University
Diagnosis Laboratory (UDL) Lahore Pakistan, and RBPT antigen
Veterinary Laboratory Agency (VLA) UK. Out of 196 samples analyzed by
in vitro RBPT antigen, RBPT antigen (UDL), RBPT antigen (VLA), and an
indirect enzyme-linked immunosorbent assay (I-ELISA) 56.63 %, 53.57%,
41.84%, 35.71% were found B. abortus positively. The sensitivity calculated
for the in vitro RBPT antigen was 96.62, while RBPT antigen (UDL) and
RBPT antigen (VLA) were 89.77, 63.91 correspondingly. However, the
specificity of the in vitro RBPT antigen was lower (77.57%), than the
commercial RBPT antigen (VLA) (79.79%).
Conclusions: A very sensitive and low-cost in vitro RBPT antigen
compared to commercial RBPT was magnificently developed in
J Clin Med Kaz 2020; 5(59):46-50
the current study. It was determined that the in vitro RBPT antigen
could substitute the available commercial RBPT antigen, which is
Corresponding author: comparatively expensive and less sensitive in the detection of brucellosis.
Mujeeb ur Rahman. Therefore, it is concluded that the in vitro RBPT antigen could be used
Email: [email protected]
for epidemiological surveillance of brucellosis.
Key words: brucella, antigen, diagnosis, serology, animals

Introduction like the Middle East and Mediterranean region, Mexico,

Brucellosis is a major globally re-emerging parts of Central and South America and South Asian
zoonosis, which mostly affects domestic animals such countries including India, Pakistan, Srilanka and China
as, cattle, goat, sheep, swine, buffalo, and dogs caused [9-11]. Although in very few economically developed
by Brucella species [1, 2]. They are Gram-negative, countries this disease is controlled, it is still an issue that
non-hemolytic, non-motile, non-spore-forming and causes a significant globally economic loss [12]. Fetal
facultative intra-cellular living, coccobacilli. However, membrane retention (FMR) and last trimester abortion
Brucellae demonstrate a preference for a certain host, e.g. are the characteristic signs in female animals whereas
Brucella Melitensis prefers small ruminants, B. abortus epididymitis and orchitis are common in males but the
bovine, the transmission of cross-species occurs when infection may remain asymptomatic and the infected
various animals are in near contact with one another [3- animals may remain undiagnosed [13]. Through milk
8]. Brucellosis is endemic in several geographical regions and vaginal secretions, the infected animals shed bacteria

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46
in the environment [14]. In animals, brucellosis is typically kill bacteria and mixed with 1% Rose Bengal. The suspension
transmitted either through direct contact or by the ingestion of was stored at 4℃.
contaminated water or feed, while in humans this usually occurs The prepared antigen and serum were kept at room
through the ingestion of contaminated milk [15,16]. Human temperature (22±4℃). Initially, 20 µl serum was added on
beings are accidental hosts for this infectious disease and can the white porcelain plate through a pipette. Then, 20 µl of that
be prevented by eradicating the disease in animals, which often antigen was mixed with the serum and shaken for four minutes.
have close interaction with humans [17,18]. The data was recorded as positive after agglutination. The
The diagnosis of brucellosis verified by isolation which I-ELISA was used as a gold standard to determine the sensitivity
is the gold standard but this process is time-consuming, and specificity of RBPT antigen.
laborious, low sensitivity and additionally there is a high risk of
infection. Hence, serological test including Rose Bengal Plate Data analysis
Test (RBPT), Enzyme-Linked Immunosorbent Assay (ELISA), The specificity and sensitivity were determined by using
Indirect Enzyme-Linked Immunosorbent Assay (iELISA) and the following formula [26]:
Complement Fixation Test (CFT) is normally used for the
diagnosis of Brucella [19-21]. As no serological test is 100% True Positive
reliable, diagnosis is usually based on two or more test results. Sensitivity = ×100
True Positive + False negative
Consequently, early testing is usually done using a screening
test, which is a highly sensitive test and perhaps less specific. True Positive
The screening tests are typically cost-effective, quick and easy Specificity = ×100
True Positive + False negative
to perform. If a positive reaction occurs during a screening
test, a confirmatory test is carried out. The confirmatory test is
a kind of test that offers good sensitivity but relatively higher
test specificity, thus eliminating some false positives reaction. Results
The majority of confirmatory tests are more complex and very Brucella isolate was grown on selective Tryptic Soy
expensive to carry out [22]. Agar for morphological examination and incubated for 2-3
The Rose Bengal plate test is a rapid test that was originally days. The Brucella colonies were characterized as smooth,
developed for screening use in veterinary medicine but is now glistening, pinpoint, bluish, honey-colored and translucent [27].
