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Validation of Different Versions of The Card or Rose-Bengal Test for The ... investigate the fitness of Rose-Bengal plate test (RBPT) formats namely the US.

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: BCT, RBPT, CFT, Brucella, cow, buffalo, ewe, goat, camel.

Correspondence to: Nour H. Abdel-Hamid, [email protected]

cows, ewes, female goats and she-camels in seven Nile Delta governorates. Most animals had a history of Brucella melitensis biovar 3 infection. This study was to investigate the fitness of Rose-Bengal plate test (RBPT) formats namely the US 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 nonbovine ruminants. Taking the complement fixation test (CFT) as the gold standard, the performance of RBPT assays and the buffered acidified plate antigen test was estimated as relative sensitivity, accuracy, kappa agreement with CFT and association with CFT 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.

cells are suspended and standardized. The test brings about agglutination of the nonagglutinogenic IgG1 distinctive of the longstanding Brucella infection (Alton et al., 1988). This adds up for more sensitivity and specificity to the test. Used after the presumptive BAPA test, the Rose-Bengal plate test (RBPT) reduces the number of positive samples demanding confirmation. Each of these tests is

1. INTRODUCTION Among the rapid agglutination assays for brucellosis surveillance are the buffered acidified plate antigen (BAPA) and the Rose-Bengal plate tests. The Rose-Bengal or brucellosis card test (BCT) is a rapid qualitative one-dilution plate agglutination at acidic pH of 3.65±0.05 attained by lactate buffered phenol saline in which inactivated Rose-Bengal stained Brucella abortus

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recommended for international trade in the OIE Terrestrial Manual (2016a).

isolates including 44 B. melitensis, 28 B. abortus and 5 B. suis isolates from milking bovines (Sayour and Sayour, 2015). B. melitensis was also recovered from Egyptian camelids (unpublished data).

There are different versions of the Rose-Bengal test worldwide depending on the Brucella epidemiological status of the country, the battery of immunoassays used, the animal species under test and whether series/ parallel interpretation of serologic results is adopted. Among the RoseBengal test versions are the American BCT using 8% packed cell volume for large ruminants (Alton et al., 1988) and 3% cells for small ruminants (Mikolon et al., 1998), the British version adjusted to give weak agglutination with the OIE International Standard Serum for Brucella abortus (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. (1994), the old French version with 4.5% cell concentration (Pilet et al., 1972; Toma et al., 1972), and a Scottish commercial version for humans (Lennette, 1985).

The aim of this investigation was to study the fitness of different Rose-Bengal test formats for the purpose of pre-confirmation of B. melitensis infection in non-bovine domestic ruminants under the brucellosis epidemiological status quo. This is part of achieving accreditation nuts and bolts of the ISO/IEC 17025:2005 concerned with the general requirements for the competence of testing and calibration laboratories.

2. MATERIAL AND METHODS 2.1. Animals A total of 543 serum samples from different ruminant species were either selected from the serum collection of the Department of Brucellosis Research, Animal Health Research Institute, Dokki, Giza, Egypt (Table 1) or otherwise collected from abattoirs. Most of these sera were serologically positive and belonged to animals in the Nile Delta governorates. Most of the bovine, ovine and caprine positive sera had a history of Brucella melitensis biovar 3 infection (Sayour and Sayour, 2015). There was no history of vaccination against brucellosis. Only some animals were reported to have late abortion and retained placenta.

The OIE guidelines (OIE Terrestrial Manual, 2016a) allow the application of serologic tests originally standardized for cattle to be used for other ruminants like buffaloes, sheep, goats and camels following proper validation. It is known that B. abortus mainly infects large ruminants, while B. melitensis usually infects small ruminants (Alton et al., 1988). This is not the case in Egypt where B. melitensis is predominant among livestock as proven by the detection of 93 B. melitenssis isolates among a total of 126

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

Cows Buffalo cows Ewes Female goats She-camels

Breed Native Friesian Hybrid Native Native Native Fellahi

Age (year)

Population

Governorate

No.

