Rev. sci. tech. Off. int. Epiz.

, 2014, 33 (3), 869-875

Effect of incubation temperature

on the diagnostic sensitivity of the glanders
complement fixation test
I. Khan (1, 2, 3)*, L.H. Wieler (3), M. Saqib (4), F. Melzer (1), V.L.D.A. Santana (5),
H. Neubauer (1) & M.C. Elschner (1)

(1) Friedrich Loeffler Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and
Zoonoses, Naumburger Strasse 96a, 07743 Jena, Germany
(2) Section of Epidemiology and Public Health, College of Veterinary and Animal Sciences, 12-Km Chiniot
Road, Jhang, 35200, Pakistan
(3) Institute of Microbiology and Epizootics, Free University Berlin, Berlin, Robert-von-Ostertag-Str. 7–13,
14163 Berlin, Germany
(4) Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad 38040, Pakistan
(5) Laboratório Nacional Agropecuário, Ministério da Agricultura, Pecuária e Abastecimento, Rua Dom Manoel
De Medeiros, S/N, Dois Irmãos, Recife, Pernambuco, 52171-030, Brazil
*Corresponding author: drkhan_uaf@yahoo.com

Summary
The complement fixation test (CFT) is the only serological test prescribed by
the World Organisation for Animal Health (OIE) for the diagnosis of glanders in
international trading of equids. However, false-positive reactions have caused
financial losses to the animal owners in the past, and false-negative tests
have resulted in the introduction of glanders into healthy equine populations
in previously glanders-free areas. Both warm (incubation at 37°C for 1 h) and
cold (overnight incubation at 4°C) procedures are recommended by the OIE for
serodiagnosis of glanders. In a comparison of the sensitivity and specificity of the
two techniques, using the United States Department of Agriculture antigen, warm
CFT was found to be significantly less sensitive (56.8%; p < 0.0005) than the cold
CFT (83.6%). Cold CFT thus increases the detection rate of glanders but a lower
diagnostic specificity has to be accepted. The immunoblot was used as the gold
standard.

Keywords
Burkholderia mallei – Complement fixation test – Equids – Glanders – Immunoblot –
Incubation temperature – Sensitivity – Serodiagnosis.

Introduction Bahrain, Kuwait and Afghanistan (3, 4, 5, 6, 7, 8, 9, 10,

11, 12).

Glanders is a zoonotic disease of solipeds and is caused

Although several conventional serological tests are currently
by the Gram-negative pathogen Burkholderia mallei. The used for diagnosis of glanders, the complement fixation
disease is notifiable to the World Organisation for Animal test (CFT), because of its ability to detect chronic carrier
Health (OIE) (1, 2). Glanders manifests as nodules on the animals, is the only serological test recommended by the
skin (farcy) and on the mucous membranes of the upper OIE for use in the international transport of equids (13, 14,
respiratory tract. It can also cause high mortality (95%) in 15, 16, 17). However, false-positive CFT results can cause
untreated humans and is still endemic in many countries, large-scale economic losses to animal owners and undue
including Turkey, Iraq, Iran, India, Pakistan, Mongolia, transboundary restrictions on valuable animals. False-
the People’s Republic of China and Brazil. Outbreaks have negative or equivocal results, on the other hand, can occur
also been reported in the United Arab Emirates, Lebanon, in tests on old, debilitated or pregnant animals and also

