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Validation of Different Versions of The Card or Rose-Bengal Test for The

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DOI: 10.5455/ajvs.266638

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 Alexandria Journal of Veterinary Sciences
www.alexjvs.com
AJVS. Vol. 53(1): 187-202. April 2017
DOI: 10.5455/ajvs.266638

Validation of Different Versions of The Card or Rose-Bengal Test for The Diagnosis of
Brucella melitensis Infection in Ruminants
Ashraf E. Sayour, Essam M. Elbauomy, Nour H. Abdel-Hamid*, Mohammad K. El-Kholi,
Rania I. Ismail and Eman I. M. Beleta
Department of Brucellosis Research, Animal Health Research Institute, Dokki, Giza 12618, Egypt

ABSTRACT
Tested was a total of 543 sera from unvaccinated ruminant species, viz. cows, buffalo
Key words: cows, ewes, female goats and she-camels in seven Nile Delta governorates. Most
BCT, RBPT, CFT, animals had a history of Brucella melitensis biovar 3 infection. This study was to
Brucella, cow, buffalo, investigate the fitness of Rose-Bengal plate test (RBPT) formats namely the US
ewe, goat, camel. brucellosis card (BCT) 8% and 3%, the UK RBPT and its modification, the Scottish
RBPT and the French RBPT 4.5% for pre-confirmation of B. melitensis infection in non-
bovine ruminants. Taking the complement fixation test (CFT) as the gold standard, the
Correspondence to: performance of RBPT assays and the buffered acidified plate antigen test was estimated
Nour H. Abdel-Hamid, e- as relative sensitivity, accuracy, kappa agreement with CFT and association with CFT
nour78_78@yahoo.com based on Pearson's chi-squared test and Phi coefficient. Almost all acidified
agglutination test formats were generally associated with CFT. The best performance of
RBPT in cows, buffalo cows, ewes, female goats and she-camels was reached by the
Scottish, the French, the French, the UK and the Scottish RBPT respectively. From the
animal inter-species perspective, the performance of the BCT 8% considerably fell in
buffaloes and goats improving greatly with its 3% version, but having extra drop in
goats. The performance of the UK RBPT dropped in sheep and drastically fell in camels
with its modification achieving some progress in buffaloes, sheep and camels, and minor
drop in cattle and goats. The Scottish RBPT slightly dropped in buffaloes but noticeably
in goats with good performance in cattle and camels. The French RBPT drastically fell
in goats and less severely in camels. It was concluded that enhancing the sensitivity of
the RBPT resulted in overall performance promotion especially in sheep, but still
insufficiently accurate requiring some intervention in goats, buffaloes and camels in
descending order of urgency.
1. INTRODUCTION cells are suspended and standardized. The test
brings about agglutination of the non-
Among the rapid agglutination assays for agglutinogenic IgG1 distinctive of the
brucellosis surveillance are the buffered acidified longstanding Brucella infection (Alton et al.,
plate antigen (BAPA) and the Rose-Bengal plate 1988). This adds up for more sensitivity and
tests. The Rose-Bengal or brucellosis card test specificity to the test. Used after the presumptive
(BCT) is a rapid qualitative one-dilution plate BAPA test, the Rose-Bengal plate test (RBPT)
agglutination at acidic pH of 3.65±0.05 attained reduces the number of positive samples
by lactate buffered phenol saline in which demanding confirmation. Each of these tests is
inactivated Rose-Bengal stained Brucella abortus

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 Sayour et al. 2017. AJVS 53(1): 187-202

recommended for international trade in the OIE isolates including 44 B. melitensis, 28 B. abortus
Terrestrial Manual (2016a). and 5 B. suis isolates from milking bovines
(Sayour and Sayour, 2015). B. melitensis was
There are different versions of the Rose-Bengal also recovered from Egyptian camelids
test worldwide depending on the Brucella (unpublished data).
epidemiological status of the country, the battery
of immunoassays used, the animal species under The aim of this investigation was to study the
test and whether series/ parallel interpretation of fitness of different Rose-Bengal test formats for
serologic results is adopted. Among the Rose- the purpose of pre-confirmation of B. melitensis
Bengal test versions are the American BCT using infection in non-bovine domestic ruminants under
8% packed cell volume for large ruminants (Alton the brucellosis epidemiological status quo. This
et al., 1988) and 3% cells for small ruminants is part of achieving accreditation nuts and bolts of
(Mikolon et al., 1998), the British version the ISO/IEC 17025:2005 concerned with the
adjusted to give weak agglutination with the OIE general requirements for the competence of
International Standard Serum for Brucella abortus testing and calibration laboratories.
(OIEISS) at 1/45 and negative result with the
OIEISS diluted at 1/55 as per the European
directive CEE 64/432 (OIE Terrestrial Manual,
2016a), the test modification by Blasco et al. 2. MATERIAL AND METHODS
(1994), the old French version with 4.5% cell 2.1. Animals
concentration (Pilet et al., 1972; Toma et al.,
1972), and a Scottish commercial version for A total of 543 serum samples from different
humans (Lennette, 1985). ruminant species were either selected from the
serum collection of the Department of Brucellosis
The OIE guidelines (OIE Terrestrial Manual, Research, Animal Health Research Institute,
2016a) allow the application of serologic tests Dokki, Giza, Egypt (Table 1) or otherwise
originally standardized for cattle to be used for collected from abattoirs. Most of these sera were
other ruminants like buffaloes, sheep, goats and serologically positive and belonged to animals in
camels following proper validation. It is known the Nile Delta governorates. Most of the bovine,
that B. abortus mainly infects large ruminants, ovine and caprine positive sera had a history of
while B. melitensis usually infects small Brucella melitensis biovar 3 infection (Sayour and
ruminants (Alton et al., 1988). This is not the Sayour, 2015). There was no history of
case in Egypt where B. melitensis is predominant vaccination against brucellosis. Only some
among livestock as proven by the detection of 93 animals were reported to have late abortion and
B. melitenssis isolates among a total of 126 retained placenta.