1-3

Small/ large herds

Sharkia/ Monofia/ Gharbia

117

1.5-4 1-4 1-3 2-3

Individuals/ small herds Individuals/ small flocks Small flocks Individuals Total animals

Beheira/ Monofia/ Gharbia Kafr El-Sheikh/ Sharkia Beheira/ Sharkia/ Giza Sharkia

104 102 112 108 543

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2.2. Antigens for serologic tests Table 2. Antigens for serologic tests Standardization by either OIEISS No No No Weak positive at 1/40 of the 4.5% OIEISS Positive at 1/45 and negative at No 1/55 of the OIEISS

Antigen

pH

US BAPA US BCT 8% US BCT 3% French RBPT 4.5%

3.7 ± 0.03 3.65 ± 0.05 3.65 ± 0.05

UK RBPT

3.65 ± 0.05

Scottish RBPT

3.6

No

Yes, details unknown

US CFT

~7

4%

50% fixation with 1/200 of the OIEISS

3.65 ± 0.05

PCV 11% 8% 3%

Producer Veterinary Serum and Vaccine Research Institute (VSVRI), Abbassia, Cairo, Egyp Department of Brucellosis Research, Animal Health Research Institute, Dokki, Giza, Egypt (Alton et al., 1988 and Mikolon et al., 1998) AHVLA (now APHA), New Haw, Addlestone, Surrey KT15 3NB, UK Omega Diagnostics Ltd., Alva, FK12 5DQ, Scotland, UK NVSL/DBL, USDA, USA

OIEISS = OIE International Standard Serum (previously the WHO Second International anti-Brucella abortus Serum)

Quality control and quality assurance were fulfilled according to the requirements of the ISO/IEC 17025:2005 and the OIE guidelines (OIE Terrestrial Manual, 2016b).

2.3. Serologic tests 2.3.1. Buffered acidified plate antigen (BAPA) and brucellosis card (BCT)/ Rose-Bengal plate (RBPT) test formats

2.4. Statistical analyses

The BAPA was performed according to Angus and Barton (1984) and the OIE Terrestrial Manual (2016a). The BCT with 8% cells for large ruminants and the BCT with 3% cells for small ruminants are currently adopted by the NVSL, USDA (NVSL Reagent Manual, 2016). Except for the modified British RBPT performed after Ferreira et al. (2003), other versions of the RoseBengal test were implemented according to Alton et al. (1988).

The following analyses were performed using IBM® SPSS® Statistics, Version 21, IBM Corporation, 2012, under the environment of Windows® 8.1, Microsoft Corporation. 2.4.1. Kappa (κ) agreement and relative sensitivity/ specificity: The kappa (κ) agreement of CFT as the gold standard with other serologic tests was used to assess the matching of results at p < 0.05. Relative sensitivity/ specificity pairs were also calculated.

2.3.2. Complement fixation test (CFT) Complement and hemolysin were prepared and 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 from an adult healthy ram serologically negative to brucellosis. These were standardized to 2% suspension in veronal buffered saline (VBS). The proper test was performed according to the current American SOP by Hennager (2015). Warm fixation of complement at 37°C was adopted as cold fixation was unacceptably slow. The positive cutoff point for CFT was ≥ 20 ICFTU/ml.

2.4.2. Receiver operating characteristic (ROC) curves: Considering the CFT as the gold standard, ROC curves expressing the sensitivity versus the false positive rate were plotted for all serologic methods. Data obtained from ROC curves included the area under the curve (AUC) representing the test method accuracy according to Hanley and McNeil (1982). The AUC measures how well the test separates the positive

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from negatives without reference to a particular decision threshold. AUCs of 0.9-1, 0.8-0.9, 0.70.8 and 0.6-0.7 indicate excellent, good, fair, and poor test respectively, while an AUC of 0.5-0.6 designates an invalid test (Hanley and McNeil, 1982).

descending dilutions of the national Egyptian standard anti-Brucella abortus bovine serum equivalent to the OIE International Standard Serum (OIEISS). The OIEISS is the current analog of the WHO Second International antiBrucella abortus Serum.

2.4.3. Pearson’s independence (χ²):

Tables 4 to 8 reveal the overall performance of all acidified plate agglutination test formats in each of 5 ruminant species, viz. cattle, buffaloes, sheep, goats and camels. The performance characteristics included relative sensitivity, accuracy, kappa agreement with CFT association with CFT based on Pearson's chi-squared test and Phi coefficient.

chi-squared

test

of

This test measures the independence or association between two categorical variables, namely the CFT as the gold standard and each of the Rose-Bengal test formats. The effect size was estimated using Phi coefficient values, φ (Cohen, 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 comparative performance criteria of every acidified agglutination test format in all ruminant species.