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in well-kept clinically healthy carriers (1, 16). Improved Complement fixation test procedures
serodiagnostic tests with higher diagnostic specificity and
sensitivity are therefore regularly called for (18). Both All serum samples were tested using the CFT method
warm and cold CFT procedures are described in the OIE according to the OIE Terrestrial Manual (18). To minimise
Manual of Diagnostic Tests and Vaccines for Terrestrial Animals the inherent errors of the CFT, a commercially available
(Terrestrial Manual) (18). According to the OIE (2008), the CFT antigen (United States Department of Agriculture,
temperature and duration of incubation can interfere with Ames, Iowa, USA) (18) and a ready-to-use complement
the analytical performance of an assay (19). Nevertheless, as and haemolytic system (Institut Virion/Serion GmbH,
the glanders CFT is a widely used and cheap test, the effect of Würzburg, Germany) were used (19).
incubation temperature does not attract sufficient attention.
No literature on the effect of temperature on the glanders The detailed procedure was as follows. Sera were
CFT is available, although there is some old literature on diluted 1/5 in veronal-buffered saline containing
the Wassermann reaction (CFT) for diagnosis of syphilis. In 0.1% gelatin and inactivated in a water bath for 30 min
1909, Satta and Donati were the first to address this subject at 58°C for horse sera and 63°C for sera from mules,
and to publish their observations (20). donkeys and non-equids. The inactivated test sera were
mixed well on a shaker before pipetting into 96-well
In the present study, the warm and cold CFT procedures round-bottomed microtitre plates. Two-fold dilutions of
were compared and the recently evaluated immunoblot was the sera were then prepared. First, 25 μl of veronal buffer
used as the gold standard (21). The immunoblot is the most was added to all rows except the second row, then 25 μl of
specific and sensitive test and can also be used to test sera diluted inactivated test serum was pipetted into the first,
that show anti-complementary activity in CFT (21, 22). second and third rows. Two-fold serial dilutions were made,
starting from the third row until the end, and discarding the
final 25 μl from the last row. Antigen (25 μl) at a working
Materials and methods dilution was then added to all rows except the first. Lastly,
25 μl of complement, diluted to the number of units required,
Origin of serum samples was added to each well. Negative control wells containing
diluent only, complement/diluent, or complement/diluent/
The 342 equid sera included in this study were divided into antigen were prepared (75 μl in each well). Positive and
two groups. In group I, 196 sera were randomly collected negative serum control wells were also prepared. Plates
(Mersenne Twister software, SPSS Inc., Chicago, USA) from were shaken for 60 s on a shaker before incubation. For
among 2,282 horse and donkey sera in Germany. These cold CFT, plates were covered and incubated at 4°C for
sera were declared true negative because glanders was 16 to 20 h; for warm CFT, plates were incubated at 37°C
eradicated in Germany more than 50 years ago and the sera for 1 h only.
were immunoblot negative (21, 23).

To compare the sensitivity of warm and cold CFT procedures, In a second step, the haemolytic system was prepared
a set of 146 true-positive sera were selected (group II). This by careful mixing of amboceptor at the working
group included 123 samples from Brazil (Pernambuco) dilution 1:1 with 1% erythrocyte suspension, followed by
and Pakistan (Punjab) that were collected during glanders incubation for 30 min in a 37°C water bath. The plates
outbreaks between 2007 and 2009 from microbiologically from the first day were also pre-warmed for 30 min in a
or clinically (mallein test) positive glanderous animals. 37°C incubator to ensure that the haemolytic system and
The Pakistani sera were collected from clinically positive plates were at the same temperature. Freshly prepared
and culture-positive equids during two separate outbreaks haemolytic system (50 μl) was added to each well and
of glanders among police horses (Faisalabad Metropolis), shaken carefully. Plates were moist-incubated at 37°C for
polo ponies (Lahore Polo Club, Lahore), animals on private 15 to 30 min.
farms (Sargodha district) and draught equines (Faisalabad
City). The samples from Brazil were collected from clinically Incubation was stopped when the complement control
positive glanderous animals in different endemic areas of wells with 2 and 1 units showed complete haemolysis.
Pernambuco State (National Animal and Plant Laboratory, Plates were centrifuged for 5 min at 670 g. The absence of
Ministry of Agriculture, Livestock and Food Supply, Recife, anti-complementary activity was checked for each serum in
Brazil). In addition, 25 sera from B. mallei-immunised the first row. The first row was regarded as a serum control
horses were collected (21): of these, 23 sera tested positive (no antigen). Sera were considered negative when 100%
in immunoblot and were included in group II; the remaining haemolysis occurred at 1:5 dilution, 25% to 75% haemolysis
two sera were immunoblot negative and were excluded from was considered equivocal and absence of haemolysis was
the study (21, 23). The animal experiment was authorised considered to indicate a positive serum (18). Complement
by the government of Thuringia, Germany (registration no. fixation test titres were defined at the step at which the
04-105/07). serum dilution showed inhibition of haemolysis.

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Statistical analysis significantly higher (one- to two-fold) antibody titres in the

cold test (p < 0.0005).
The diagnostic specificity and sensitivity of the warm
and cold CFT procedures were calculated using standard Overall, in group I the warm CFT showed 100% specificity
formulae (24) and software R. The McNemar test was (CI 0.98; 1) and the cold CFT, 97.95% (CI 0.95; 0.99)
used for comparison of the two CFT procedures and the specificity. In group II, the warm CFT showed 56.8%
Wilcoxon test for comparison of the CFT titres (Predictive sensitivity (CI 0.48; 0.65) and the cold CFT, 83.6%
Analysis is Software, version 17.0 SPSS). Confidence sensitivity (CI 0.76; 0.89), which was significantly higher
intervals (CI) were calculated. (p < 0.0005).

