Journal of Life Sciences Volume 8 Number (4)
JLS
Journal of Life Sciences
Volume 8, Number 10, October 2014 (Serial Number 78)
Contents
Microbiology
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
Tsveteslava Veselinova Ignatova-Ivanova and Radoslav Iliev Ivanov 805
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
Srwa Ali Muhammed 811
Seroprevalence of Human Brucellosis in Kuku Dairy Scheme, Khartoum State, Sudan
Tamador-Elkhansaa Elnour Angara, Adil Abdel Rahman Ali Ismail and Nageeb Suliman Saeed
Botany and Zoology
Detection of Mycoplasma synoviae Infection in Broiler Breeder Farms of Morocco Using Serological Assays and Real Time PCR
Nassik Saâdia, Aboukhalid Rachid, Azzam Falak, Rahmatallah Naoufal, Lahlou-Amine Idriss, Fassi-Fihri Ouafaa and El Houadfi Mohammed
Prebiotic Isomalto-oligosaccharide Production from Thai Rice
Premsuda Saman, Achara Chaiongkarn, Somporn Moonmangmee and Chantra Poonsiri 828
Influence of the Type of Fertilization on Nitrogen Balance of the Mountain Meadow
Piotr Kacorzyk, Miros ław Kasperczyk and Wojciech Szewczyk 835
Effect of Phosphonate Fertilizers on the Growth of Soil Fungi
Samer Samir Mohd Habash, Mohammad Saleh Al-Bess, Ahmad Saleh Al-Bess and Luma Shareef AL Banna
Interdisciplinary Researches
Ecological Research of Former Brown-coal Quarry—the Most Lake in the Czech Republic
Martin Neruda, Ladislava Filipová, Jana Říhová Ambrožová, Iva Machová, Karel Kubát, Michal Holec and Diana Holcová
Limited Efficacy of Aspirin in Patients with Peripheral Arterial Occlusive Disease
Pavel Poredoš 857
The Comparison of Urinalysis Parameters in Pregnant and Non-pregnant Women in Berat
Bakaj (Çizmja) Aurora and Lika (Çekani) Mirela
Journal of Life Sciences 8 (2014) 799-804
doi: 10.17265/1934-7391/2014.10.001
DAVID PUBLISHING
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
Tsveteslava Veselinova Ignatova-Ivanova and Radoslav Iliev Ivanov Department of Biology, University of Shumen, Shumen 9712, Bulgaria
Received: September 17, 2014 / Accepted: October 19, 2014 / Published: October 30, 2014.
Abstract: Biocorrosion processes at metal surfaces are associated with microorganisms, or the products of their metabolic activities including enzymes, exopolymers, organic and inorganic acids, as well as volatile compounds such as ammonia or hydrogen sulfide. It was proved that strain Lactobacillus delbrueckii B5 constituted biofilms in the presence of different amounts of carbohydrates (5% sucrose and a mixture of 5% lactose, 5% fructose and 5% maltose). The obtained information was used in a study treating the anticorrosive properties of microbial biofilms synthesized by the latter strain. The study of the corrosive stability of steel samples was conducted on the gravimetrique method. The rate of corrosion, the degree of protection, and coefficient of protection has been calculated. The structure of layer over steel plates was analyzed by Scanning Electron Microscopy (SEM) JSM 5510.
Key words: Biofilm, corrosion, inhibitor, lactic acid bacteria, SEM.
I. Introduction volatile compounds such as ammonia or hydrogen sulphide can affect cathodic and/or anodic reactions,
Corrosion is the deterioration of a material, thus altering electrochemistry at the biofilm/metal especially metal, as a result of chemical or interface. This phenomenon is often referred to as electrochemical reactions on the metal/solution “biocorrosion” or “microbially influenced corrosion”. interface [1, 2]. The metallic corrosion is a The adoption of preventive measures that reduce or spontaneous process which consists of the inverse of eliminate corrosion is financially costly and time metallurgical processes, where the processed metals consuming. In fact, corrosion prevention and revert to their natural state of lower free energy, i.e., treatment consume more than 20% of a typical chemical compounds or minerals [1]. The metallic
industrial budget.
corrosion represents an economic burden for many It was found that exopolysaccharides produced by industry sectors [3, 4]. According to the World bacteria were suitable candidates for such Corrosion Organization [5], the annual cost of investigation [6]. Accelerated corrosion in which corrosion is greater than 3% of global GDP (Gross metal substrates were colonized by bacteria had been Domestic Product); however, governments and noted [7, 8]. However, in some cases, biofilms, industries pay little attention to corrosion, except in composed of a secreted polymeric substance high-risk areas, like aircrafts and pipelines. Kinetics of containing microbial populations, have shown to corrosion processes of metals, mineral and polymeric inhibit corrosion in metals [9, 10]. In most cases, the materials can be influenced by biofilms. Products of metal was submerged in an aqueous medium their metabolic activities including enzymes, inoculated by a bacterial species. The bacteria exopolymers, organic and inorganic acids, as well as colonised the metal substrate and produced a
Corresponding author: Tsveteslava Veselinova heterogenous extracellular polymeric substance that Ignatova-Ivanova, Ph.D., associate professor, research field:
adhered to metal surfaces facilitated by the functional microbiology. E-mail: radi_cvet@abv.bg.
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
groups of the exopolymer substance. Corrosion
2. Materials and Methods
inhibition was usually measured by loss-in-weight
2.1 Strain
calculation and generally attributed to the diffusion barrier provided by the bacterial biofilm.
Strain Lactobacillus delbrueckii B5 was obtained Work by Stadler et al. [11] concentrated on
from Collection of Department of General and corrosion by sulphate-reducing bacteria but suggested
Applied Microbiology, Sofia University. that dextrans could prevent corrosion on metals. Van
Leuven et al. [12] found an (1 → 3), (1 → 6)—linked D-glucan produced by Lactobacillus
2.2 Media Used
The strain cultivated in media of MRS (de Mann reuteri that inhibited corrosion while dispersed in a
Rogosa Sharpe, Biolife 272-20128, Milano, Italia) in electrolyte solution rather that as a coating [13]. In the
composition, per liter: Tween 80—1; pepton from authors’ previous studies [14-16], it showed how at
casein—10.0; meat extract—8.0; yeast extract—4.0; the presence of high concentration of lactose (5 to
K 2 HPO 4 —2.0; sodium acetat—5.0; amonium 15%), high concentration of sucrose 4%, and mixture
citrate—2.0; MgSO 4 .7H 2 O—0.2 and MnSO 4 —0.05 of sucrose 4% and 2% maltose, the strains g/L. The pH of media was adjusted to 6.5 with 1N Lactobacillus delbrueckii B5, L. delbrueckii K27, L.