Results are summarized in Tables 3 to 8 and Figures 1 to 12. Table 3 reveals the analytical sensitivities determined by testing certain

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 National anti-Brucella standard serum dilutions in ICFTU/ml

US BAPA

5 10 15 18 20 22 25 30 40 50

0 0 0 + + + + + + +

US BCT 8% 0 0 0 0 0 0 + + + +

US BCT 3% 0 0 0 + + + + + + +

UK RBPT

UK RBPT, modified

Scottish RBPT

0 0 0 0 0 + + + + +

0 0 0 + + + + + + +

0 0 0 0 + + + + + +

French RBPT 4.5% 0 0 0 0 0 + + + + +

US CFT 0 1/2.5 3/2.5 2.5 1/5 1/5 2/5 3/5 1/10 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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French RBPT 4.5%

Scottish RBPT

UK RBPT, modified

UK RBPT

US BCT 3%

US BCT 8%

US BAPA

Table 4. Performance of the BAPA and Rose-Bengal test formats versus the CFT in cows

-ve BAPA +ve BAPA -ve BCT 8% +ve BCT 8% -ve BCT 3% +ve BCT 3% -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT 4.5% +ve RBPT 4.5%

No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT

US CFT -ve CFT +ve CFT 13 1 1.1% 52.0% (rSp) 12 91 48.0% 98.9% (rSe) 21 6 6.5% 84.0% (rSp) 4 86 16.0% 93.5% (rSe) 17 1 1.1% 68.0% (rSp) 8 91 32.0% 98.9% (rSe) 20 9 9.8% 80.0% (rSp) 5 83 20.0% 90.2% (rSe) 16 1 1.1% 64.0% (rSp) 9 91 36.0% 98.9% (rSe) 20 1 1.1% 80.0% (rSp) 5 91 20.0% 98.9% (rSe) 19 2 2.2% 76.0% (rSp) 6 90 24.0% 97.8% (rSe)

Agreement κ

Accuracy

Pearson’s chi square *

Phi coefficient

0.606 ± 0.096

0.755

53.820

0.678

0.753 ± 0.074

0.887

66.476

0.754

0.745 ± 0.080

0.835

73.195

0.791

0.664 ± 0.083

0.851

51.990

0.667

0.712 ± 0.084

0.815

62.654

0.732

0.838 ± 0.064

0.895

88.775

0.871

0.784 ± 0.073

0.869

72.752

0.789

* 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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French RBPT 4.5%

Scottish RBPT

UK RBPT, modified

UK RBPT

US BCT 3%

US BCT 8%

US BAPA

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

-ve BAPA +ve BAPA -ve BCT 8% +ve BCT 8% -ve BCT 3% +ve BCT 3% -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT 4.5% +ve RBPT 4.5%

No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT

US CFT -ve CFT +ve CFT 14 2 2.6% 50.0% (rSp) 14 74 50.0% 97.4% (rSe) 24 24 31.6% 85.7% (rSp) 4 52 14.3% 68.4% (rSe) 20 1 1.3% 71.4% (rSp) 8 75 28.6% 98.7% (rSe) 25 26 34.2% 89.3% (rSp) 3 50 10.7% 65.8% (rSe) 20 1 1.3% 71.4% (rSp) 8 75 28.6% 98.7% (rSe) 20 1 1.3% 71.4% (rSp) 8 75 28.6% 98.7% (rSe) 24 1 1.3% 85.7% (rSp) 4 75 14.3% 98.7% (rSe)

Agreement κ

Accuracy

Pearson’s chi square *

Phi coefficient

0.548 ± 0.096

0.737

35.268

0.582

0.442 ± 0.082

0.771

24.129

0.482

0.761 ± 0.074

0.851

62.417

0.775

0.437 ± 0.079

0.775

24.835

0.489

0.761 ± 0.074

0.851

62.417

0.775

0.761 ± 0.074

0.851

62.417

0.557

0.874 ± 0.055

0.922

79.819

0.876

* 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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French RBPT 4.5%

