Pakistan J. Zool., vol. 45(6), pp. 1751-1756, 2013.

Prevalence of Burkholderia mallei in Equids of Remount Depot,

Sargodha, Pakistan
Iahtasham Khan1,2*, Shahzad Ali1,3, Mayada Gwida1,4, Mandy C. Elschner1, Muhammad Ijaz2, Aftab
Ahmad Anjum2 and Heinrich Neubauer1
1
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute for Bacterial Infections
and Zoonoses, Naumburger Str.96a, D-07743 Jena, Germany
2
University of Veterinary and Animal Sciences, Lahore, Pakistan
3
Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan
4
Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University, Mansoura
35516, Egypt
Abstract.- Glanders is a highly contagious disease of solipeds caused by Burkholderia mallei. Nodules and
ulcers are either seen in the upper respiratory tract and lungs (i.e. glanders) or the skin (i.e. farcy). Infection always
results in persistence of the agent and intermittent shedding. Progressive loss of efficiency and fatal outcome resulting
in massive economical losses forced veterinary authorities worldwide to start disease control including mass testing
using complement fixation test and/or malleinisation, and culling of positives. In the last decade, the number of
outbreaks in Asia and South America has been steadily increasing and glanders proved to be a re-emerging
transboundary disease again. Pakistan has demonstrably been an endemic country for the last 120 years. Actual data
however on the presence of disease among Pakistani army equid are absent. A seroprevalence study of equids rearing
establishment, Remount Depot, Sargodha Pakistan having the densest working equine population from Sargodha
district was made in the year 2009. A total of 920 (horses: 75; mules: 844; donkey: 01) serum samples were collected
from apparently healthy paddock equids. The complement fixation test (CFT) and the highly specific and newly
validated immunoblot (IB) technique were used for serodiagnosis. No positive animal (horse, mule and donkey) was
found. Glanders seems to be restricted to remote, sporadic pockets of endemicity and may cause outbreaks after being
introduced in native populations by asymptomatic shedders. The diagnostic specificity of the ccPro antigen based CFT
was 68.39%, and of the CIDC antigen based CFT 65.87%.
Key words: Seroprevalence, Complement fixation tests, Equidae, glanders.

INTRODUCTION

landers is caused by the bacterium

Burkholderia (B.) mallei a Gram-negative, nonmotile, obligate intracellular mammalian zoonotic
pathogen. Nodules and ulcers are either seen in the
upper respiratory tract and lungs (i.e., glanders) or
the skin (i.e. farcy). Infection always results in
persistence of the agent and intermittent shedding.
The disease appears mostly in chronic form in
horses and as an acute form in donkeys and mules.
Most cases of glanders remain nonclinical or latent
(Dvorak et al., 2008; Khan et al., 2013; OIE, 2008).
Progressive loss of efficiency and fatal outcome
resulting in massive economical losses forced
__________________________
*

Corresponding author: [email protected]

0030-9923/2013/0006-1751 $ 8.00/0
Copyright 2013 Zoological Society of Pakistan

veterinary authorities worldwide to start disease

control including mass testing using complement
fixation test or malleinisation, and culling of
positives. Following this, glanders was eradicated
from Western Europe and North America in the
1950s. However, the number of outbreaks in Asia
and South America has been steadily increasing in
the last decade and glanders again proved to be a reemerging transboundary disease. Pakistan is a good
example for a country which has demonstrably been
endemic at least for the last 120 years. The glanders
and Farcy Act of the British government in 1899
made B. mallei infection a notifiable disease
involving culling of positive equidae and camels. In
Pakistan, low indemnity paid to the owner it is the
same amount as in 1899 - and lack of stringent
implementation of this act have limited the owners
preparedness to destroy their diseased animals
(Khan et al., 2013; Muhammad et al., 1998; Wahid,
2011) and hinder eradication of the disease. A