NaOH. The basic media was sterilized by autoclaving delbrueckii B8, L. delbrueckii O43, L. delbrueckii K3,
at 121 ºC for 20 min, and carbohydrates supplemented L. delbrueckii K17 and L. delbrueckii K15 synthesized
were sterilized using 0.22 µm filters (Manisart ® ). The exopolysaccharides which can be used as corrosion
basic MRS broth was supplemented with 5% sucrose inhibitor. It is well known that some lactobacillus
and 5% lactose; 5% sucrose and 5% fructose and 5% strains as well genus Leuconostoc secreted sucrose and 5% maltose to be tested. transglucosidases after cultivation in the presence of
2.3 Study of the Corrosive Stability sucrose. Glucose polymers and their corresponding
oligosaccharides can be synthesized through enzymic The study of the corrosive stability of steel samples transglucosylation by a wide variety of glycansucrases
was conducted on the gravimetrique method [18]. capable of using sucrose as substrate. Among this
Before use, steel panels (10 × 4 × 0.2 mm) were treated group, dextransucrases (EC 2.4.1.5) are with 70% C 2 H 5 OH, washed with water and dried in an glucansucrases (GSs) able to produce dextran, a
oven, cooled in a desiccator, weighed on a balance and glucose polymer linked mainly through a1-6 bonds,
kept in a desiccator unit used. The weight of the although a1-3, a1-6, a1-4 and a1-2 bonds are also
samples was measured using analytical balances. The found, in both the main chain and the branching
dimensions of the samples measured with micrometer. linkages. Furthermore, glucose can also be transferred
Three types of experimental series were performed: to molecules that can act as nucleophile acceptors; in
(1) Cultivation of the studied strain in mMRS the case of sugars such as maltose, a wide variety of
media with 5% of sucrose and 5% fructose low-molecular-mass glucose oligosaccharides are
(2) In mMRS media with 5% sucrose and 5% produced [17].
maltose
In this paper, data on the effect of EPS on corrosion (3) In mMRS media with 5% sucrose and 5% of steel produced by Lactobacillus delbrueckii B5,
lactose
cultivated on media with sucrose as donor and maltose, Initially the steel samples were added in two fructose, and lactose as acceptor are presented and
variants: deproteinised supernatant and free cell discussed.
supernatant. Then the steel samples were added in 10%
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
HCl as control probe and as inhibitor of the corrosion
3. Results and Discussion
were added dilution (3:100) of the cultural media of Strain L. delbrueckii B5 was cultivated in a media the studied strain. The duration of the procedure was containing 5% sucrose and 5% fructose; 5% sucrose 120 h at 18 ºC. After the treatment the steel samples and 5% maltose and in a media containing 5% sucrose were washed with water and dried to constant weight. and 5% lactose for 12 h. After the cultivation the cells The structure of layer over steel plates was analyzed were removed and the obtained supernatants by Scanning Electron Microscopy (SEM) JSM 5510. (deproteinized supernatant and cell free supernatants)
2.4 Parameters of Corrosion were used for protection of the steel plates. The steel Parameters of corrosion after retrieval, the corrosion
plates were placed in 10% HCl as control probe and as products were removed using washed with water. They
inhibitor of the corrosion were added dilution (3:100) were dried in an oven. After the removal of corrosion,
of the cultural media of the studied strain. The steel plates were cleaned and reweighed as above to
received results are presented on Table1 and Table 2. estimate weight loss.
From the presented data in Table 1 the protective The rate of corrosion, the degree of protection, and
effect in all studied cases was proved. The coefficient coefficient of protection were calculated.
of the protection of corrosion varied between 9.25 and The corrosion rates 2 К (g/cm .h) presented as
2.64. From the obtained results is clear that the follows:
protection of corrosion was higher for strain L. К = ΔG/ S.τ, g/cm 2 .h;
delbrueckii B5 in the presence of 5% sucrose and 5% Where: К is the corrosion rate; ΔG—losses of mass
maltose were used during the fermentations process but consequence of corrosion, g; S—is the area of plates,
were in the presence of the cells. The protection of m 2 ; τ—is duration of the corrosion, h.
corrosion was higher for strain L. delbrueckii B5 in the For track out of inhibitor properties of EPS,
presence of 5% sucrose and 5% lactose were used synthesized in media has been calculated the degree of
during the fermentations process but were without the protection (Z) and coefficient of protection ( γ) using
cells.
formulas: From the presented data in Table 2 the protective
Z = (K 0 –K i )/K 0 × 100%;
effect in all studied cases was proved. When used as
inhibitor of the protection strain L. delbrueckii B5 Where: K 0 is the corrosion rate in control media;
γ=K 0 /K i ;
cultivated in the presence of 5% sucrose and 5% K i —the corrosion rate in test media.
lactose the protection of corrosion was highest.
Table 1 Characterization of the protective properties in cultural media.
Type of the tested inhibitor
K.10 -5 , g/cm 2 .h Z %
Cell free supernatants
2. Deproteinised 5% lactose supernatant 0.13 82.43 5.69 3. 5% sucrose
Cell free supernatants
4. Deproteinised 5% fructose supernatant 0.25 66.22 2.96 5. 5% sucrose
Cell free supernatants
6. Deproteinised 5% maltose supernatant 0.28 62.16 2.64 7. control
- *The steel plates were photographed after washing. Results are mean ± SEM of three separate trails.
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
Table 2 Characterization of the protective properties in HCl with added supernatant.
sample Media
The quantity of the supernatant in 10% HCl
K.10 -5 , g/cm .h Z %
5% sucrose 1. +
3.0 4.04 87.78 8.18 5% lactose 5% sucrose
2. + 3.0 9.23 72.07 3.58 5% fructose 5% sucrose
3. + 3.0 6.58 80.09 5.02 5% maltose 4. control
*The steel plates were photographed after washing. Results are mean ± SEM of three separate trails.