Scottish RBPT

UK RBPT, modified

UK RBPT

US BCT 3%

US BCT 8%

US BAPA

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

-ve BAPA +ve BAPA -ve BCT 8% +ve BCT 8% -ve BCT 3% +ve BCT 3% -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT 4.5% +ve RBPT 4.5%

No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT

US CFT -ve CFT +ve CFT 8 1 1.1% 53.3% (rSp) 7 86 46.7% 98.9% (rSe) 11 3 3.4% 73.3% (rSp) 4 84 26.7% 96.6% (rSe) 8 1 1.1% 53.3% (rSp) 7 86 46.7% 98.9% (rSe) 10 4 4.6% 66.7% (rSp) 5 83 33.3% 95.4% (rSe) 9 2 2.3% 60.0% (rSp) 6 85 40.0% 97.7% (rSe) 11 3 3.4% 73.3% (rSp) 4 84 26.7% 96.6% (rSe) 12 2 2.3% 80.0% (rSp) 3 85 20.0% 97.7% (rSe)

Agreement κ

Accuracy

Pearson’s chi square *

Phi coefficient

0.625 ± 0.119

0.761

43.307

0.652

0.719 ± 0.100

0.849

52.768

0.719

0.625 ± 0.119

0.761

43.307

0.652

0.638 ± 0.111

0.810

41.624

0.639

0.649 ± 0.114

0.789

44.274

0.659

0.719 ± 0.100

0.849

52.768

0.719

0.799 ± 0.087

0.889

65.231

0.800

* 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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French RBPT 4.5%

Scottish RBPT

UK RBPT, modified

UK RBPT

US BCT 3%

US BCT 8%

US BAPA

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

-ve BAPA +ve BAPA -ve BCT 8% +ve BCT 8% -ve BCT 3% +ve BCT 3% -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT 4.5% +ve RBPT 4.5%

No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT

US CFT -ve CFT +ve CFT 23 1 1.6% 47.9% (rSp) 25 63 52.1% 98.4% (rSe) 26 2 3.1% 54.2% (rSp) 22 62 45.8% 96.9% (rSe) 22 1 1.6% 45.8% (rSp) 26 63 54.2% 98.4% (rSe) 32 2 3.1% 66.7% (rSp) 16 62 33.3% 96.9% (rSe) 26 2 3.1% 54.2% (rSp) 22 62 45.8% 96.9% (rSe) 26 1 1.6% 54.2% (rSp) 22 63 45.8% 98.4% (rSe) 24 1 1.6% 50.0% (rSp) 24 63 50.0% 98.4% (rSe)

Agreement κ

Accuracy

Pearson’s chi square *

Phi coefficient

0.494 ± 0.077

0.732

35.004

0.559

0.538 ± 0.077

0.755

38.111

0.583

0.574 ± 0.077

0.721

32.943

0.542

0.659 ± 0.071

0.818

52.382

0.684

0.538 ± 0.077

0.755

38.111

0.583

0.556 ± 0.075

0.763

41.486

0.609

0.515 ± 0.076

0.742

37.114

0.576

* 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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French RBPT 4.5%

Scottish RBPT

UK RBPT, modified

UK RBPT

US BCT 3%

US BCT 8%

US BAPA

Table 8. Performance of the BAPA and Rose-Bengal test formats versus the CFT in she camels

-ve BAPA +ve BAPA -ve BCT 8% +ve BCT 8% -ve BCT 3% +ve BCT 3% -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT +ve RBPT -ve RBPT 4.5% +ve RBPT 4.5%

No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT No. of animals % within CFT

US CFT -ve CFT +ve CFT 13 1 1.2% 48.1% (rSp) 14 80 51.9% 98.8% (rSe) 23 6 7.4% 85.2% (rSp) 4 75 14.8% 92.6% (rSe) 15 1 1.2% 55.6% (rSp) 12 80 44.4% 98.8% (rSe) 23 28 34.6% 85.2% (rSp) 4 53 14.8% 65.4% (rSe) 15 2 2.5% 55.6% (rSp) 12 79 44.4% 97.5% (rSe) 24 2 2.5% 88.9% (rSp) 3 79 11.1% 97.5% (rSe) 21 6 7.4% 77.8% (rSp) 6 75 22.2% 92.6% (rSe)