Table 1. Epidemiologic data of ruminant sera included in the current study

Age
Animal spp. Breed Population Governorate No.
(year)
Native
Cows Friesian 1-3 Small/ large herds Sharkia/ Monofia/ Gharbia 117
Hybrid
Buffalo cows Native 1.5-4 Individuals/ small herds Beheira/ Monofia/ Gharbia 104
Ewes Native 1-4 Individuals/ small flocks Kafr El-Sheikh/ Sharkia 102
Female goats Native 1-3 Small flocks Beheira/ Sharkia/ Giza 112
She-camels Fellahi 2-3 Individuals Sharkia 108
Total animals 543

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 Sayour et al. 2017. AJVS 53(1): 187-202

2.2. Antigens for serologic tests

Table 2. Antigens for serologic tests

Standardization by either
Antigen pH Producer
PCV OIEISS
US BAPA 3.7 ± 0.03 11% No Veterinary Serum and Vaccine Research
US BCT 8% 3.65 ± 0.05 8% No Institute (VSVRI), Abbassia, Cairo, Egyp
US BCT 3% 3.65 ± 0.05 3% No Department of Brucellosis Research, Animal
French RBPT Weak positive at 1/40 of the Health Research Institute, Dokki, Giza, Egypt
3.65 ± 0.05 4.5%
4.5% OIEISS (Alton et al., 1988 and Mikolon et al., 1998)
Positive at 1/45 and negative at AHVLA (now APHA), New Haw, Addlestone,
UK RBPT 3.65 ± 0.05 No
1/55 of the OIEISS Surrey KT15 3NB, UK
Omega Diagnostics Ltd., Alva, FK12 5DQ,
Scottish RBPT 3.6 No Yes, details unknown
Scotland, UK
50% fixation with 1/200 of
US CFT ~7 4% NVSL/DBL, USDA, USA
the OIEISS
OIEISS = OIE International Standard Serum (previously the WHO Second International anti-Brucella abortus Serum)

2.3. Serologic tests Quality control and quality assurance were

fulfilled according to the requirements of the
2.3.1. Buffered acidified plate antigen (BAPA) ISO/IEC 17025:2005 and the OIE guidelines (OIE
and brucellosis card (BCT)/ Rose-Bengal plate Terrestrial Manual, 2016b).
(RBPT) test formats
2.4. Statistical analyses
The BAPA was performed according to Angus
and Barton (1984) and the OIE Terrestrial Manual The following analyses were performed using
(2016a). The BCT with 8% cells for large IBM® SPSS® Statistics, Version 21, IBM
ruminants and the BCT with 3% cells for small Corporation, 2012, under the environment of
ruminants are currently adopted by the NVSL, Windows® 8.1, Microsoft Corporation.
USDA (NVSL Reagent Manual, 2016). Except
for the modified British RBPT performed after 2.4.1. Kappa (κ) agreement and relative
Ferreira et al. (2003), other versions of the Rose- sensitivity/ specificity:
Bengal test were implemented according to Alton
et al. (1988). The kappa (κ) agreement of CFT as the gold
standard with other serologic tests was used to
2.3.2. Complement fixation test (CFT) assess the matching of results at p < 0.05.
Relative sensitivity/ specificity pairs were also
Complement and hemolysin were prepared and calculated.
preserved according to Alton et al. (1988) and
coping with Hennager (2004) (H. E. Stowell,
personal communication, November 22, 2010).
Sheep RBCs were collected on Alsever’s solution 2.4.2. Receiver operating characteristic (ROC)
from an adult healthy ram serologically negative curves:
to brucellosis. These were standardized to 2% Considering the CFT as the gold standard, ROC
suspension in veronal buffered saline (VBS). The curves expressing the sensitivity versus the false
proper test was performed according to the positive rate were plotted for all serologic
current American SOP by Hennager (2015). methods. Data obtained from ROC curves
Warm fixation of complement at 37°C was included the area under the curve (AUC)
adopted as cold fixation was unacceptably slow. representing the test method accuracy according
The positive cutoff point for CFT was ≥ 20 to Hanley and McNeil (1982). The AUC
ICFTU/ml. measures how well the test separates the positive

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 Sayour et al. 2017. AJVS 53(1): 187-202

from negatives without reference to a particular descending dilutions of the national Egyptian
decision threshold. AUCs of 0.9-1, 0.8-0.9, 0.7- standard anti-Brucella abortus bovine serum
0.8 and 0.6-0.7 indicate excellent, good, fair, and equivalent to the OIE International Standard
poor test respectively, while an AUC of 0.5-0.6 Serum (OIEISS). The OIEISS is the current
designates an invalid test (Hanley and McNeil, analog of the WHO Second International anti-
1982). Brucella abortus Serum.