It could be underlined that 5% sucrose mixed with the morphology of microbial cells and colonies, their 5% maltose, or with 5% lactose in the media
distribution on the surface, the presence of EPS (Fig. stimulated the process of protection of corrosion.
1A and 1B) and the nature of corrosion products The structure of the layer over the steel plates was
(crystalline or amorphous; Fig. 1C). They can also analyzed by Scanning Electron Microscopy.
reveal the type of attack (e.g., pitting or uniform The results from this procedure are shown in Fig. 1.
corrosion) by visualizing changes in microstructure The pictures in Fig. 1A show that there’s a biofilm
and surface features after removal of the biofilm and formed on the steel surface which is an indicator of
corrosion products (Fig. 1C).
the good adhesive capacity of L. delbrueckii B5 type. The role of EPS in MIC of stainless steel remains The biofilm makes it not easily corrodible in 10%
obscure. It has been postulated that they are not HCl, supplemented with cultivated ambient from the
sufficient to induce biocorrosion of stainless steel same strain grown in a composite of 5% sucrose and
unless aided by the presence of a biocatalyst of 5% lactose (Fig. 1B). Fig. 1C shows a picture of a
oxygen reduction, which could be oxido-reductase steel surface sample treated directly with 10% HCL.
enzymes entrapped in the biofilm [20]. EPS has even The observed lamellae are most probably FeCl 2 been suggested to protect metal surfaces from
crystals, product of the corrosion.
corrosion.
A bacterial consortium consisting of a thermophilic the interaction of microorganisms with metals are
The forms of corrosion which can be promoted by
sp. and Deleya marina produced numerous, including general pitting, crevice attack,
Bacillus
metalbinding EPS that reduced the rate of corrosion of stress corrosion cracking, enhancement of carbon steel by 94% [21]. Such a mechanism may be corrosionfatigue, intergranular stress cracking and
responsible for the protection microorganisms afford hydrogen embrittlement and cracking. Most cases of
to mild steel under certain conditions [22].
microbially-influenced corrosion (MIC) are associated
4. Conclusions
with localized attack. The complexity of MIC reactions means that a broad range of techniques must
From received results it was evident that mixture of
be employed to relate the corrosion processes to the 5% sucrose mixed with 5% maltose, or with 5% microbial activities at surfaces [19].
lactose stimulated the formation of microbial biofilm Microscope techniques provide information about
inhibiting the corrosion of steel.
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
Fig. 1 Biofilm formed by L. delbrueckii B5 on the surface of mild steel, visualized using SEM. A) steel plates after corrosion in mixed 5% sucrose and 5% lactose; B) steel plates after corrosion in 10% HCl with inhibitor supernatant obtained of mixed 5% sucrose and 5% lactose; C) control—steel plates after corrosion in 10% HCl.
Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel
Acknowledgments
Produced by the Lactobacillus reuteri Strain 180 Glucansucrase GTS180 Enzyme.” Carbohydr Res. 343:
The contributors express their gratitude for the
1237-1250.
funding by the project by Shumen University project [13] Penninga, N., Kraly, S., Euverink, G. J., van der Maarel, M., van Geel-Shutten, I., and Dijkhuizen, L. 2002.
RD 08-213/10.03.2014. “GTF180: A Glycosiltransferase Producing a Glucan with Anti-corrosive properties.” In Proceedings of the
References
Nederlandse Vereniging voor Medische Microbiologie, [1] Gentil, V. 2007. Corrosão. 5th ed. Rio de Janeiro, S. A.:
Livros Técnicos e Científicos. [14] Ignatova-Ivanova, Ts., Ivanov, R., Iliev, I., and Ivanova, I. [2] Shi, X., Xie, N., and Gong, J. 2011. “Recent Progress in
2009. “Study Anticorrosion Effect of EPS from Now the Research on Microbially Influenced Corrosion: A
Strains Lactobacillus delbruecii.” Biotechnol. & Bird’s Eye View Through The engineering Lens.” Recent
Biotechnol. EQ Special edition/on line 23 (1): 705-708. Patents on Corrosion Science. 11: 118-131.
[15] Ignatova-Ivanova, Ts., Ivanov, R., Iliev, I., and Ivanova, I. [3] Demadis, K. D., Mantzaridis, C., and Lykoudis, P. 2006.
2011. “Study of Anticorrosion Effect of “Effects of Structural Differences on Metallic Corrosion
Exopolysaccharides Produced Lactobacillus delbrueckii Inhibition by Metalpolyphosphonate Thin Films.”
B5 Cultivated on Different Carbohydrates.” Biotechnol. & Industrial & Engineering Chemistry 45: 7795-7800.
Biotechnol. EQ Special edition/on line 26 (1): 224-227. [4] Hansson, C. M. 2010. “The Impact of Corrosion on
[16] Ignatova-Ivanova Ts., Ivanov, R. 2013. “Anticorrosion Society.” Metallurgical & Materials Transactions 42:
Effect of Biofilm Forming by Lactobacillus Strains on 2952-2962.
Metal Surfaces.” Bulgarian Journal of Agricultural [5] Geroge, F. H. 2012. Now is the Time. World Corrosion
Science 19 (2): 83-85.
Organization. [17] Clarita Olvera, Jose´ Luis Ferna´ndez-Va´ zquez, Luis [6] Finkenstadt, V., Cote, G., and Willett, J. 2008.
Ledezma-Candanoza and Agustı´n Lo´ pez-Munguı´a. “Agricultural Polymers for Corrosion Protection of
2007. “Role of the C-terminal Region of Dextransucrase Metals.” 236th National Meeting of the American
from Leuconostoc Mesenteroides IBT-PQ in Cell Chemical Society.
Anchoring.” Microbiology 153 (12): 3994-4002. [7] Beech, I. B., and Sunner, J. 2004. “Biocorrosion:
[18] Raychev, R., Fachikov, L., and Zaprjanova, V. 2002. Towards Understanding Interaction Between Biofilms
Corrosion and Protection of the Materials—Handbook and Metals.” Curr. Opin. Biotechnol. 15: 181-186.
for Laboratorial Exercises. Sofia.