Agreement κ

Accuracy

Pearson’s chi square *

Phi coefficient

0.559 ± 0.096

0.735

39.502

0.605

0.759 ± 0.072

0.889

62.368

0.760

0.629 ± 0.091

0.772

47.348

0.662

0.390 ± 0.080

0.753

20.817

0.439

0.606 ± 0.093

0.765

43.028

0.631

0.875 ± 0.054

0.932

82.739

0.875

0.704 ± 0.080

0.852

53.481

0.704

* 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

195

100

100

95

95

90

90

85

Sensitivity

80

80

US BAPA US BCT 8% US BCT 3% UK RBPT UK RBPT, modified French RBPT 4.5% Scottish RBPT

75

70

10

20

30

40

75

70

65

50

10

20

30

1 - S pecificity

100

95

95

90

90

Sensitivity

85

80

85

80

75

75

70

70

65 20

30

40

50

10

20

30

100

100

R elative sensitivity R elative specificity Agreement k Accuracy P earson chi-square P hi value

90

90

Performance characteristics

95

US BAPA US BCT 8% US BCT 3% UK RBPT UK RBPT, modified French RBPT 4.5% Scottish RBPT

85

80

75

70

80

70

60

50

40

30

B

uf fa lo

1 - S pecificity

m el s

he -c a

50

w es

40

E

30

C

20

ow s

20 10

co w s

S ensitivity

50

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

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

65

40

1 - S pecificity

1 - S pecificity

oa ts

Sensitivity

100

10

50

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

Figure 1. ROC curves of Rose-Bengal test versions in cows

65

40

1 - S pecificity

S

65

85

G

Sensitivity

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

US B AP A performance in ruminants

Figure 5. ROC curves of Rose-Bengal test versions in she camels

Figure 6. US BAPA performance in ruminants

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90

90

40

Figure 8. US BCT 3% performance in ruminants. See legend in Figure 12. 100

90

90

40

C

ow s

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

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Figure 12. French RBPT 4.5% performance in ruminants.

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Antibody reacts with antigen primarily by combination between the variable region of the Fab portion of the antibody and its corresponding antigenic epitope in a step known as sensitization (Tizard, 2004). Secondarily, antibody bridges, presumably among the Fc portions, lead to the formation of macroscopically visible clumps of antigen-antibody aggregates or lattice (Steward, 1984). This two-stage agglutination reaction is a practical in vitro serologic means for brucellosis monitoring by detection of antibodies. The current work was to study the fitness for purpose of different Rose-Bengal agglutination test formats to select the most suitable pre-confirmatory diagnostic tool for Brucella melitensis infection in non-bovine domestic ruminants based on the test performance under the brucellosis epidemiological status quo.

determined the selectivity of the test mainly for IgG1 by enhancement of its agglutination ability (Blasco et al., 1994), where the hydronium ions resulting from the acidic pH allowed for certain bonding changes of antibodies which affected the aggregation capacity of IgG1 directly but that of IgM inversely. Low pH presumably modified the protein 3D structure of IgG1 by rotation of its bonds leading to some sort of elasticity, and hence, enhancement of its agglutination ability. Having resolved in favor of IgG1 as the most characteristic long-lasting antibody of Brucella infection (Ducrotoy et al., 2016), the selectivity of the test boosted both its sensitivity and specificity simultaneously due to this very fact and the point that IgM could be a potential non-specific agglutinin (Levieux, 1978).

3.1. Determinants of sensitivity in the RoseBengal test formats

3.1.1.2. Final cell concentration of the antigen Another influential antigen-bound factor that resulted in noticeable variation in sensitivity was the difference in concentration of antigen expressed as packed cell volume (PCV). These PCV values are revealed in Table 2, but what really matter were the final antigen cell concentrations after mixing with serum, being 4%, 1.5%, less than 4%, less than 2% and less than 2% for the BCT 8%, BCT 3%, UK RBPT, modified UK RBPT, Scottish RBPT and old French RBPT respectively. The higher the cell volume, the lower the sensitivity (Mikolon et al., 1998) and the higher the limit of detection of the test (Table 3). Whether the antigen was standardized based on either the packed cell volume or against the OIEISS was a sensitivity element. Based on the ROC curves (Table 4-8) and (Figures 1-5), the Rose-Bengal versions with the advantage of lower packed cell volume (BCT 3% and French RBPT 4.5%), standardized against the OIEISS (Scottish RBPT) or both (modified UK RBPT) had higher sensitivity and/ or accuracy. Compared to the slow tube agglutination format, the Rose-Bengal as a plate variety with higher final concentration of antigen that compensate for the short incubation time is supposed to be more tolerant to prozone phenomenon resulting from too much antibodies that compete on antigenic determinant sites (Tizard, 2004), thus preventing stage two of agglutination mentioned earlier. Still, prozone formation can sometimes lead to false negative reactions in the Rose-Bengal test formats (Herr, 1982).