2.4.3. Pearson’s chi-squared test of Tables 4 to 8 reveal the overall performance of all
independence (χ²): acidified plate agglutination test formats in each
of 5 ruminant species, viz. cattle, buffaloes, sheep,
This test measures the independence or goats and camels. The performance
association between two categorical variables, characteristics included relative sensitivity,
namely the CFT as the gold standard and each of accuracy, kappa agreement with CFT association
the Rose-Bengal test formats. The effect size was with CFT based on Pearson's chi-squared test and
estimated using Phi coefficient values, φ (Cohen, Phi coefficient.
1988), where values of 0.1, 0.3 and 0.5 match up
to small, medium and large effects respectively. Figures 1 to 5 display the receiver operating
characteristics curves (ROCs) of all acidified
agglutination test versions in every ruminant
species.
3. RESULTS AND DISCUSSION
Figures 6 to 12 graphically represent the
Results are summarized in Tables 3 to 8 and comparative performance criteria of every
Figures 1 to 12. Table 3 reveals the analytical acidified agglutination test format in all ruminant
sensitivities determined by testing certain species.

Table 3. Minimum analytical sensitivities of serologic methods using known dilutions of the national
Egyptian standard anti-Brucella abortus bovine serum equivalent to the OIEISS
US US French
National anti-Brucella standard US UK UK RBPT, Scottish
BCT BCT RBPT US CFT
serum dilutions in ICFTU/ml BAPA RBPT modified RBPT
8% 3% 4.5%
5 0 0 0 0 0 0 0 0
10 0 0 0 0 0 0 0 1/2.5
15 0 0 0 0 0 0 0 3/2.5
18 + 0 + 0 + 0 0 2.5
20 + 0 + 0 + + 0 1/5
22 + 0 + + + + + 1/5
25 + + + + + + + 2/5
30 + + + + + + + 3/5
40 + + + + + + + 1/10
50 + + + + + + + 2/10
BAPA = buffered acidified plate antigen test, BCT 8% = brucellosis card test with 8% packed cell volume for large ruminants,
BCT 3% = brucellosis card test with 3% packed cell volume for small ruminants, RBPT = Rose-Bengal plate test, RBPT 4.5%
= RBPT with 4.5% packed cells, US CFT = American complement fixation test

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 Sayour et al. 2017. AJVS 53(1): 187-202

Table 4. Performance of the BAPA and Rose-Bengal test formats versus the CFT in cows
US CFT Pearson’s chi
Agreement κ Accuracy Phi coefficient
-ve CFT +ve CFT square *
No. of animals 13 1
-ve BAPA
BAPA

% within CFT 52.0% (rSp) 1.1%

US

0.606 ± 0.096 0.755 53.820 0.678

No. of animals 12 91
+ve BAPA
% within CFT 48.0% 98.9% (rSe)
No. of animals 21 6
US BCT

-ve BCT 8%
% within CFT 84.0% (rSp) 6.5%
8%

0.753 ± 0.074 0.887 66.476 0.754

No. of animals 4 86
+ve BCT 8%
% within CFT 16.0% 93.5% (rSe)
No. of animals 17 1
US BCT

-ve BCT 3%
% within CFT 68.0% (rSp) 1.1%
3%

0.745 ± 0.080 0.835 73.195 0.791

No. of animals 8 91
+ve BCT 3%
% within CFT 32.0% 98.9% (rSe)
No. of animals 20 9
-ve RBPT
RBPT

% within CFT 80.0% (rSp) 9.8%

UK

0.664 ± 0.083 0.851 51.990 0.667

No. of animals 5 83
+ve RBPT
% within CFT 20.0% 90.2% (rSe)
No. of animals 16 1
modified

-ve RBPT
RBPT,

% within CFT 64.0% (rSp) 1.1%

UK

0.712 ± 0.084 0.815 62.654 0.732

No. of animals 9 91
+ve RBPT
% within CFT 36.0% 98.9% (rSe)
No. of animals 20 1
Scottish

-ve RBPT
RBPT

% within CFT 80.0% (rSp) 1.1%

0.838 ± 0.064 0.895 88.775 0.871
No. of animals 5 91
+ve RBPT
% within CFT 20.0% 98.9% (rSe)
No. of animals 19 2
-ve RBPT 4.5%
French
RBPT
4.5%

% within CFT 76.0% (rSp) 2.2%

0.784 ± 0.073 0.869 72.752 0.789
No. of animals 6 90
+ve RBPT 4.5%
% within CFT 24.0% 97.8% (rSe)
* Phearson’s chi-squared test (χ²) = measures the independence between the CFT and each of the Rose-Bengal test formats at P value < 0.05, BAPA = buffered acidified plate
antigen test, BCT 8% = brucellosis card test with 8% packed cells for large ruminants, BCT 3% = BCT with 3% cells for small ruminants, RBPT = Rose-Bengal plate test, RBPT
4.5% = RBPT with 4.5% cells, US CFT = American complement fixation test, rSe = relative sensitivity, rSp = relative specificity

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 Sayour et al. 2017. AJVS 53(1): 187-202

Table 5. Performance of the BAPA and Rose-Bengal test formats versus the CFT in buffalo cows

US CFT Pearson’s chi

Agreement κ Accuracy Phi coefficient
-ve CFT +ve CFT square *
No. of animals 14 2
-ve BAPA
BAPA

% within CFT 50.0% (rSp) 2.6%

US

0.548 ± 0.096 0.737 35.268 0.582

No. of animals 14 74
+ve BAPA
% within CFT 50.0% 97.4% (rSe)
No. of animals 24 24
US BCT