[8] Beech, I. B., and Gaylarde, C. C. 1991. “Microbial [19] Iwona, B., Beech, Christine, C., and Gaylarde 1999. Polysaccharides and Corrosion.” Int. Biodeterior 27:
“Recent Advances in the Study of Biocorrosion. An 95-107.
Overview.” Revista de Microbiologia 30: 177-190. [9] Chongdar, S., Gunasekaran, G., and Kumar, P. 2005.
[20] Lai, M. E., Scotto, V., and Bergel, A. 1999. “Analytical “Corrosion Inhibition of Mild Steel by Aerobic Biofilm.”
Characterization of Natural Marine Biofilms.” Presented Elecrtrochim Acta 50: 4655-4665.
at 10th Internat. Congress on Marine Corrosion and [10] Fang, H. H. P., Xu, L. C., Chan, K. Y. 2002. “Effects of
Fouling, Melbourne, Australia.
Toxic Metals and Chemicals on Biofilm and [21] Edyvean, R. G. J., Benson, J., Thomas, C. J., Beech, I. B., Biocorrosion.” Water Res. 36: 4709-4716.
and Videla, H. 1998. “Biological Influences on Hydrogen [11] Stadler, R., Fuerbeth, W., Harneit, K., Grooters, M.,
Effects in Steel in Seawater.” Mat. Perform 37: 40-44. Woellbrink, M., and Sand, W. “Electrochim.” Acta 54:
[22] Soracco, R. J., Berger, L. R., Berger, J. A., Mayack, L. A., 91-99.
Pope, D. H., and Wilde, E. W. 1984. “Microbiologically [12] van Leeuwen, S., Kraly, S., Van Geel-Shutten, I., Gerwig,
Mediated Reduction in the Pitting of Mild Steel Overlaid G. J., Dijkhuizen, L., and Kamering, J. P. 2008.
with Plywood.” In Proceedings of NACE Corrosion 84, “Structural Analysis of the a-D-glucan (EPS180)
NACE, Houston, TX, 98.
Journal of Life Sciences 8 (2014) 805-810
doi: 10.17265/1934-7391/2014.10.002
DAVID PUBLISHING
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
Srwa Ali Muhammed Department of Microbiology, Faculty of Science and Health, Koya University, Daniel Mitterrand Boulevard, Koya KOY45 AB64 46017, Kurdistan Region, Iraq
Received: September 18, 2014 / Accepted: October 13, 2014 / Published: October 30, 2014.
Abstract: The study was conducted for the detection of Listeria monocytogenes bacteria from different sources (CSF and blood) obtained from patients in Pediatric hospital and from food sources like (yogurt, raw vegetables and raw milk, sausage). Ten isolates were isolated from 150 specimens one of them from CSF and one isolate isolated from blood samples the others isolated from food specimens 6 isolates isolated from sausage and 2 from raw vegetables. Isolates were identified traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties, identification by Api listeria was done. Determine the isolates that produce biofilm by tissue culture plate method. The highest biofilm forming strains of Listeria monocytogenes isolates appear in No. (D10, E1, E5 and E7) OD reading each of them is (0.13, 0.09, 0.11 and 0.19) respectively, the lowest or poor biofilm forming strains appear in No. (D11, D12, E2, E3, E4 and E6) that optical density (OD) reading are (0.04, 0.03, 0.05, 0.04, 0.05 and 0.03) respectively by comparing with control prepared in well (A12) that stained by crystal violate without putting any isolates and the OD reading is (0.003). Confirmation by PCR was done only for four isolates that produce biofilm (D10 and E1) that obtained from CSF and blood sample and for (E5 and E7) that obtained from food samples.
Key word: Listeria monocytogenes, biofilm, Api, PCR, OD.
1. Introduction people [2, 3].
L. monocytogenes successfully contaminates Listeria monocytogenes is the bacterium that causes processed foods because it persists on food-processing the infection listeriosis. It is a facultative anaerobic surfaces in the form of biofilms [4]. Unlike most other bacterium, capable of surviving in the presence or food-borne pathogens, L. monocytogenes grows absence of oxygen. It can grow and reproduce inside
during refrigeration.
the host’s cells and is one of the most virulent Thus, a small inoculums at the time of packaging food-borne pathogens, with 20 to 30 percent of can lead to a significant burden of organisms by the clinical infections resulting in death [1].
time it reaches the consumer [4].
Listeria monocytogenes is a gram-positive Biofilm-coated surfaces are particularly difficult to food-borne pathogen that causes life-threatening decontaminate, since bacteria in biofilms are more infections in fetuses, newborns, and resistant to detergents, biocides, and antibiotics than immunocompromised people. It also causes a severe are their planktonic counterparts [5, 6]. flu-like illness in pregnant women and self-limited Very little is known about the molecular gastrointestinal infections in immune-suppressed mechanisms of L. monocytogenes biofilm formation.
Flagellum-mediated motility is important for biofilm Corresponding author: Srwa Ali Muhammed, M.D., research fields: bacteriology, microbial genetic and the effect of
formation by several gram-negative bacteria [7]. In medicinal plants on bacterial virulence factors. E-mail:
contrast, flagella are implicated as surface adhesions srwaali@ymail.com.
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
early in L. monocytogenes surface attachment, but a Ultra-Pure DNase/RNase-Free distilled water: 1 μL of role for motility in biofilm formation has not been
Mgcl; 1 μL of dNTps; 4 μL of reaction buffer. Then examined [8].
added one single colony of the sample and put under
95 ˚C for 5 min for destroying the cell wall. After that
2. Materials and Methods
put 1 μL of DNA polymerase, the DNA amplification
2.1 Specimens’ Collection reactions were performed in thermal cycler. The
A total of 150 samples were collected from the cycling conditions for PCR illustrated in Table 2. The presence of genomic DNA in all prepared
different sources of human infections (CSF and Blood)
obtained from patients that bedded in Pediatric samples was confirmed by 0.8% agarose gel electrophoresis followed by staining with ethidium
hospital in Sulaimania city, and also isolated from food sources like (yogurt, raw vegetables andraw milk,
bromide [10]. Listeria monocytogenes isolates were sausages).
detected by PCR assay using primer sequence mentioned in Table 1.