Results
A total of 342 equid sera were evaluated in warm and cold 300
CFTs (Table I). In group I (true negatives; 196 sera), all sera
250
(100%) were negative in the warm CFT; in the cold CFT
192 sera (97.95%) tested negative and four (2.04%) were 200
positive. CFT titre
150

In group II (146 sera), 74 sera (50.68%) tested positive, 100

nine sera (6.16%) were equivocal and 63 (43.15%) M
* * * * * * *
were negative in the warm CFT. In the cold CFT, 50
117 (80.13%) tested positive, five (3.42%) were equivocal 0
and 24 (16.44%) were negative (Table I). In further -1 0 1.5 3 4 5 6 7 8 9 10 11 12 12.5 13 14 16 18 20 22 24 26 28 30 34
calculations the equivocal samples were considered Weeks
positive. Weekly samples collected from an immunised Warm CFT Cold CFT
horse up to six weeks after the application of mallein tested
negative in the warm CFT but positive in the cold test CFT: complement fixation test
M: application of mallein
(Fig. 1). In the group of immunised horse sera, differences in *immunisation weekly for 7 weeks
cold CFT titres between 12 and 13 days post-immunisation Fig. 1
showed a difference of only one two-fold dilution step. Most Comparison of warm and cold complement-fixation titres of
group II sera that tested positive in the warm CFT showed serum from a horse immunised against B. mallei

Table I
Warm and cold procedures for complement-fixation testing of sera from glanders-free (Group I) and glanderous
and immunised animals (Group II)

Nature/ No. of samples Warm CFT Cold CFT

origen of the sera Horse Donkey Mule Positive Equivocal Negative Positive Equivocal Negative
Group I

True negative 195 1 0 0 0 196 4 (a) 0 192

(n = 196)

Group II

Positive glanderous animals 117 2 4 57 (b) 8 (c) 58 (d) 95 (e) 4 (f) 24 (g)
(mallein test)
(n = 123)

Positive immunised animals 23 0 0 17 1 5 22 1 0

(immunoblot)
(n = 23)

CFT: complement fixation test c) 8 horses f) 4 horses

a) 4 horses d) 56 horses; 1 donkey; 1 mule g) 23 horses; 1 donkey
b) 53 horses; 1 donkey; 3 mules e) 90 horses; 1 donkey; 4 mules

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Discussion infected animals up to one week earlier than the warm

CFT. Similarly, Ris (26) found that sera with titres between
1:10 and 1:20 in the cold CFT had no measurable titres in
Both temperature and duration of the primary incubation of the warm test. He concluded that in ovine brucellosis an
the test serum have an important effect on the performance estimated 1.6% of sera tested false positive with cold CFT.
of the CFT. This effect was first demonstrated more than
90 years ago in the Wassermann reaction, a CFT for syphilis
In the present study, among the 196 true-negative sera in
diagnosis. In most syphilitic sera, primary incubation for
group I, no serum sample tested false positive in the warm
18 h at 2°C to 10°C resulted in a more efficient and sensitive
CFT; however, in the cold CFT four (2%) sera tested false
complement fixation reaction than water-bath or incubator
positive. One possible reason for the slightly increased
incubation at 38°C for 1 h (25). It was later experimentally
percentage of false-positive results in the cold CFT might
proven that more complement is gradually fixed at 0°C than
be cross-reactivity with other Gram-negative bacteria, such
at 37°C. This finding is based on the assumption that the
as Burkholderia cepacia and Pseudomonas spp. (28, 29, 30).
process depends on the formation of an antigen antibody
complement complex that creates a triple adsorption
compound. The smaller particles in the cold mixture
present a larger surface for adsorption and therefore the Conclusion
amount of fixed complement increases (20).
The CFT has the advantage of being cheap and easy to
While working on brucellosis, Ris demonstrated in handle, and this should be borne in mind as current budget
1974 that diagnostic sensitivity in the tube CFT could be cuts in veterinary public health are a concern not only
increased by incubation at 4°C for 16 h. In a preliminary for developing countries. However, further investigation
study, Ris also found that animals that shed brucellosis should focus on the production of better-quality antigens
organisms in their semen tested negative in the warm CFT for use in the test. The cold CFT, with its greater diagnostic
but positive in the cold test (26). Burgess and Norris (27), also sensitivity, is recommended for surveillance and screening
working on brucellosis, were able to demonstrate that the cold programmes; nevertheless, its use depends on the epidemic
CFT was more sensitive than the warm test. On examining situation of a country and on the availability of laboratory
11,922 sera for brucellosis, they observed that 187 (1.57%) capacity for identification of the higher number of expected
were positive in the cold CFT but only 38 (0.32%) were false positives. For import and export testing, the CFT
positive in the warm test. should be supported with use of the highly specific
immunoblot in order to avoid inconvenience for private
In the present paper, overnight (16 h to 20 h) incubation at owners and public veterinary health authorities.
4°C was used to achieve maximum fixation of complement
and the results are in line with other findings indicating that
cold CFT is indeed more sensitive (83.6%) than the warm Acknowledgements
test (56.8%) in the diagnosis of glanders. The differences I.K. is indebted to the Islamic Development Bank,
observed between the two tests in terms of antibody titres Kingdom of Saudi Arabia, for granting a foreign PhD merit
of positive sera are also in agreement with the findings of scholarship. I.K. is also grateful to the Friedrich Loeffler
Ris (26) (Fig. 1). Most of the sera that were positive in both Institute in Germany for providing the diagnostic facilities
CFTs showed one- to two-fold higher antibody titres in to complete this study. All the authors thank Dr Lisa Sprague
the cold test. Moreover, four immunoblot-positive samples for critical reading of the manuscript and Dr Roland Diller
collected from a horse after malleinisation tested negative in for statistical support.
the warm CFT but positive in the cold test. This observation
was also made by Burgess and Norris (27), who found that
cold CFT detected brucellosis antibodies in experimentally