also used to diagnose human brucellosis [23,24]. Its high Microscopically, the culture smear appeared as gram-negative
sensitivity, ease of use and affordable prices make it extremely coccobacilli in Gram staining and red-stained coccobacilli in
common in hospital emergency departments for the diagnosis of modified Z-N staining. Therefore, these finding shows the
febrile syndromes. resemblance with bacteria characteristic of Brucella [28]. The
The present study aimed to develop RBPT antigens from isolate was positive to Oxidase, Catalase, and Urease tests and
Brucella abortus strain 99, compared with commercial PBPT negative to Methyl Red, Indole production, Simmon Citrate
antigens University Diagnosis Laboratory (UDL) Lahore, utilization and Voges-Proskauer tests, these results were in
Pakistan and Veterinary Laboratory Agency (VLA) UK and agreement with the previous study [29]. The in vitro prepared
finally, its specificity and sensitivity were determined. RBPT antigen was tested by mixing 20 µl of the sample with 20
µl of prepared antigen on a porcelain plate. In the presence of
Material and methods distinct agglutination the reaction was declared positive (Figure
This research study was conducted at Animal Sciences 1).
Institute, National Agriculture Research Center Islamabad,
Figure 1 - Rose Bengal Plate Test. Figure A representing a
Pakistan from October 2015 to December 2016. Blood samples
strong agglutination reaction.
were collected from goats and sheep at the slaughterhouse in
Islamabad. Approximately 5–7 mL of blood was obtained in a
transparent tube without any anticoagulant and placed on ice
immediately. All the specimens collected for animals had either
aborted or reproductive disorder such as infertility. Samples
were transported to the Animal Sciences Institute, bacteriology
laboratory after proper labelling and sealing. The sera were
isolated by centrifugation of blood samples at 1500 rpm for 10
mins and preserved at -20 ℃ for further analysis.
The culture of B. abortus strain 99 was inoculated on
Tryptic Soy Agar (TSA) media for three days at 37 ℃. Initially,
the Gram stain and then Ziehl-Neelsen (Z-N) staining was carried
out. Then, biochemical tests, such as Urease, Oxidase, Catalase,
Indole production, Methyl Red, Simmon Citrate utilization and Positive Reaction Negative Reaction
Voges-Proskauer tests were conducted for further confirmation.
The B. abortus strain 99 was used to prepare the RBPT The in vitro RBPT antigen was compared with RBPT
antigen. The antigen was prepared following the previous antigen (UDL), a total number of 196 samples were processed,
literature by Office International des Epizooties (2009) [25]. and the result explored that in vitro RBPT antigen was 111
Briefly, 4-5 colonies of B. abortus were inoculated into TSA (RBPT+) and 85 were (RBPT-), while the RBPT antigen (UDL)
broth media and incubated at 37 ℃ for 48 hours. The liquid showed that 105 were (RBPT+) and 91 were (RBPT-), as
media was centrifuged to get an isolated organism. The pellet mentioned in Table 1.
was re-suspended in 0.5% phenol saline. The mixture was
discarded, and the pellet was heated at 80℃ for 90 minutes to
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47
 Percentage of in vitro RBPT antigen and RBPT specificity of the brucellosis diagnosis [10]. The I-ELISA has
Table 1
antigen (UDL) been regarded as a gold standard by many researchers to compare
the results for brucellosis diagnosis [30]. In this study, I-ELISA
Sample In vitro Percentage RBPT antigen Percentage was used as a gold standard to calculate the specificity and
RBPT Ag (UDL)
sensitivity of in vitro RBPT antigen and two other commercial
RBPT (+) 111 56.63 105 53.57 RBPT antigens.