2.2 Isolation and Identification
2.4 Screening of Biofilm Production Microscopic examination with Gram stain was done.
Biochemical test (Catalase test, Oxidase test, Isolates were sub cultured in tryptic soy broth (TSB) heamolysis, Sugar Fermentation test) in addition to
for 18 h at 37 ºC. Before the experiments, all the biochemical testes Api for listeria also was done. For
strains were vortex for 5 min and the optical density confirmation used colony PCR, PCR identification,
adjusted to 1-1.5 at 600 nm by spectrophotometer Gel Electrphorsis detection.
according to McFarland scale to the overnight culture was standardised to a concentration of 3 × 10 8 Cfu/mL
2.3 Confirmation of L. monocytogenes by Colony PCR
(PRO-LAB Diagnostics).
Preparation of colony PCR production for L. All isolates were studied for their capability to monocytogenes PCR identification, 2 primers were
produce biofilm in a modified microplate quantitative selected based on the invasive association protein
assay. These strains were previously isolated from gene (hlyA) [9] as shown in Table 1.
cerebrospinal fluid, blood and foods. Then each All PCR reactions were performed in a final
strains diluted by obtain 50 µL of sample to 950 µL volume of 24 μL using 2 μL of distilled water was
broth aliquots of 200 µL transferred to pre-sterilized, added to one PCR tube as negative control. Each
96-well polystyrene microtiter plates commercially reaction mixture contained 1 μL (10 µM) of forward
available (Deltalab S. L., Spain), then incubated for 24 primer; 1 μL (10 µM) of reverse primer and 15 μL of
h at 37 ºC. After incubation, discard the bacterial
Table 1 Primer sequence for detection of L. monocytogenes.
Primers Target gene
Reference hlyA-F
Length
Primer sequence
Amplication product (bp)
(Ritu Arora et al., 2007) hlyA-R
Table 2 Thermal cycling protocols for detection of L. monocytogenes.
Species Initial Denaturation Denaturation Annealing Extension Extension Final Hold
72 ˚C L. monocytogenes
Repeated for 25 cycles
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
suspension totally, each well was washed with 200 µL monocytogenes breaks down the esculin in the sterile phosphate buffer solution (PBS) to remove the
medium to glucose and esculetin which reacts with planktonic cells, then put on the hot plate for one hour
ferric citrate producing a brownish black precipitate at 60 ºC for fixation, 25 µL of 1% Crystal Violet was
around the colonies of Listeria monocytogenes [12]. added to each well, shaking the plates three times to
Biochemical tests: listeria species are positive for help the colorant to get the bottom of the well. After
catalase test, and give a typical umbrella growth
15 min at room temperature, each well was washed pattern in the motility test medium which is semisolid with 200 µL sterile PBS to remove the planktonic
medium near the microaerophllic subsurface of the cells and stain not adhered to the well. Only the
medium at (25 °C) [13]. Green colour colonies adhered bacteria forming the biofilm were kept on
surrounded by a black zone on PALCAM agar plates, the surface of the well. To determine the degree of
indicating esculinase positive, were detected for the biofilm formation, the absorbance was determined at
hemolytic activity, by 330 streaked on 7% blood agar wave length (450 to 630) nm in an ELISA microtiter
plates, and appearance of a clear zone around the (Botic England). Controls were performed with
growth area after incubation, was considered as a , Crystal Violet binding to the wells exposed only to the
positive reaction [14]. All Listeria species hydrolyzed culture medium without bacteria. The data obtained
esculin to esculetin [15]. Listeria species are were used to classify the strains as high producers
differentiated on the bases of sugar fermentation [16]. (OD between 0.19 and 0.09) or poor producers (OD
The result showed that L. monocytogenes fermented lower than 0.09) [11].
salcin, lactose, galactose and rhamnose, and did not
3. Results
ferment manitol, xylose, sucrose, and melibiose. L. monocytogenes produced narrow zone of β-hemolysis
3.1 Isolation and Identification of Listeria Species [17], in addition to biochemical test Api of Listeria
150 samples were obtained from hospitalized spp. also was done as shown in Fig. 1. 6010 according patients in pediatric hospital in sulaimanya city and
Api listeria system for the identification of listeria. also from different food sources. The samples obtained
Thus according of above study 10 isolates was and transferred to the laboratory and L. monocytogenes
obtained from 150 samples one isolate isolated from were identified by colony identification. Palcam
CSF and one from blood sample, six isolated from listeria selective agar is recommended for the isolation
sausage and two isolates isolated from raw vegetable. of Listeria monocytogenes from foods, while
3.2 PCR Identification of L. monocytogenes inhibiting Gram-negative and most of the
Gram-positive accompanying bacteria. The selectivity Four isolates of L. monocytogenes were identified of this medium results from its content of polymyxin,
by biochemical tests and produced biofilm subjected acriflavin, ceftazidime, and lithium chloride. L.
to PCR, and these four isolates were successfully
Fig. 1 Result of Api listeria (6010) test used for identification of of Listeria spp.
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
amplified the desired amplicon of 456 bp for listeria microtiter (Botic England) as show in Fig. 3. Fig. 3 monocytogenes (E1 and E4) that isolated from CSF
show the capability of L. monocytogenes isolates and blood sample and for 8 and E10 that isolated from
from clinical sources and food appear in No. (D10, food samples as shown in Fig. 2. The PCR was
E1, E5 and E7) OD of each of them (0.13, 0.09, 0.11 performed using hlyA primer pair of gene. Listeria
and 0.19) respectively as shown in Table 3, the monocytogenes has several important virulence lowest or poor biofilm forming strains appear in markers. Among them, Listeriolysin O (LLO) is one
No. (D11, D12, E2, E3, E4 and E6) that optical of the important markers encoded by hlyA gene and is
density (OD) reading are (0.04, 0.03, 0.05, 0.04, 0.05 essential for disruption of phagocytic vacuole and
and 0.03) respectively by comparing with control release of bacteria into cytoplasm [9].
No.1 prepared in well (A12) that stained by crystal violate without putting any isolates and the OD
3.3 Screenning of (L. monocytogenes) Isolates That
reading is (0.003).