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Effet de la température d’incubation sur la sensibilité

diagnostique de l’épreuve de fixation du complément
pour la détection de la morve
I. Khan, L.H. Wieler, M. Saqib, F. Melzer, V.L.D.A. Santana, H. Neubauer
& M.C. Elschner

Résumé
L’épreuve de fixation du complément est la seule épreuve sérologique prescrite
par l’Organisation mondiale de la santé animale (OIE) pour le diagnostic
de la morve aux fins des échanges internationaux d’équidés. Néanmoins,
des réactions faussement positives ont fait perdre beaucoup d’argent aux
propriétaires de chevaux, tandis que des réactions faussement négatives ont eu
pour conséquence d’introduire la morve dans des populations équines saines
de régions précédemment indemnes de morve. Le protocole recommandé par
l’OIE pour le diagnostic sérologique de la morve prévoit une incubation à chaud
(à 37 °C pendant 1 heure) ou à froid (pendant une nuit à 4 °C). Dans une étude
comparative sur la sensibilité et la spécificité de ces deux techniques, utilisant un
antigène préparé par le Département de l’agriculture des États-Unis d’Amérique,
la méthode de fixation du complément à chaud s’est avérée significativement
moins sensible (56,8 % ; p < 0,0005) que la méthode à froid (83,6 %). La méthode
de fixation du complément à froid accroît le taux de détection de la morve, mais
au prix d’une spécificité diagnostique moindre. L’immunoblot a été utilisé comme
épreuve de référence.

Mots-clés
Burkholderia mallei – Diagnostic sérologique – Équidés – Immunoblot – Morve –
Sensibilité – Température d’incubation – Test de fixation du complément.

Influencia de la temperatura de incubación en la sensibilidad

de diagnóstico de la prueba de fijación del complemento para
detectar el muermo
I. Khan, L.H. Wieler, M. Saqib, F. Melzer, V.L.D.A. Santana, H. Neubauer
& M.C. Elschner

Resumen
La técnica de fijación del complemento (FdC) es la única prueba serológica
prescrita por la Organización Mundial de Sanidad Animal (OIE) para diagnosticar
el muermo en operaciones de comercio internacional de équidos. Sin embargo,
ya ha habido falsas reacciones positivas que han causado pérdidas económicas
a los propietarios de animales, y falsos resultados negativos que se han saldado
con la introducción del muermo en poblaciones equinas sanas de zonas que
hasta entonces estaban libres de la enfermedad. Para el diagnóstico serológico
del muermo la OIE recomienda tanto el procedimiento en caliente (1 hora de
incubación a 37°C) como en frío (una noche de incubación a 4°C). Tras comparar
la sensibilidad y especificidad de ambas técnicas empleando el antígeno del
Departamento de Agricultura de los Estados Unidos, se observó que la prueba

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de FdC era significativamente menos sensible en caliente (56,8%; p < 0,0005) que
en frío (83,6%). El método de FdC en frío incrementa pues el índice de detección
del muermo, si bien a costa de aceptar una menor especificidad de diagnóstico.
Como referencia absoluta se utilizó la prueba de inmunoblot.

Palabras clave
Burkholderia mallei – Diagnóstico serológico – Équidos – Inmunoblot – Muermo – Prueba
de fijación del complemento – Sensibilidad – Temperatura de incubación.

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