The sensitivities of the Rose-Bengal panel of test formats were mainly dependent on reagent, animal and human associated factors that might even interfere with test results. 3.1.1. Reagents (antigenic preparations) All Rose-Bengal test formats were performed using antigens prepared from Brucella abortus biovar 1 strain 99 stained with Rose-Bengal and suspended in lactate buffer at a pH of 3.65 ± 0.05 (OIE Terrestrial Manual, 2016a). It should be noted that the almost constant incubation of the Rose-Bengal test formats at room temperature range of 22°C ± 4°C allowed for almost equal chances for both specific IgM and IgG to participate in the reaction. However, the nature of the antigen is not the only factor that could influence reagent dynamics. The test positive and negative controls could also have an effect. 3.1.1.1. Final hydrogen ion concentration of the test format The initial pH of all Rose-Bengal antigens cannot be incriminated alone for sensitivity differences among test versions. After mixing antigen with serum samples, a final pH of around 3.8 was reached, being slightly higher (around 4) in case of the modified British RBPT as serum and antigen were not mixed in a ratio of 1:1, but rather 3:1 to boost sensitivity by increasing the potential amount of antibodies if present. The final acidic pH

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longer than 13.7 and 12 of sheep (Smith et al., 1976) and goats (Humphrey and Turk, 1961) respectively. Postoret et al. (1998) reviewed that cattle produce serum concentrations of neutral-pH agglutinins of IgM (3.05 mg/ml) and IgG2 (9.2 mg/ml) higher than those produced by sheep (1.5-3 and 5-7 mg/ml respectively), but sheep produce almost double the concentration of IgG1 acid agglutinins (16-22 mg/ml) as compared to cattle (11.2 mg/ml). The antibody class, concentration and half life differences between bovines and small ruminants explain the need for Rose-Bengal test format of enhanced sensitivity for the latter animals. Infections with antigenically similar Gram negative bacteria like Yersinia enterocolitica O9 may cause false agglutination (Gerbier et al., 1997). Recent immunization with smooth Brucella vaccines usually leads to false positive results (Nielsen, 2010), a case which does not apply to the current investigation where all animals studied were not vaccinated.

3.1.1.3. Brucella species nature of the antigen As far as whole cell antigens are concerned, the outermost parts sterically exposed are those immunogenically dominant (Ducrotoy et al., 2016). Accordingly, the O chain of the surface LPS is the main part involved in serologic reactions with Brucella antibodies. The O chain is a homopolymer of some hundred residues of N-formyl-Dperosamine that bind together in two slightly different linkage orders (Iriarte et al., 2004) leading to two chemically related structures known as A and M epitopes. By no means, A and M stand for abortus and melitensis as both entities are present with different ratios on the surface of all smooth brucellae (Corbel and Banai, 2005). Brucella species/ biovars arranged in descending order of dominance among livestock in Egypt are Brucella melitensis biovar 3, B. abortus biovar 1, B. melitensis biovar 2 and B. suis biovar 1 (Sayour and Sayour, 2015), all of which are A-dominant strains except for the former that has both A and M structures (Iriarte et al., 2004). More importantly is the fact that smooth brucellae highly cross react with each other due to the presence of a common epitope C (Corbel and Banai, 2005), hence, the relatively low virulent B. abortus has been globally accepted for antigen production to detect antibodies not only to B. abortus, but also to its smooth counterparts (OIE Terrestrial Manual, 2016a).

Unlike normal mammals and in addition to the possession of classic antibodies with heavy and light chains, camel sera have unique highly effective antibodies of the IgG2 and the IgG3 classes lacking the light chains (Pastoret et al., 1998). Camel IgG subclasses of IgG1, IgG2a, IgG2b and IgG3 are provoked in response to B. melitensis and B. abortus infections (Abbady et al., 2011). Camel IgG3 is both agglutinating and complement fixing antibody (Pastoret et al., 1998). The biological activity and interaction of camel antibody classes and subclasses in immunoassays require further investigation.