Produce Biofilm Using Trypticase Soy Broth (TSB Difco) with 1% Glucose
4. Discussion
Preparing the samples for detecting biofilm Ten isolates L. monocytogenes were identified by according [18] the absorbance was determined at
biochemical tests and four of them that produced wave length (450 to 630) nm in an ELISA biofilm subjected to PCR, and all these isolates were
Fig. 2 Agarose gelelectrophoresis of the PCR amplication products of L. monocytogenes colonies amplified hlyA 456 bp sequence. Lane 1: 1Kb DNA ladder; Lane 2: negative control; Lane (3-4-6-7): samples positive for L. monocytogenes with hlyA 456 bp.
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
Fig. 3 Biofilm formation of L. monocytogenes were isolated from hospitalized patients and different food sources by using TSB.
Table 3 Determination of biofilm with a micro ELISA
5. Conclusions
using Microtitre plate and reader using TSB medium supplemented with 1% glucose for (L. monocytogenes).
The results show that the Listeria monocytogenes Isolates
isolated from CSF and blood sample in pediatric L. monocytogenes
Well No.
OD value
hospital and also isolated from food samples specially 1 D10
isolated from sausage samples. The results show that 2 D11
3 D12
0.03 Listeria monocytogenes produce biofilm from 10
4 E1 0.09 isolates four of the isolates produce high amount of
5 E2 0.05 biofilm. 6 E3 0.04
7 E4 0.05 References
8 E5 0.11 [1] Ramaswamy, V., Cresence, V. M., Rejitha, J. S., Lekshmi, 9 E6 0.03 M. U., Dharsana, K. S., Prasad, S. P., and Vijila, H. M. 10 E7 0.19 2007. “Listeria—Review of Epidemiology and
Control A12
Pathogenesis.” J. Microbiol. Immunol. Infect 40 (1): 4-13. PMID 17332901.
successfully amplified the desired amplicon of 456 bp [2] Farber, J. M., and Peterkin, P. I. 1991. “Listeria as shown in Fig. 2. The PCR was performed using
monocytogenes, a Food-borne Pathogen.” Microbiol. Rev. primer pair of hlyA gene, there are many studies used 55: 476-511. [3] Lorber, B. 2005. “Listeria monocytogenes.” In Mandell, hlyA gene as primer [19], used hlyA gene as
Douglas, and Bennett’s Principles and Practice of PCR—target for the species specific detection of L.
Infectious Diseases, edited by Mandell, G. L., Bennett, J. monocytogenes from fish samples.
E., and Dolin, R. Philadelphia, PA: Elsevier Churchill Livingstone.
The present study determined the ability of Listeria [4] Moretro, T., and Langsrud, S. 2004. “Listeria
monocytogenes for producing biofilms. There are monocytogenes: Biofilm Formation and Persistence in many studies that determined the ability of listeria to
Food-processing Environments.” Biofilms 1: 107-121. produce biofilm [20] was treated, the development of
[5] Hogan, D., and Kolter, R. 2002. “Why are Bacteria Refractory to Antimicrobials?” Curr. Opin. Microbiol. 5:
microbial biofilms of three pathogens (Listeria
472-477.
monocytogenes, Pseudomonas aeruginosa and [6] Fux, C. A., Costerton, J. W., Stewart, P. S., and Stoodley, Candida albicans).
P. 2005. “Survival Strategies of Infectious Biofilms.”
810
Biofilm Determination of Listeria monocytogenes That Isolated from Different Sources
Trends Microbiol. 13: 34-40. [14] Lazar, V. 2003. Microbial Adherence. Bucharest: [7] O’Toole, G., Kaplan, H. B., and Kolter, R. 2000. “Biofilm
Romanian Academy Publishing House. Formation as Microbial Development.” Annu. Rev.
[15] Feresu, S., and Jones, D. 1988. “Taxonomic Studies on Microbiol. 54: 49-79.
Brochottrix, Erysipelothrix, Listeria and Atypical [8] Vatanyoopaisarn, S., Nazli, A., Dodd, C. E., Rees, C. E.,
Lactobacilli.” J. Gen. Microbiol. 134: 1165-1183. and Waites, W. M. 2000. “Effect of Flagella on Initial
[16] Pagotto, F., Daley, E., Fraber, J., and Warburton, D. 2001. Attachment of Listeria monocytogenes to Stainless Steel.”
“Isolation of Listeria monocytogenes from All Food and Appl. Environ. Microbiol. 66: 860-863.
Environmental Samples.” In Health Products and Food [9] Ritu, A., Alka, P., and Sant P. 2006. “A Comparative
Branch Ottawa, Canada, MFHPB-30 method. Study of Conventional Culture and PCR Method the
[17] Robinson, R. K., Batt, C. A., and Patel, P. D. 2000. Detection of Listeria monocytogenes from Artificially
Encyclopedia of Food Microbiology. San Diego, CA: Inoculated Milk.” Indian Journal of Dairy Science 60 (5).
Academic Press.
[10] Dmitriy, V., Joseph, G., Christine, A., Robert, E. D., and [18] Mendez, C., Garza, E., Gulati, P., Morris, P. A., and Anthony, D. H. 2006: “Discovery of Natural Atypical
Allen, C. A. 2005. “Isolationand Identification of Nonhemolytic Listeria seeligeri Isolates.” Applied and
Micro-organisms in JSC Mars-1 Simulant Soil.” Lunar Environ. Microbiol. 72: 2439-2448. and Planetary Science 36: 1-2.
[11] Danhorn, R. M., Hentzer, M., Givskov, M. R., Parsek, [19] Swetha, C. S., Madhava Rao, T., Krishnaiah, N., and and Fuqua, C. 2004. “Phosphorus Limitation enhances
Kumar, A. V. 2012. “Detection of Listeria Biofilm Formation of the Plant Pathogen Agrobacterium
monocytogenes in Fish Samples by PCR Assay.” Annals Tumefaciens Through the PhoR-PhoB Regulatory
of Biological Research 3 (4): 1880-1884. ISSN System.” J. Bacteriol. 1186: 4492.
0976-1233 CODEN (USA): ABRNBW. [12] Walter, F. 2000. “Food Borne Listeriosis.” Clinic Infect
scholarsresearchlibrary.com.
Disease 31: 770-775. [20] Maxleene, S., 2008. “The Effect of Plant Extracts on [13] Brugere-Picoux, O. 2008. “Ovine Listeriosis.” Small
Microbial Biofilm Formation and Development.” Master Ruminant Research 76: 12-20.
Thesis, Tshwane University of Technology.