-ve BCT 8%
% within CFT 85.7% (rSp) 31.6%
8%

0.442 ± 0.082 0.771 24.129 0.482

No. of animals 4 52
+ve BCT 8%
% within CFT 14.3% 68.4% (rSe)
No. of animals 20 1
US BCT

-ve BCT 3%
% within CFT 71.4% (rSp) 1.3%
3%

0.761 ± 0.074 0.851 62.417 0.775

No. of animals 8 75
+ve BCT 3%
% within CFT 28.6% 98.7% (rSe)
No. of animals 25 26
-ve RBPT
RBPT

% within CFT 89.3% (rSp) 34.2%

UK

0.437 ± 0.079 0.775 24.835 0.489

No. of animals 3 50
+ve RBPT
% within CFT 10.7% 65.8% (rSe)
No. of animals 20 1
modified

-ve RBPT
RBPT,

% within CFT 71.4% (rSp) 1.3%

UK

0.761 ± 0.074 0.851 62.417 0.775

No. of animals 8 75
+ve RBPT
% within CFT 28.6% 98.7% (rSe)
No. of animals 20 1
Scottish

-ve RBPT
RBPT

% within CFT 71.4% (rSp) 1.3%

0.761 ± 0.074 0.851 62.417 0.557
No. of animals 8 75
+ve RBPT
% within CFT 28.6% 98.7% (rSe)
No. of animals 24 1
-ve RBPT 4.5%
French
RBPT
4.5%

% within CFT 85.7% (rSp) 1.3%

0.874 ± 0.055 0.922 79.819 0.876
No. of animals 4 75
+ve RBPT 4.5%
% within CFT 14.3% 98.7% (rSe)
* Phearson’s chi-squared test (χ²) = measures the independence between the CFT and each of the Rose-Bengal test formats at P value < 0.05, BAPA = buffered acidified plate
antigen test, BCT 8% = brucellosis card test with 8% packed cells for large ruminants, BCT 3% = BCT with 3% cells for small ruminants, RBPT = Rose-Bengal plate test, RBPT
4.5% = RBPT with 4.5% cells, US CFT = American complement fixation test, rSe = relative sensitivity, rSp = relative specificity

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 Sayour et al. 2017. AJVS 53(1): 187-202

Table 6. Performance of the BAPA and Rose-Bengal test formats versus the CFT in ewes

US CFT Pearson’s chi

Agreement κ Accuracy Phi coefficient
-ve CFT +ve CFT square *
No. of animals 8 1
-ve BAPA
BAPA

% within CFT 53.3% (rSp) 1.1%

US

0.625 ± 0.119 0.761 43.307 0.652

No. of animals 7 86
+ve BAPA
% within CFT 46.7% 98.9% (rSe)
No. of animals 11 3
US BCT

-ve BCT 8%
% within CFT 73.3% (rSp) 3.4%
8%

0.719 ± 0.100 0.849 52.768 0.719

No. of animals 4 84
+ve BCT 8%
% within CFT 26.7% 96.6% (rSe)
No. of animals 8 1
US BCT

-ve BCT 3%
% within CFT 53.3% (rSp) 1.1%
3%

0.625 ± 0.119 0.761 43.307 0.652

No. of animals 7 86
+ve BCT 3%
% within CFT 46.7% 98.9% (rSe)
No. of animals 10 4
-ve RBPT
RBPT

% within CFT 66.7% (rSp) 4.6%

UK

0.638 ± 0.111 0.810 41.624 0.639

No. of animals 5 83
+ve RBPT
% within CFT 33.3% 95.4% (rSe)
No. of animals 9 2
modified

-ve RBPT
RBPT,

% within CFT 60.0% (rSp) 2.3%

UK

0.649 ± 0.114 0.789 44.274 0.659

No. of animals 6 85
+ve RBPT
% within CFT 40.0% 97.7% (rSe)
No. of animals 11 3
Scottish

-ve RBPT
RBPT

% within CFT 73.3% (rSp) 3.4%

0.719 ± 0.100 0.849 52.768 0.719
No. of animals 4 84
+ve RBPT
% within CFT 26.7% 96.6% (rSe)
No. of animals 12 2
-ve RBPT 4.5%
French
RBPT
4.5%

% within CFT 80.0% (rSp) 2.3%

0.799 ± 0.087 0.889 65.231 0.800
No. of animals 3 85
+ve RBPT 4.5%
% within CFT 20.0% 97.7% (rSe)
* Phearson’s chi-squared test (χ²) = measures the independence between the CFT and each of the Rose-Bengal test formats at P value < 0.05, BAPA = buffered acidified plate
antigen test, BCT 8% = brucellosis card test with 8% packed cells for large ruminants, BCT 3% = BCT with 3% cells for small ruminants, RBPT = Rose-Bengal plate test, RBPT
4.5% = RBPT with 4.5% cells, US CFT = American complement fixation test, rSe = relative sensitivity, rSp = relative specificity

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 Sayour et al. 2017. AJVS 53(1): 187-202

Table 7. Performance of the BAPA and Rose-Bengal test formats versus the CFT in goats

US CFT Pearson’s chi

Agreement κ Accuracy Phi coefficient
-ve CFT +ve CFT square *
No. of animals 23 1
-ve BAPA
BAPA

