Recovery of <em>Brucella melitensis</em> from Artificially Infected Dromedaries

Special Article - Brucella

J Bacteriol Mycol. 2018; 5(6): 1080.

Recovery of Brucella melitensis from Artificially Infected Dromedaries

Johnson B¹, Kinne J¹, Jose Sh¹, Pfeffer M², Shanmugaraja R¹, Pandarakandy Sh¹, Ali F¹, Maio E¹, Caveney MR¹, Söllner NK¹ and Wernery U¹*

¹Central Veterinary Research Laboratory, Dubai, United Arab Emirates

²Institute for Animal Hygiene, University Leipzig, Germany

*Corresponding author: Wernery U, Central Veterinary Research Laboratory, Dubai, United Arab Emirates

Received: August 27, 2018; Accepted: September 26, 2018; Published: October 03, 2018


Fourteen serological positive dromedaries intratracheally and intranasally infected with Brucella melitensis were euthanased 12 months after infection. A full necropsy was performed on all 14 dromedaries and 43 different organ samples from each dromedary tested for the presence of the pathogen using 2 selective Brucella media and 3 culture techniques as well as RT PCR.

From a total of 43 different organs 21 (49%) were culture negative and 22 (51%) positive. The pathogen resided mainly in body lymph nodes. The highest culture result was achieved when the enrichment method was used. However, it is proposed, to use all 3 culture methods (direct, concentrated, enriched) as few samples were also negative in the enrichment method but positive in the other methods. BHI is the optimal agar because the Brucella colonies are easier to identify than on Farrell’s agar. RT PCR is not sensitive enough to identify Brucella directly from organ samples, as the pathogen concentration is very low. Culture is still the “gold standard’’ for the diagnosis of brucellosis.

Keywords: Brucella melitensis; Dromedaries; Samples; Isolation


Dromedary brucellosis is widespread in camel rearing countries and is preliminary caused by Brucella melitensis. The pathogen has been mainly isolated from aborted fetuses, milk, hygromas [1], but rarely from dromedary organs of infected dams [2]. In connection with a serological investigation performed at CVRL, Dubai [3,4], we had the opportunity to culture the pathogen from a great number of organ tissues from 14 dromedaries infected with B. melitensis. The results of these investigations are presented here.

Materials and Methods

Fourteen non pregnant female dromedaries (Animal ID C1-C14) of different age (14-23 years) were intratracheally and intranasally infected with a B. melitensis strain, referred to as EM2 [3] belonging to the genetic group East Mediterranean (former African group). This strain was genotyped with multiple-locus variable number tandem repeat (MLVA) [5]. It was previously isolated from a dromedary placenta. The purpose of this infection experiment was the evaluation of serological tests for the in vivo diagnosis of dromedary brucellosis. All 14 infected dromedaries became serologically as well as blood culture positive and were euthanased 12 months after infection. A full necropsy was performed on all camels and 43 different organ samples were taken from each dromedary. Each panel of samples included different lymphoid tissues, internal organs, neuronal tissues (brain and spinal cord), joint fluids of both tarsal and carpal joints, and all four udder cisterns. Each tissue sample was tested for the presence of B. melitensis, using 3 methods; the direct, the concentrated and the enrichment culture methods explained hereafter.

Two types of selective Brucella agars were used

Farrell’s media (Brucella medium base CM0169, Oxoid, supplemented with filtered horse serum SHS100, E and O Laboratories, UK and Brucella selective supplement SR0083A, Oxoid) and Brain-Heart-Infusion agar (Brain Heart Infusion CM 1135, Oxoid, with 1% bacteriological agar and supplemented with filtered horse serum SHS100, E and O Laboratories, UK and Brucella selective supplement, SR0083A, Oxoid).

Direct culture method

The cut surface of organs and lymph nodes was streaked on the surface of the selective Brucella agar plates and 0.1ml of joint fluids was cultured by spread plate method on the Brucella selective agars mentioned above.

Concentration culture method

Organs and lymph nodes were finely minced and transferred into a sterile filter bag (Bag Page, Inter Science. France). 30ml of sterile PBS was added to it, then blended and homogenized in a Lab Blender Mixer (Inter Science, France) for 6 minutes at high speed. The filtrate was decanted into sterile 50ml Falcon tubes and centrifuged at 3000g for 30min. The supernatant was discarded and 0.1ml of the sediment was streaked on the selective Brucella agar plates.

Enrichment culture method

A 1.0ml aliquot of the sediment which was used for the concentration culture method described above was transferred into 7ml of Trypticase Soy Broth (Merck 1.05459.0500) with Brucella selective supplement SR0083A (Oxoid) in a Greiner tube for incubation at 37°C for 6 days. After direct culture, the remaining joint fluid was also enriched in Trypticase Soy Broth with Brucella supplements.


All streaked plates and inoculated broth tubes were incubated at 37°C in an atmosphere of 5% CO2 for 6 days. After 6 days incubation, all plates were examined for the growth of typical Brucella colonies.

The enrichment broth was well homogenized and then 0.1ml of broth was quadrant streaked on selective Brucella agars. The streaked plates were incubated for another 6 days at 37°C in an atmosphere of 5% CO2. After 6 days of incubation, the plates were examined for growth of typical Brucella colonies.


A PCR for the detection of Brucella antigen was only performed on the original samples and not on the concentrated or enriched samples. Briefly, a small piece of tissue about 200mg was placed in an Eppendorf tube containing 2mm glass beads (Sigma, US) and 20ul of proteinase K (20mg/ml concentration from Qiagen protease, Germany) and 200μl ATL buffer (Qiagen, Germany) were added. Sample tube was vortexed thoroughly and incubated at 56°C for 1hr. 300μl buffer AL (Qiagen, Germany) was added and sample tube vortexed for 15secs after which 500μl of the sample was transferred to the MagNA Pure automated extraction platform (Roche Diagnostics Ltd, UK). DNA was extracted using MagNA Pure LV DNA extraction kit according to the manufacturer’s instructions. The DNA was finally eluted with 100μl of Magna pure elution buffer. The PCR was performed according to method described by Probert [6], using ABI 7500 DX machine.


The results of our investigations are shown in Tables 1, 2 and 3

Table 1 summarizes the results of B. melitensis culture and PCR from 43 different dromedary organs of 14 artificially infected dromedaries. From a total of 43 different organs cultured for Brucella bacteria, 21 (49%) were negative in culture and 22 (51%) harbored the bacteria. Following artificial infection with the pathogen, Brucella bacteria resided mainly in body lymph nodes (21%, 9/43), udder tissues and lymph nodes (16%, 7/43) and other tissues (12%, 5/43). An exception was one tarsal joint fluid, from which Brucella bacteria were isolated in numbers too numerous to count.