Journal of Life Sciences 8 (2014) 811-814 doi: 10.17265/1934-7391/2014.10.003
Seroprevalence of Human Brucellosis in Kuku Dairy Scheme, Khartoum State, Sudan
Tamador-Elkhansaa Elnour Angara 1 , Adil Abdel Rahman Ali Ismail 2 and Nageeb Suliman Saeed 3
1. Department of Development Studies & Extension, Sudan University of Science and Technology (SUST), Khartoum 11111, Sudan 2. Department of Preventive Veterinary Medicine, University of Bahri, Khartoum 11111, Sudan 3. Department of Microbiology, Faculty of Medicine, University of Khartoum, Khartoum 11111, Sudan
Received: May 20, 2014 / Accepted: October 28, 2014 / Published: October 30, 2014.
Abstract: A seroprevalence investigation of human brucellosis was carried out in Kuku Dairy Scheme, Sudan. A total of 176 serum samples were collected and screened by Rose Bengal Plate Test (RBPT). The positive sera were further examined using Tube Agglutination Test (TAT) and c-Elisa. The seropositivity was 15.9%, 14.8% and 11.4% using RBPT, TAT and c-Elisa respectively. Whereas, the active infection based on seropositivity and clinical signs were 4.6%, 4.6% and 2.3% in case of RBPT, TAT and c-Elisa respectively. Based on c-Elisa result the infected individuals were further subjected to clinical examination and treated with streptomycin and doxocycline for six weeks.
Key words: Malta fever, in contact persons, agricultural workers, Kuku Scheme.
1. Introduction
Brucellosis is one of the most common zoonosis in the world [1]. The disease is caused by organisms belonging to the genus Brucella, gram-negative, non-spore-forming, facultative, intracellular bacteria [2]. The centers for Disease Control and Prevention (CDC) classify B. abortus, B. melitensis and B. suis as “agents of mass destruction” and as category B organisms [3]. The occurrence of the disease in human is largely dependent on animal reservoir [4]. The disease is transmitted to humans by ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols [5]. Cattle are the most important livestock source of human brucellosis [6, 7]. Although the definite diagnosis of brucellosis is made by isolation of the organism from blood samples or other clinical specimens [8]. Human brucellosis is diagnosed on the basis of clinical findings and laboratory studies [9].
Corresponding author: Tamador-Elkhansaa Elnour Angara, Ph.D., associate professor, research field: public health economics. Email: angaratamador@gmail.com.
Serodiagnosis is most commonly made on the basis of the Tube Agglutination Test (TAT). Other assays, including the (RBT) and the anti-brucella Coombs test. The ELISA is more sensitive than the TAT in diagnosis of brucellosis [10]. Studies on the infection in Saudi Arabia and Oman revealed that 20% of the population had serological evidence of exposure in southern Saudi Arabia [11] while 1% of healthy residents of Dhofar, Oman (mainly children) had serologic evidence of exposure [12]. The disease in agricultural workers was investigated in Bari, Southern Italy by standard tube agglutination test. None of the subjects examined had antibodies to Brucella [13]. In Castellon, Spain, the seroprevalence of brucellosis in agricultural workers was 3.1% based on Wright and Coombs tests; all sera were negative to Rose Bengal [14].
A 12.7% prevalence rate was reported in nomads community in Darfur states [15] using serological and bacteriological tests. In Chad brucellosis was investigated in three nomadic communities of Chad (Fulani cattle breeders, and Arab camel and cattle
DAVID PUBLISHING
Seroprevalence of Human Brucellosis in Kuku Dairy Scheme, Khartoum State, Sudan
breeders) by indirect ELISA, the seropositivity were Bengal Plate Test (RBT), was used as screening test 3.8% [16]. The combination therapies recommended
as described by Alton et al. [20]. The positive sera by WHO for treatment of brucellosis are doxycycline
were subject to Tube Agglutination Tests (TAT) as plus rifampicin or doxycycline plus streptomycin [17].
described by Meyer [21] and Competitive Sudan was proved to be endemic with bovine
Enzyme-Linked Immunosorbent Assay (cELISA) brucellosis; Kuku Scheme was not an exception.
tests using SVANOVIR®Brucella-Ab C-ELISA test There was no formal control strategy adopted to
kits. Actively infected persons were interviewed in control the disease in cattle. The probability of the
depth and subjected to further clinical examination transmission of the disease to at risk population is
and treated with streptomycin and doxocycline for very high; however, no data regarding human
six weeks and followed up for 6 months for any brucellosis in the scheme is available. This work
relapse.
aimed at providing data on human brucellosis among
3. Results
in contact individuals and the risk factors associated with the disease to highlight the seriousness of the
A total of 176 sera were tested by using RBPT as problem and to draw the attention of the policy
screening test, the result is shown in Table 1 where a makers towards controlling the disease.
total of 28 (15.9%) serum samples reacted positive to the test. To confirm this result the positive sera were
2. Materials and Methods
subject to TAT and c-Elisa. The seropositive samples
2.1 The Study Area were 26 (14.8%) and 20 (11.4%) based on TAT and Kuku Scheme covers an area of about 2600 acre
c-Elisa respectively.
stretching out from the old riverain cultivation area on The positive sera of symptomatic individuals were the Blue Nile banks, east of Khartoum North [18]. The
8 (4.6%), 8 (4.6%) and 4 (2.3%) in case of RBPT, project was established in 1963 by American aid to
TAT and c-Elisa respectively.
settle the semi nomadic tribes and to increase milk The study revealed that one individual who reacted supply to the capital town by up grading the local
positively to c-Elisa had symptoms of fever, headache, cattle breeds. Currently the scheme is a part of East
arthralgia, whereas another one suffered from fever, Nile locality one of the seven localities of Khartoum
headache, arthralgia and night sweat and two individuals State [19].
showed symptoms of fever, headache, arthralgia, night sweat and, fatigue as shown in Table 2.
2.2 Sample Collection and Testing:
4. Discussion
A total of 176 out of 649 at risk were examined for brucellosis. Blood samples were collected using 5 cc
The result based on confirmatory c-Elisa and
disposable syringes. Case recording forms were used clinical signs indicated that the scheme is endemic to collect data on the history and clinical symptoms.
with brucellosis and the infection rate was 2.3%. This The blood samples were transferred to The National
infection occurred as a result of direct contacts with Health Laboratory for serological examination. Rose
herds in the scheme. The infection rate among cattle
Table 1 Seroprevalence of human brucellosis Kuku Dairy Scheme.