% within CFT 47.9% (rSp) 1.6%

US

0.494 ± 0.077 0.732 35.004 0.559

No. of animals 25 63
+ve BAPA
% within CFT 52.1% 98.4% (rSe)
No. of animals 26 2
US BCT

-ve BCT 8%
% within CFT 54.2% (rSp) 3.1%
8%

0.538 ± 0.077 0.755 38.111 0.583

No. of animals 22 62
+ve BCT 8%
% within CFT 45.8% 96.9% (rSe)
No. of animals 22 1
US BCT

-ve BCT 3%
% within CFT 45.8% (rSp) 1.6%
3%

0.574 ± 0.077 0.721 32.943 0.542

No. of animals 26 63
+ve BCT 3%
% within CFT 54.2% 98.4% (rSe)
No. of animals 32 2
-ve RBPT
RBPT

% within CFT 66.7% (rSp) 3.1%

UK

0.659 ± 0.071 0.818 52.382 0.684

No. of animals 16 62
+ve RBPT
% within CFT 33.3% 96.9% (rSe)
No. of animals 26 2
modified

-ve RBPT
RBPT,

% within CFT 54.2% (rSp) 3.1%

UK

0.538 ± 0.077 0.755 38.111 0.583

No. of animals 22 62
+ve RBPT
% within CFT 45.8% 96.9% (rSe)
No. of animals 26 1
Scottish

-ve RBPT
RBPT

% within CFT 54.2% (rSp) 1.6%

0.556 ± 0.075 0.763 41.486 0.609
No. of animals 22 63
+ve RBPT
% within CFT 45.8% 98.4% (rSe)
No. of animals 24 1
-ve RBPT 4.5%
French
RBPT
4.5%

% within CFT 50.0% (rSp) 1.6%

0.515 ± 0.076 0.742 37.114 0.576
No. of animals 24 63
+ve RBPT 4.5%
% within CFT 50.0% 98.4% (rSe)
* Phearson’s chi-squared test (χ²) = measures the independence between the CFT and each of the Rose-Bengal test formats at P value < 0.05, BAPA = buffered acidified plate
antigen test, BCT 8% = brucellosis card test with 8% packed cells for large ruminants, BCT 3% = BCT with 3% cells for small ruminants, RBPT = Rose-Bengal plate test, RBPT
4.5% = RBPT with 4.5% cells, US CFT = American complement fixation test, rSe = relative sensitivity, rSp = relative specificity

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 Sayour et al. 2017. AJVS 53(1): 187-202

Table 8. Performance of the BAPA and Rose-Bengal test formats versus the CFT in she camels
US CFT Pearson’s chi
Agreement κ Accuracy Phi coefficient
-ve CFT +ve CFT square *
No. of animals 13 1
-ve BAPA
BAPA

% within CFT 48.1% (rSp) 1.2%

US

0.559 ± 0.096 0.735 39.502 0.605

No. of animals 14 80
+ve BAPA
% within CFT 51.9% 98.8% (rSe)
No. of animals 23 6
US BCT

-ve BCT 8%
% within CFT 85.2% (rSp) 7.4%
8%

0.759 ± 0.072 0.889 62.368 0.760

No. of animals 4 75
+ve BCT 8%
% within CFT 14.8% 92.6% (rSe)
No. of animals 15 1
US BCT

-ve BCT 3%
% within CFT 55.6% (rSp) 1.2%
3%

0.629 ± 0.091 0.772 47.348 0.662

No. of animals 12 80
+ve BCT 3%
% within CFT 44.4% 98.8% (rSe)
No. of animals 23 28
-ve RBPT
RBPT

% within CFT 85.2% (rSp) 34.6%

UK

0.390 ± 0.080 0.753 20.817 0.439

No. of animals 4 53
+ve RBPT
% within CFT 14.8% 65.4% (rSe)
No. of animals 15 2
modified

-ve RBPT
RBPT,

% within CFT 55.6% (rSp) 2.5%

UK

0.606 ± 0.093 0.765 43.028 0.631

No. of animals 12 79
+ve RBPT
% within CFT 44.4% 97.5% (rSe)
No. of animals 24 2
Scottish

-ve RBPT
RBPT

% within CFT 88.9% (rSp) 2.5%

0.875 ± 0.054 0.932 82.739 0.875
No. of animals 3 79
+ve RBPT
% within CFT 11.1% 97.5% (rSe)
No. of animals 21 6
-ve RBPT 4.5%
French
RBPT
4.5%

% within CFT 77.8% (rSp) 7.4%

0.704 ± 0.080 0.852 53.481 0.704
No. of animals 6 75
+ve RBPT 4.5%
% within CFT 22.2% 92.6% (rSe)
* Phearson’s chi-squared test (χ²) = measures the independence between the CFT and each of the Rose-Bengal test formats at P value < 0.05, BAPA = buffered acidified plate
antigen test, BCT 8% = brucellosis card test with 8% packed cells for large ruminants, BCT 3% = BCT with 3% cells for small ruminants, RBPT = Rose-Bengal plate test, RBPT
4.5% = RBPT with 4.5% cells, US CFT = American complement fixation test, rSe = relative sensitivity, rSp = relative specificity

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 Sayour et al. 2017. AJVS 53(1): 187-202

100 100

95 95

90 90
Sensitivity

85

Sensitivity
85

80 80

US BAPA
75 US BCT 8% 75
US BCT 3%
UK RBPT
70 UK RBPT, modified 70
French RBPT 4.5%
Scottish RBPT
65 65
10 20 30 40 50 10 20 30 40 50

1 - S pecificity 1 - S pecificity

Figure 1. ROC curves of Rose-Bengal test Figure 2. ROC curves of Rose-Bengal tes
versions in cows versions in buffalo cows. See legend in Figure 1.
100 100

95 95

90 90
Sensitivity

85
Sensitivity

85

80 80

75 75

70 70

65 65
10 20 30 40 50 10 20 30 40 50

1 - S pecificity 1 - S pecificity

Figure 3. ROC curves of Rose-Bengal test Figure 4. ROC curves of Rose-Bengal test
versions in ewes. See legend in Figure 1. versions in goats. See legend in Figure 1.