3.1.2. Animal species and stage of infection/ vaccination Animal-associated factors that might directly affect the Rose-Bengal test sensitivity include the species and the stage of Brucella infection. These factors influence the nature, concentration and duration of antibody isotypes produced and consequently the test selectivity and sensitivity. All mammals possess the five classes of immunoglobulins IgM, IgG, IgA, IgE and IgD (Tizard, 2004) of which the first two are the major antibodies in blood sera detected by brucellosis serologic assays (Ducrotoy et al., 2016) including the Rose-Bengal test (Stryszak, 1986). The ten antigen binding sites of the pentameric IgM are credentials for a way better agglutinin than the monomeric IgG (Steward, 1984). On the other hand, the lingering IgG serum concentration is way higher than that of the early IgM (Tizard, 2004). The IgG half life in days of bovines is 17.4 (Nielsen et al., 1978) which is similar to 16.3 of camels (Hüselbusch, 2000), but

3.1.3. Human operator variation Apart from potential technical human errors, the Rose-Bengal agglutination reaction was subject to individual human variation due to the very fact that the test was read by the naked eye. An analyst with healthy eye sight of 6/6 might misidentify several repetitions of a single weak positive sample to include some or few false negative ones. Additionally, individual humans might often have different perspectives regarding the cutoff between positive and negative samples based on visual matching of the test result with the positive and negative controls. This interpretation difference might be attributed to the fact that the smallest resolvable dot (visible agglutination element) the human eye can see is about 0.1 mm at the minimum comfortable viewing distance of 20-25 cm adopted

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revealed by Pearson’s chi-squared test of independence at P value < 0.05 as well as Phi coefficient values. This reflects the fact that all these tests, as well as the CFT, are selective for the same antibody (IgG1). Figures 6-12 graphically represent the comparative performance criteria of every acidified agglutination test version in all ruminant species. The BAPA exhibited relatively lower performance in buffalo cows and goats (Figure 6). A similar picture was revealed by the BCT 8% with even more pronounced fall in buffaloes (Figure 7) that greatly improved by the use of its 3% version, but with extra drop in goats (Figure 8). The overall performance of the UK RBPT dropped in ewes and drastically fell in shecamels (Figure 9) with its modified form achieving some progress in buffaloes, sheep and camels and minor drop in cows and goats (Figure 10). The Scottish RBPT slightly dropped in buffalo cows but noticeably in goats with good performance in cows and she-camels (Figure 11). The French RBPT drastically fell in goats and less severely in shecamels (Figure 12). Goats achieved the worst behavior with Rose-Bengal test formats. Less badly were buffaloes and even less badly were she-camels. This reflects the fact mentioned earlier that unlike in goats, the antibody pattern of buffaloes and camels is relatively closer to that of cattle (Postoret et al., 1998; Tizard, 2004).

in inspection purposes laboratories (Yanoff and Duker, 2009) under normal lighting conditions (light source of about 1000 lumens at a height of 6070 cm with a viewing angle of about 35°). By increasing the viewing distance to 40 cm, the smallest resolvable dot seen by the healthy naked eye increases. Thus, the human factor can affect both the repeatability and reproducibility of the test. A zone of unreliability forms around the threshold limit including false positives, inconclusive results and false negatives, below, roughly at and above the cutoff point respectively. 3.2. Ruminant intra-species performance of Rose-Bengal test formats The relative sensitivity cannot be taken as a standalone criterion for performance comparison of test formats intended as screening pre-confirmatory ones. What really matters beside sensitivity is agreement and independence/ association with the confirmatory CFT as well as accuracy (Tables 4-8 and Figures 1-5). Starting with cows (Table 4 and Figure 1), the best overall performance (rSe, accuracy, κ agreement with CFT and large association with CFT based on Pearson's chisquared test at P value < 0.05 as well as Phi coefficient) was achieved by the Scottish RBPT. Other test formats, viz. US BAPA, US BCT 3%, and 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 Figure 2) had similar high rSe of 98.7% for the French RBPT (4.5%), the Scottish RBPT, the UK modified RBPT and the US BCT (3%). However, the best overall performance was attained by the French version. Then again, the overall best performance of RBPT versions in ewes, as shown in Table 6 and Figure 3, was accomplished by the French (4.5%) RBPT followed by the Scottish RBPT and US BCT (8%). The best performance in caprines (Table 7 and Figure 4) was scored by the UK RBPT followed by the Scottish RBPT and the US BCT (8%). In she-camels, the best performance was documented by the Scottish RBPT followed by the US BCT (8%) as shown in Table 8 and Figure 5.