Total people investigated
Test used
Positive reactors
Symptomatic positive reactors
RBT
TAT
cELISA
Seroprevalence of Human Brucellosis in Kuku Dairy Scheme, Khartoum State, Sudan
Table 2 Symptoms of individuals found serologically positive to c-Elisa.
Description (Symptoms)
Percent (%) Fever, headache and arthralgia
No infected
1.0 0.6 Fever, headache, arthralgia and night sweating
1.0 0.6 Fever, headache, arthralgia, night sweat, fatigue
2.0 1.1 Total symptomatic people
population in the scheme was 24.9% based on c-Elisa
5. Conclusions
[22]. The study proved that the scheme is endemic with The result obtained in this study is slightly less than brucellosis and that many cases passed undiagnosed. the result of 12.7% reported in nomadic tribes in The recommendations were raising the awareness of Southern Darfur-Sudan by Musa [15]. On the other in contact individuals, test the in contact population hand the current result disagrees with the RBPT result routinely, and formulation of a control strategy in obtained from Agricultural workers living in the
animal population.
coastal areas of Castellon, Spain [14] where all the participants reacted negative to RBPT in contrast to
Acknowledgments
the current result where 15.9% of the participant Prof. Musa Mohammed Khair from Faisal reacted positive to the test. On the other hand the Professional Private Clinic was the Physician who seroprevalence of 3.8% obtained from three nomadic examined the patients; Miss Zenab Fadlelmola who communities of Chad by Schelling et al. [16] was assisted in blood samples collection. much lower than that obtained in this study.
Comparing the four-seroprevalence results of the
Ethical Considerations
agricultural workers: the current work, Darfur This study carried out in The National Health (nomads), Chad (nomads), Castellon, Spain and in Laboratory. The infected people were further Bari, Southern Italy. The prevalence rates obtained clinically examined and treated in Faisal Professional from Sudan are much alike in spite of the different
Private Clinic.
production systems: sedentary in Kuku and nomadic in Darfur. The authors expect that the prevalence rates
References
of the two nomadic communities in Darfur and Chad [1] Caporale, V., Giovannini, A., Calistri, P., and Tittarelli, to be much alike than those of Kuku and Darfur. The
M. 2009. “An Approach to Developing Coordinated and two former communities are more similar alike with
Harmonised Actions for the Control of Brucellosis.” regard to geographical condition, pattern of life, type Presented at the 10th conference of the OIE Regional Commission for the Middle East Doha, Qatar, 26-29.
of cattle breed and animal husbandry practices, [2] Ko, J., and Splitter, G. A. 2003. “Molecular whereas the latter differ much in these aspects. The
Host-pathogen Interaction in Brucellosis: Current results of Spain and Italy differ largely from the
Understanding and Future Approaches to Vaccine results of Sudan and Chad this may be attributed to Development for Mice and Humans.” Clinical Microbiology Reviews 16 (1): 65-78.
different factors including human behavior, and public [3] Elzer, P. H. 2002. “Brucellosis Vaccines for the 21st health measures and control of the disease in animal.
Century” In Proceedings of NIAA Annual Meeting, Unlike the case of Southern Saudi Arabia [11], the
Louisiana State University Ag Center and School of seroprevalence result of brucellosis in the general Veterinary Medicine, Baton Rouge, LA. [4] World Health Organization (WHO) 1997. “Brucellosis.”
population in Dofar, Oman [12] is less than those in Fact sheet N173. Geneva, Switzerland. Accessed April 2, the agricultural workers.
2014. http://www.thailabonline.com/sec8brucellosis.htm.
Seroprevalence of Human Brucellosis in Kuku Dairy Scheme, Khartoum State, Sudan
[5] Gerald, E. M. 2001. “Brucella Infection.”
205-209.
eMedicine-Brucellosis: Article Medicine, Department of [14] Villamarin-Vazquez, J. L., hiva-Nebot, and Arnedo-Pena, Veterinary Science, Jr, DO 111 Dalrymple Building,
F. C. 2002. “Seroprevalence of Brucellosis in Baton Rouge, LA. Accessed April 8, 2014.
Agricultural Workers Living in the Coastal Areas of ftp://ftp.cgiar.org/ilri/ICT/Theme%203/assam/consumer
Castelon, Spain.” Salu Publica de Mexico 44 (2): 1-3. %20survey/eMedicine%20-%20CBRNE%20- %20Brucel
[15] Musa, M. T. 1995. “The Magnitude and the Problem of losis%20%20Article%20by%20Gerald%20E%20Malone
Brucellosis in Darfur States and the Methods of y,%20Jr,%20DO.htm.
Diagnosis and Control.” Ph.D. Thesis, Khartoum, Sudan. [6] Collard, P. 1962. “Antibodies Against Brucellae in the
[16] Schelling, E., Diguimbaye, C., Daoud, S., Nicolet, J., Sera of Healthy Persons in Various Parts of Nigeria.” The
Boerlin, P., Tanner, M., and Zinsstag, J. 2003. WAMJ 89: 172-177.
“Brucellosis and Q-fever Seroprevalence of Nomadic [7] Alausa, K. O., and Awoseyi, A. 1976 “Brucellosis: the
Pastoralists and Their Livestock in Chad.” J. Preventive Situation in Western Nigeria.” Trop. geogr. Med. 28
Veterinary Medicine 61 (December): 279-293. (March): 54-59.
[17] Karabay, O., Sencan, I., Kayas, D., and Sahim, I. 2004. [8] Espinosa, B. J., Chacaltana, J., Mulder, M., Franco, M.,
“Oflaxacin Plus Rifampicin Versus Doxycycline Plus P., Blazes, D. L., Gilman, R. H., Smits, H. L., and Hall, E.
Rifampicin in the Treatment of Brucellosis; A R. 2009. “Comparison of Culture Techniques at Different
Randomized Clinical Trial.” Bio. Med. Central BMC Stages of Brucellosis.” Am. J. Trop. Med. Hyg. 80 (April):
Infectious Diseases 4:18. doi:10.1186/1471-2334-4-18. 625-627.