1752

I. KHAN ET AL.

cascade of outbreaks in civilian equids have been

reported from 1999 to 2007 in the Pakistani Punjab
and various strains of B. mallei have been isolated.
Fifteen of these were characterised by variable
number tandem repeat (VNTR) typing (Hornstra et
al., 2009). It appears that there exists specific B.
mallei lineages (genotypes) of the Pakistani Punjab
and the disease is spread via communal stables and
water troughs. Glanders outbreaks have been
reported from Indian army equids during the past 30
years (Verma, 1981). An outbreak of glanders was
also noticed in the equid breeding establishment
Mona Remount Depot (RD), Sargodha, Pakistan in
2006-2007. A preliminary study was carried out is
equids rearing establishment RD, Sargodha to assess
the clinical manifestations of glanders and anti-B.
mallei antibodies using complement fixation test
(CFT) (OIE, 2008; Elschner et al., 2011) and
immunoblot (IB) technique (Elschner et al., 2011;
Khan et al., 2011). No seroprevalence study has
been conducted so far to examine anti-B. mallei
antibodies in apparently healthy Pakistani army
equids. The main objectives of this study was to
determine the seroprevalence of glanders in
apparently healthy equid of Pakistan Army and to
comparatively evaluate CFT (using two different
antigens: c.c.pro, Germany and CIDC, The
Netherlands) and IB for the diagnosis of glanders.

issues, guidelines were followed made for the

welfare of livestock from which blood is being
harvested for commercial and research purposes by
the Animal Advisory Committee, Ministry of
Agriculture, Wellington (1996), New Zealand
(NAEAC, 2009). Basic data on animals were
gathered with a questionnaire (open and closed
questions) for each animal. All sera were tested with
the OIE prescribed CFT method (OIE, 2008) using
two different antigens: ccpro (ccpro GmbH,
Oberdola, Germany) and CIDC (Central Veterinary
Institute of Wageningen UR, The Netherlands).
Both these antigen were extracted from B. mallei
cultures in phosphate buffer saline and the
compound contained 0.5% phenol as a preservative.
Working dilution in Veronal buffer (VB) was 1:40.
For CIDC B. mallei strains were heat killed for 3 h
in a Kochs steamer at 100 C and the working
dilution in VB is 1:600.
CFT procedure was adopted as described by
Khan et al. (2011). All CFT positive/suspicious and
anti-complementary sera were retested with the
confirmatory IB (Elschner et al., 2011). Antigen
preparation and test procedures were performed as
described by Elschner et al. (2011) with little
modification (Khan et al., 2011).

MATERIALS AND METHODS

Table I shows the information retrieved

through questionnaires of 920 study animals. The
equines were divided into North, East and West
charges and each charge was several kilometres
away from each other. Each charge was further
divided into different paddocks. According to the
questionnaires, 920 blood samples belonged to
equines of different age, sex and breeds. All animals
were non-vaccinated. Two hundred and fifty four
female animals (horses: 01 mules: 253) were nonpregnant by history but no pregnancy test was
performed onsite. Six hundred and sixty six (horse:
74; mules: 591; donkey: 01) male equids were
castrated. All animals were well managed with their
pedigree records, were well fed and showed good
health status. Animals used common feeding and
water troughs. Mules were separated from horses
into different paddocks. The age of mules was from
3-16 years while the age of horses was 3-5 years.

A total of 920 (horses: 75; mules: 844;

donkey: 01) equine blood samples were collected
from the Pakistani Army equine rearing
establishment, RD, Sargodha. Pink spray (Komi
Phrama, Korea) was used for marking the animal
after blood sampling and as antisepsis at the site of
blood collection. All blood samples were collected
during the hot summer season i.e. from April to
July, 2009. The temperature ranged from 4049C.
During field conditions, blood samples were stored
at 4C and immediately transferred to local
laboratory for sera isolation. The samples were
centrifuged at 1300 rpm for 15 minutes at RT
(800D, Jiangsu Zhengji Instruments, China). For
long-term storage, the serum samples were kept at 20C and shipped to the Friedrich-Loeffler-Institut,
Jena, Germany on dry ice. For animals ethical

RESULTS

BURKHOLDERIA MALLEI IN EQUIDS OF PAKISTAN

No equid was tested positive in the IB but

depending upon the use of antigen (ccPro/CIDC/
both antigens) 5/9/10 horse sera and 88/112/139
mules sera were diagnosed false positive by CFT.
The sera of fifty five equids (horse: 10; mules: 45)
showed anti-complementary effect with both
antigens in CFT. CFT and 1B results of 920 animals
have been summarized Table II. Excluding 55 anticomplementary sera, the diagnostic specificity of
the ccPro antigen based CFT was 68.39%, and of
the CIDC antigen based CFT was 65.87%.
Table I.-

Main
information
retrieved
questionnaires of 920 study animals.

through

Sr. No.