100 100
R elative sensitivity
R elative specificity
90
95 Agreement k
Accuracy
P earson chi-square
Performance characteristics

80
90 US BAPA P hi value
US BCT 8%
US BCT 3% 70
UK RBPT
S ensitivity

85
UK RBPT, modified
60
French RBPT 4.5%
Scottish RBPT
80
50

75
40

70 30

20
65
ts
s

es

10 20 30 40 50
ow

el
oa
w
co

m
C

G
E

1 - S pecificity
lo

-c
fa

he
uf

S
B

US B AP A performance in ruminants
Figure 5. ROC curves of Rose-Bengal test Figure 6. US BAPA performance in ruminants
versions in she camels

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100 100

90 90

Performance characteristics
Performance characteristics

80 80

70 70

60 60

50 50

40 40

30 30

20 20

ts
s

es

s
ts
s

es

ow
s

el
oa
ow

el

w
oa

co

m
w
co

G
E

a
C

G
E

lo
a

-c
lo

-c

fa

he
fa

he

uf
uf

S
B
S
B

US B C T 8% performance in ruminants US B C T 3% performance in ruminants

Figure 7. US BCT 8% performance in Figure 8. US BCT 3% performance in
ruminants. See legend in Figure 12. ruminants. See legend in Figure 12.

100 100

90 90
Performance characteristics

Performance characteristics

80 80

70 70

60 60

50 50

40 40

30 30

20 20
ts
s

es

ts
s

es

s
ow

el

ow

el
oa

oa
w
co

w
m

co

m
C

G
E

G
E
a

a
lo

lo
-c

-c
fa

he

fa

he
uf

uf
S

S
B

UK R B P T performance in ruminants UK modified R B P T performance in ruminants

Figure 9. UK RBPT performance in Figure 10. UK modified RBPT performance in
ruminants. See legend in Figure 12. ruminants. See legend in Figure 12.

100 100

90 90
Performance characteristics
Performance characteristics

80 80

70 70

60 60

50 50

R elative sensitivity
40 40
R elative specificity
Agreement k
30 30 Accuracy
P earson chi-square
P hi value
20 20
ts
s
ts

es
s

s
s

es

ow

w
ow

el
w

el

oa
oa

w
w

co
co

m
m

C
C

G
E
G
E

a
a

lo
lo

-c
-c

fa
fa

he
he

uf
uf

S
S

B
B

S cottish R B P T performance in ruminants F rench R B P T 4.5% performance in ruminants

Figure 11. Scottish RBPT performance in Figure 12. French RBPT 4.5% performance in
ruminants. See legend in Figure 12. ruminants.

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 Sayour et al. 2017. AJVS 53(1): 187-202

Antibody reacts with antigen primarily by determined the selectivity of the test mainly for IgG1
combination between the variable region of the Fab by enhancement of its agglutination ability (Blasco
portion of the antibody and its corresponding et al., 1994), where the hydronium ions resulting
antigenic epitope in a step known as sensitization from the acidic pH allowed for certain bonding
(Tizard, 2004). Secondarily, antibody bridges, changes of antibodies which affected the
presumably among the Fc portions, lead to the aggregation capacity of IgG1 directly but that of
formation of macroscopically visible clumps of IgM inversely. Low pH presumably modified the
antigen-antibody aggregates or lattice (Steward, protein 3D structure of IgG1 by rotation of its bonds
1984). This two-stage agglutination reaction is a leading to some sort of elasticity, and hence,
practical in vitro serologic means for brucellosis enhancement of its agglutination ability. Having
monitoring by detection of antibodies. The current resolved in favor of IgG1 as the most characteristic
work was to study the fitness for purpose of long-lasting antibody of Brucella infection
different Rose-Bengal agglutination test formats to (Ducrotoy et al., 2016), the selectivity of the test
select the most suitable pre-confirmatory diagnostic boosted both its sensitivity and specificity
tool for Brucella melitensis infection in non-bovine simultaneously due to this very fact and the point
domestic ruminants based on the test performance that IgM could be a potential non-specific agglutinin
under the brucellosis epidemiological status quo. (Levieux, 1978).