4. CONCLUSION According to the analytical and diagnostic performance parameters obtained under conditions of this study, the authors concluded that the BAPA behaved better than any other acidified plate agglutination format tried showing minor performance drops in goats and buffaloes. The behavior of the classic Rose-Bengal test formats is satisfactory in cattle. Sensitivity enhancement of the Rose-Bengal test via re-standardization based on the final concentration of cells, the final pH of the antigen, or the OIEISS dilution largely added up to the test performance especially in sheep. Still, the test requires further adaptation in goats, buffaloes and camels in descending order of urgency. More attention should be given to camels due to the unique nature of their antibodies and seroconversion.

3.3. Ruminant inter-species performance of Rose-Bengal test formats Generally speaking, almost all acidified agglutination test formats were largely associated with CFT in ruminant species (Tables 4-8) as

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Herr, S. 1982. Prozones and delayed reactions in the Rose Bengal test for bovine brucellosis. Ondestepoort J. Vet. Res. 49: 53-55. Humphrey, J.H., Turk, J.L. 1961. Immunological unresponsiveness in Guinea pigs. I. Immunological unresponsiveness to heterologous serum proteins. Immunology 4: 301-309. Hüselbusch, C.G. 2000. Immunoglobulin-G status of camels during six months post-natum. Revue d'Elevage et de Médecine Vétérinaire en Pays Tropicaux 53: 105110. Iriarte, M., González, D., Delrue, R.M., Monreal, D., Conde, R., López-Goñi, I., Letesson, J.J., Moriyón, I. Brucella lipopolysaccharide: structure, biosynthesis and genetics. In. López-Goñi I, Moriyón I (Editors). Brucella Molecular and Cellular Biology. England: Horizon Bioscience; 2004, p. 152-182. ISO/IEC 17025: 2005. General Requirements for the Competence of Testing and Calibration Laboratories. Lennette, E. H. 1985. Manual of Clinical Microbiology. 4th Ed. Washington DC: American Society for Microbiol. 1985. Levieux, D. 1978. Bovine immunoglobulins and brucellosis 3. Activity of IgG1, IgG2 and IgM versus different commercial batches of rose bengal antigen. Ann. Rech. Vét. 9 (3): 489-493. Mikolon, A.B., Gardner, I.A., Hietala, S.K., Anda, J.H.de, Pestana, E.C., Hennager, S.G., Edmondson, A.J. 1998. Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats. J. Clin. Microbiol. 36 (6): 1716-1722. National Veterinary Services Laboratories (NVSL) Reagent Manual, price list 2017, USDA, USA. http://www.aphis.usda.gov/animal_health/lab_info_ser vices/downloads/AmesReagentManualCurrent.pdf (accessed February 26, 2017) Nielsen, K. 2010. Serological Diagnosis of Brucellosis. Contributions, Sec. Biol. Med. Sci. 1: 65-89. Nielsen, K., Sheppard, J., Holmes, W., Tizard, I. 1978. Changes in the catabolism of serum immunoglobulins and complement components in infected cattle. Immunology 35: 811-816. OIE Terrestrial Manual (2016a). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter 2.1.4. Brucellosis (Brucella abortus, B. melitensis and B. suis) (Infection with B. abortus, B. melitensis and B. suis). OIE, Paris. OIE Terrestrial Manual (2016b). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter 1.1.5. Quality Management in Veterinary Testing Laboratories (2012). OIE, Paris. Pastoret P, Griebel P, Bazin H, Govaerts A (Editors). Handbook of vertebrate immunology. California: Academic Press; 1998. Pilet, Ch., Toma, B., Andre, G. 1972. Diagnostic sérologique de la brucellose par l'épreuve a l'antigène tamponné (E.A.T.) ou card test. Cah. Méd. Vèt. 41: 519.

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