Characteristics of 920
study animals

Numeric / comments

1.

Animal code

To identify the animal

2.

Species
Age

3.
4.

Sex (male/female)
Breed

5.

In case of female
(pregnant or
non-pregnant)
Vaccination (if any)

Horse/mule/donkey
Varies from 3-16
years
(666/254)
Thorough
breed/Arabian/Sufflok
All female animals
(254) were nonpregnant
All animals were nonvaccinated
No

6.
7.
8.
9.
10.

Previous history of
Glanders
Any case report of
Strangels
Any case report of
Melioidosis
Feeding and drinking
behavior

11.

Serodiagnosis of glanders

12.

Malleinisation

53 horses
unknown
Use common water
troughs for drinking
Use common mengers
for feeding
No serodiagnosis was
performed
All animals were nonmalleinised

DISCUSSION
Recently, a glanders outbreak in Kabul,
Afghanistan was attributed to illegally imported
horses from Pakistan. In Pakistan B. mallei strains
were frequently isolated from glanderous equids
from the Punjab districts of Faisalabad, Lahore and
Sargodha during 1999-2007 (Wahid, 2011)

1753

demonstrating the permanent presence of B. mallei

in the equid population of this country. In disease
free periods common stables and their surroundings
seem to be active reservoirs for glanders. B. mallei
is cleared from wet surfaces or slurry within 100
days in temperate climates (Loeffler and Schutz,
1882). We assume that B. mallei is inactivated with
a few days or even hours by the dry heat and harsh
sunlight during Pakistani summer. Thus, the role of
public water troughs and stables for the spread of B.
mallei may only be of seasonal importance. Based
on a literature study we also assume that especially
healthy carriers i.e. sub-clinically infected animals
play the most important role for persistence and
propagation of disease (Manzoor et al., 2008;
Pritchard, 1995). In disease free periods i.e. when no
outbreak is obvious these animals should at least be
detected by serology. Astonishingly no clinical
signs of chronic infection i.e. scars as marks of local
infection or acute glanders or anti B. mallei
antibodies were registered. Our current study shows
that glanders was not detectable in equids, RD,
Sargodha having outbreaks in the past. It can be
supposed that glanders only re-emerged in the form
of local epidemics when glanderous equids were
introduced in native populations. The authors
suppose that only small local foci of glanders exist
in remote areas of the Punjab from which glanders
spilled over to other equine population only
occasionally. This assumption is supported by the
finding that a glanders outbreak happened in a
Lahore Polo Club (2005) after the introduction of
two glanderous horses brought from a Sargodha
district farm to participate in competitions (Wahid,
2011). Another explanation for the absence of
glanderous animals in our investigation is that most
diseased animals may succumb to glanders during
the stressful summer season. The army population
of equines tested were well monitored and
obviously glanders free army animals. This group
should serve as a local negative control group for
test evaluation as done before by Pakistani workers
(Naureen et al., 2007). So it was not astonishing that
no positive cases were found. However, the
high number of CFT positives in this group was
surprising. A total of 363 (39.5%) sera tested
positive/suspicious with both CFT procedures. It is
well known that CFT may produce a considerable

1754

I. KHAN ET AL.

Table II.- Complement fixation tests and immunoblot results summary of 920 animals.
Species (No
of samples)
Horse (5)
Horse (9)
Horse (10)
Mule (88)
Mule (112)
Mule (139)
Horse (10)
Mule (45)
Horse (41)
Mule (460)
Donkey (1)

A/M/F/B+

CFT (c.c.pro)

CFT (CIDC++)

Both CFTs

IB+++

Comment

3-5/4/1/A,S
3-5/9/0/A,S
3-5/10/0/A,S
3-16/56/32/H
3-16/69/43/H
3-16/117/22/H
3-5/10/0/A,S
3-16/33/12/H
3-5/41/0/A,S,T
3-16/316/144/H
6/1/0/ND

Positive/suspicious
Negative
Positive/suspicious
Negative
ACA
ACA
Negative
Negative
Negative

Negative
Positive/suspicious
Negative
Positive/suspicious
ACA
ACA
Negative
Negative
Negative