3.1. Determinants of sensitivity in the Rose- 3.1.1.2. Final cell concentration of the antigen
Bengal test formats
Another influential antigen-bound factor that
The sensitivities of the Rose-Bengal panel of test resulted in noticeable variation in sensitivity was the
formats were mainly dependent on reagent, animal difference in concentration of antigen expressed as
and human associated factors that might even packed cell volume (PCV). These PCV values are
interfere with test results. revealed in Table 2, but what really matter were the
final antigen cell concentrations after mixing with
3.1.1. Reagents (antigenic preparations) serum, being 4%, 1.5%, less than 4%, less than 2%
and less than 2% for the BCT 8%, BCT 3%, UK
All Rose-Bengal test formats were performed using RBPT, modified UK RBPT, Scottish RBPT and old
antigens prepared from Brucella abortus biovar 1 French RBPT respectively. The higher the cell
strain 99 stained with Rose-Bengal and suspended volume, the lower the sensitivity (Mikolon et al.,
in lactate buffer at a pH of 3.65 ± 0.05 (OIE 1998) and the higher the limit of detection of the test
Terrestrial Manual, 2016a). It should be noted that (Table 3). Whether the antigen was standardized
the almost constant incubation of the Rose-Bengal based on either the packed cell volume or against
test formats at room temperature range of 22°C ± the OIEISS was a sensitivity element. Based on the
4°C allowed for almost equal chances for both ROC curves (Table 4-8) and (Figures 1-5), the
specific IgM and IgG to participate in the reaction. Rose-Bengal versions with the advantage of lower
However, the nature of the antigen is not the only packed cell volume (BCT 3% and French RBPT
factor that could influence reagent dynamics. The 4.5%), standardized against the OIEISS (Scottish
test positive and negative controls could also have RBPT) or both (modified UK RBPT) had higher
an effect. sensitivity and/ or accuracy. Compared to the slow
3.1.1.1. Final hydrogen ion concentration of the tube agglutination format, the Rose-Bengal as a
test format plate variety with higher final concentration of
antigen that compensate for the short incubation
The initial pH of all Rose-Bengal antigens cannot be time is supposed to be more tolerant to prozone
incriminated alone for sensitivity differences among phenomenon resulting from too much antibodies
test versions. After mixing antigen with serum that compete on antigenic determinant sites (Tizard,
samples, a final pH of around 3.8 was reached, 2004), thus preventing stage two of agglutination
being slightly higher (around 4) in case of the mentioned earlier. Still, prozone formation can
modified British RBPT as serum and antigen were sometimes lead to false negative reactions in the
not mixed in a ratio of 1:1, but rather 3:1 to boost Rose-Bengal test formats (Herr, 1982).
sensitivity by increasing the potential amount of
antibodies if present. The final acidic pH

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 Sayour et al. 2017. AJVS 53(1): 187-202

3.1.1.3. Brucella species nature of the antigen longer than 13.7 and 12 of sheep (Smith et al.,
1976) and goats (Humphrey and Turk, 1961)
As far as whole cell antigens are concerned, the respectively. Postoret et al. (1998) reviewed that
outermost parts sterically exposed are those cattle produce serum concentrations of neutral-pH
immunogenically dominant (Ducrotoy et al., 2016). agglutinins of IgM (3.05 mg/ml) and IgG2 (9.2
Accordingly, the O chain of the surface LPS is the mg/ml) higher than those produced by sheep (1.5-3
main part involved in serologic reactions with and 5-7 mg/ml respectively), but sheep produce
Brucella antibodies. The O chain is a homopolymer almost double the concentration of IgG1 acid
of some hundred residues of N-formyl-D- agglutinins (16-22 mg/ml) as compared to cattle
perosamine that bind together in two slightly (11.2 mg/ml). The antibody class, concentration
different linkage orders (Iriarte et al., 2004) leading and half life differences between bovines and small
to two chemically related structures known as A and ruminants explain the need for Rose-Bengal test
M epitopes. By no means, A and M stand for format of enhanced sensitivity for the latter animals.
abortus and melitensis as both entities are present Infections with antigenically similar Gram negative
with different ratios on the surface of all smooth bacteria like Yersinia enterocolitica O9 may cause
brucellae (Corbel and Banai, 2005). Brucella false agglutination (Gerbier et al., 1997). Recent
species/ biovars arranged in descending order of immunization with smooth Brucella vaccines
dominance among livestock in Egypt are Brucella usually leads to false positive results (Nielsen,
melitensis biovar 3, B. abortus biovar 1, B. 2010), a case which does not apply to the current
melitensis biovar 2 and B. suis biovar 1 (Sayour and investigation where all animals studied were not
Sayour, 2015), all of which are A-dominant strains vaccinated.
except for the former that has both A and M
structures (Iriarte et al., 2004). More importantly is Unlike normal mammals and in addition to the
the fact that smooth brucellae highly cross react possession of classic antibodies with heavy and light
with each other due to the presence of a common chains, camel sera have unique highly effective
epitope C (Corbel and Banai, 2005), hence, the antibodies of the IgG2 and the IgG3 classes lacking
relatively low virulent B. abortus has been globally the light chains (Pastoret et al., 1998). Camel IgG
accepted for antigen production to detect antibodies subclasses of IgG1, IgG2a, IgG2b and IgG3 are
not only to B. abortus, but also to its smooth provoked in response to B. melitensis and B. abortus
counterparts (OIE Terrestrial Manual, 2016a). infections (Abbady et al., 2011). Camel IgG3 is
both agglutinating and complement fixing antibody
3.1.2. Animal species and stage of infection/ (Pastoret et al., 1998). The biological activity and
vaccination interaction of camel antibody classes and subclasses
in immunoassays require further investigation.
Animal-associated factors that might directly affect
the Rose-Bengal test sensitivity include the species 3.1.3. Human operator variation
and the stage of Brucella infection. These factors
influence the nature, concentration and duration of Apart from potential technical human errors, the
antibody isotypes produced and consequently the Rose-Bengal agglutination reaction was subject to
test selectivity and sensitivity. All mammals individual human variation due to the very fact that
possess the five classes of immunoglobulins IgM, the test was read by the naked eye. An analyst with
IgG, IgA, IgE and IgD (Tizard, 2004) of which the healthy eye sight of 6/6 might misidentify several
first two are the major antibodies in blood sera repetitions of a single weak positive sample to
detected by brucellosis serologic assays (Ducrotoy include some or few false negative ones.
et al., 2016) including the Rose-Bengal test Additionally, individual humans might often have
(Stryszak, 1986). The ten antigen binding sites of different perspectives regarding the cutoff between
the pentameric IgM are credentials for a way better positive and negative samples based on visual
agglutinin than the monomeric IgG (Steward, 1984). matching of the test result with the positive and
On the other hand, the lingering IgG serum negative controls. This interpretation difference
concentration is way higher than that of the early might be attributed to the fact that the smallest
IgM (Tizard, 2004). The IgG half life in days of resolvable dot (visible agglutination element) the
bovines is 17.4 (Nielsen et al., 1978) which is human eye can see is about 0.1 mm at the minimum
similar to 16.3 of camels (Hüselbusch, 2000), but comfortable viewing distance of 20-25 cm adopted