RBPT (-) 85 43.37 91 46.43 A study showed that out of 856 sera, 31.66% was positive
Total 196 196 by CFT using commercial RBPT and in-house RBPT detected
30.84% positive animals respectively [31]. However, the current
Ag=Antigen, RBPT=Rose Bengal plate test antigen, UDL=University Diagnosis
study indicates that out of 196 sera, 37.24 % was found to be
Laboratory, VLA=Veterinary Laboratory Agency
positive by I-ELISA , while the in vitro RBPT antigen, RBPT
antigen (UDL) and RBPT antigen (VLA) detected 56.63%,
The in vitro RBPT antigen was further compared with the 53.57%, 41.83%, positive animals, respectively. Moreover, we
RBPT antigen (VLA), and the results showed that 82/196 were observed that 85 (in vitro RBPT), 91 (RBPT antigen (UDL), 114
(RBPT+) and (RBPT-) was 114/196 (Table 2). (RBPT antigen (VLA) sera were negative with in vitro RBPT
while some of them were positive with I-ELISA. The false-
negative result of the RBPT antigen could be the “Zoning effect”
Percentage of in vitro RBPT antigen and RBPT
Table 2 in acidified RBPT antigen [32]. An additional factor which may
antigen (VLA)
lead to false-negative result can be the outdated RBPT antigen.
The sensitivity of the RBPT antigen could be lost during the
Sample In vitro Percentage RBPT antigen Percentage
RBPT Ag (VLA) storage process by the improper addition of reagents.
A study reported that the in-house RBPT was (85.24%)
RBPT (+) 111 56.63 82 41.84
sensitive as compared to commercial RBPT (78.59%), but the
RBPT (-) 85 43.37 114 58.16
commercial RBPT (97.77%) was more specific than the in-
Total 196 196 house RBPT (94.36%) [31]. The present study explored that
the in vitro antigen (96.62%) was more sensitive compared to
Finally, in vitro RBPT antigen was compared with the RBPT antigen (UDL) (89.77%) and RBPT antigen (VLA)
I-ELISA, the results were as follows; ELISA+ 73/196, and (63.91%). Nevertheless, the RBPT antigen (VLA) (79.79%)
ELISA- 123/196 (Table 3). is more specific than the in vitro RBPT antigen (77.57%).
However, we reported 111 (in vitro antigen), 105 (RBPT antigen
Percentage of in vitro RBPT antigen and (UDL) and 82 (RBPT antigen VLA) out of total sera samples
Table 3 were RBPT positive but some of them were I-ELISA negative.
I-ELISA
These false-positive results may be due to the vaccination of
Sample In vitro Percentage I-ELISA Percentage B. abortus strain 19 or exposure to a gram-negative organism
RBPT Ag
having lipopolysaccharide (LPS) O-chain similar to Brucellae
RBPT (+) 111 56.63 73 37.24 species, like E. coli O:157, V. cholerae O1, Y. enterocolitica
RBPT (-) 85 43.37 123 62.76 O:9 and Salmonella group N (O:30) [33]. The current study
Total 196 196 suggested that the sensitivity of the in vitro antigen was more
significant than previous studies, which were 85.2% and
90.1% respectively [31,34]. The possible reason may be the
The sensitivity and specificity of in vitro RBPT antigen, optimization methods achieved using international imported
RBPT antigen (UDL) and RBPT antigen (VLA) were tested. B. abortus serum to determine the specificity and sensitivity of
The results showed that the sensitivity of in vitro RBPT antigen developed in vitro antigen.
was 96.62 %, which was higher than UDL 89.77 %, while the
specificity was high potent in VLA 79.79 % than in vitro RBPT
Conclusion
Ag 77.57 % (Table 4).
This study suggests that the in vitro RBPT antigen which
is a low cost, rapid and has high sensitivity as compared to the
Specificity and sensitivity of in vitro RBPT Ag,
Table 4
RBPT antigen (UDL) and RBPT antigen (VLA) commercial RBPT antigens. Finally, this diagnostic technique
recommended replacing the commercially available RBPT
S. No Antigens Sensitivity (%) Specificity (%) antigen, which is relatively costly and less sensitive in the
1 In vitro RBPT 96.62 77.57 identification of brucellosis in sheep and goats.
Ag
2 RBPT antigen 89.77 73.83 Disclosures: There is no conflict of interest for all authors.
(UDL)
3 RBPT antigen 63.91 79.79 Acknowledgement: The authors are very thankful to the
(VLA) Animal Sciences Institute (ASI), National Agriculture Research
Center (NARC) Islamabad, Pakistan, for their collaboration.
Discussion Funding: none
Brucellosis is a zoonotic bacterial disease caused by
Brucella genus. In the present study, RBPT antigen was prepared
from B. abortus strain 99. There are many serological tests uses
for the determination of brucellosis, but we prefer RBPT, which
has considerably high sensitivity while I-ELISA used for the
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48
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