Positive/suspicious
Positive/suspicious
-

Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Not tested
Not tested
Not tested

False positives
False positives
False positives
False positives
False positives
False positives
Negative
Negative
True negatives
True negatives
True negatives

A/M/F/B+: age/male/female/breed
c.c.pro: CFT antigen from c. c. po GmbH, Germany
ACA : anticomplementary activity
CIDC++: CFT antigen from Central Veterinary
Institute of Wageningen UR, The Netherlands

number of false-positive results. False-positive CFT

results were attributed to cross-reactions to other
bacteria e.g. Streptococcus equi (Ijaz et al., 2010;
Misra and Arora, 1990). A high prevalence of anti
S. equi antibodies may be supposed as the reason for
the observed cross reactions in our study
populations. It is well known that strangles is
endemic in Pakistani army horses (Ijaz et al., 2010)
and the civilian equid population of Sargodha and
Lahore (Ashraf, 2000; Manzoor et al., 2008).
Furthermore, Ijaz et al. (2010) reported highest
(2.6%) incidence of strangles from the end of
January to the beginning of May, thus shortly before
or in the beginning of the sampling period. Two
other Pakistani working groups reported 32.2%
strangles in mules less than 2 years of age and
35.4% in mules of more than 2 years of age in
Pakistan and 54% strangles in foals in Punjab,
Pakistan (Ijaz et al., 2010, 2011). Melioidosis can
not be ignored in differential diagnosis of glanders.
No report on animals melioidosis, (Burkholderia
pseudomallei) which shows 98% genetic
resemblance with B. mallei in Pakistani Punjab, is
available. Fifty five (6%) serum samples
predominantly from
mules
showed anticomplementary activity. Mule and donkey sera are
more prone to anti-complementary activity
(Naureen et al., 2007). The reason for this high

CFT - : Complement fixation test

IB+++: Immunoblot
- Not Applicable
A, S, H, T, ND: Arabian, Suffolk, Hybrid,
Thorough-breed, Non-descript

number is not clear. Local strains can improve the

sensitivity of tests for glanders detection (Sprague et
al., 2009). To proof our preliminary data which are
biased towards sera from an army establishment, a
bigger seroprevalence study among the equids of the
Pakistani Punjab is highly recommended in the
future using CFT and IB containing also composite
mixtures of local B. mallei strains.
Our study showed that it may be very
difficult to control glanders in a country like
Pakistan. The only effective control option for
glanders lacking effective vaccines is testing and
culling of positives. It has to be stressed that a
number of false positives will always be produced
by serological tests and that destruction of these few
healthy animals is unavoidable. This line of action is
the only way to stop the establishment of B. mallei
in niches and also to avoid animal to human transfer
(Sprague et al., 2004). Testing has to be carried on
even after the disease has obviously become rare
and countermeasures have to be set into action to
avoid reintroduction of disease by healthy carriers.
It has to be stressed that it is impossible to
demonstrate absence of disease in equid in every
case. An element of risk of spreading the disease
still remains even if all precautions have been
strictly followed. Public veterinary health actions
have to be flanked by a wise re-imbursement policy

BURKHOLDERIA MALLEI IN EQUIDS OF PAKISTAN

to get full acceptance by the animal owners.

However, all equines of the country have to be
registered in advance and have to be made available
for testing. Safe destruction of carcasses,
decomposition of manure and disinfection of
premises will round up the control program. It is of
eminent importance to bear special social or
regional particularities in mind to gain acceptance of
the countermeasures at the best.
CONCLUSIONS
Glanders seems to be restricted to remote,
sporadic pockets of endemicity and may cause
epidemics after being introduced in naive
populations by (asymptomatic) shedders. In
serodiagnosis, animals tested false-positives and
false-negatives should be retested with a highly
sensitive and specific test like immunoblot to avoid
the loss of healthy animals and introduction of
glanderous animals in healthy populations,
respectively.
ACKNOWLEDGEMENTS
The authors are thankful to the Islamic
Development Bank, Kingdom of Saudi Arabia to
grant PhD merit scholarship to I.K. We are also
thankful to Prof. Dr. Lothar H. Wieler, Head
Institute of Microbiology and Epizootics, Free
University Berlin, Germany to guide us for the
accomplishment of this research project.
Conflict of interest
No conflict of interest exists.
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