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 Sayour et al. 2017. AJVS 53(1): 187-202

in inspection purposes laboratories (Yanoff and revealed by Pearson’s chi-squared test of

Duker, 2009) under normal lighting conditions independence at P value < 0.05 as well as Phi
(light source of about 1000 lumens at a height of 60- coefficient values. This reflects the fact that all
70 cm with a viewing angle of about 35°). By these tests, as well as the CFT, are selective for the
increasing the viewing distance to 40 cm, the same antibody (IgG1). Figures 6-12 graphically
smallest resolvable dot seen by the healthy naked represent the comparative performance criteria of
eye increases. Thus, the human factor can affect every acidified agglutination test version in all
both the repeatability and reproducibility of the test. ruminant species. The BAPA exhibited relatively
A zone of unreliability forms around the threshold lower performance in buffalo cows and goats
limit including false positives, inconclusive results (Figure 6). A similar picture was revealed by the
and false negatives, below, roughly at and above the BCT 8% with even more pronounced fall in
cutoff point respectively. buffaloes (Figure 7) that greatly improved by the
use of its 3% version, but with extra drop in goats
3.2. Ruminant intra-species performance of (Figure 8). The overall performance of the UK
Rose-Bengal test formats RBPT dropped in ewes and drastically fell in she-
camels (Figure 9) with its modified form achieving
The relative sensitivity cannot be taken as a stand- some progress in buffaloes, sheep and camels and
alone criterion for performance comparison of test minor drop in cows and goats (Figure 10). The
formats intended as screening pre-confirmatory Scottish RBPT slightly dropped in buffalo cows but
ones. What really matters beside sensitivity is noticeably in goats with good performance in cows
agreement and independence/ association with the and she-camels (Figure 11). The French RBPT
confirmatory CFT as well as accuracy (Tables 4-8 drastically fell in goats and less severely in she-
and Figures 1-5). Starting with cows (Table 4 and camels (Figure 12). Goats achieved the worst
Figure 1), the best overall performance (rSe, behavior with Rose-Bengal test formats. Less badly
accuracy, κ agreement with CFT and large were buffaloes and even less badly were she-camels.
association with CFT based on Pearson's chi- This reflects the fact mentioned earlier that unlike in
squared test at P value < 0.05 as well as Phi goats, the antibody pattern of buffaloes and camels
coefficient) was achieved by the Scottish RBPT. is relatively closer to that of cattle (Postoret et al.,
Other test formats, viz. US BAPA, US BCT 3%, and 1998; Tizard, 2004).
UK modified RBPT fulfilled the same high relative
sensitivity, but with slightly lower figures in the
other performance characteristics. The
corresponding picture in buffalo cows (Table 5 and 4. CONCLUSION
Figure 2) had similar high rSe of 98.7% for the
French RBPT (4.5%), the Scottish RBPT, the UK According to the analytical and diagnostic
modified RBPT and the US BCT (3%). However, performance parameters obtained under conditions
the best overall performance was attained by the of this study, the authors concluded that the BAPA
French version. Then again, the overall best behaved better than any other acidified plate
performance of RBPT versions in ewes, as shown in agglutination format tried showing minor
Table 6 and Figure 3, was accomplished by the performance drops in goats and buffaloes. The
French (4.5%) RBPT followed by the Scottish behavior of the classic Rose-Bengal test formats is
RBPT and US BCT (8%). The best performance in satisfactory in cattle. Sensitivity enhancement of
caprines (Table 7 and Figure 4) was scored by the the Rose-Bengal test via re-standardization based on
UK RBPT followed by the Scottish RBPT and the the final concentration of cells, the final pH of the
US BCT (8%). In she-camels, the best performance antigen, or the OIEISS dilution largely added up to
was documented by the Scottish RBPT followed by the test performance especially in sheep. Still, the
the US BCT (8%) as shown in Table 8 and Figure 5. test requires further adaptation in goats, buffaloes
and camels in descending order of urgency. More
3.3. Ruminant inter-species performance of attention should be given to camels due to the
Rose-Bengal test formats unique nature of their antibodies and
seroconversion.
Generally speaking, almost all acidified
agglutination test formats were largely associated
with CFT in ruminant species (Tables 4-8) as
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 Sayour et al. 2017. AJVS 53(1): 187-202

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