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Research ArticleIsolation and Molecular Characterization ofBrucella Isolates in Cattle Milk in Uganda
Denis Rwabiita Mugizi1 Shaman Muradrasoli2 Sofia Boqvist2
Joseph Erume1 George William Nasinyama1 Charles Waiswa1
Gerald Mboowa3 Markus Klint4 and Ulf Magnusson5
1College of Veterinary Medicine Animal Resources and Bio-Security Makerere University PO Box 7062 Kampala Uganda2Division of Bacteriology and Food Safety Department of Biomedical Sciences and Veterinary Public HealthFaculty of Veterinary Medicine and Animal Science Swedish University of Agricultural Sciences PO Box 702875007 Uppsala Sweden3College of Health Sciences Makerere University PO Box 7062 Kampala Uganda4Section of Clinical Bacteriology Department of Medical Sciences Uppsala University 75185 Uppsala Sweden5Division of Reproduction Department of Clinical Sciences Faculty of Veterinary Medicine and Animal ScienceSwedish University of Agricultural Sciences PO Box 7054 75007 Uppsala Sweden
Correspondence should be addressed to Denis Rwabiita Mugizi dmugizicovabmakacug
Received 1 July 2014 Revised 22 October 2014 Accepted 23 October 2014
Academic Editor Jacques Cabaret
Copyright copy 2015 Denis Rwabiita Mugizi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Brucellosis is endemic in livestock and humans inUganda and its transmission involves amultitude of risk factors like consumptionof milk from infected cattle To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic andmolecular approaches to delineate the Brucella species biovars and genotypes shed in cattle milk Brucella abortuswithout a biovardesignation was isolated from eleven out of 207 milk samples from cattle in UgandaThese isolates had a genomic monomorphismat 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with otherAfrican strains or other B abortus biovars from other parts of the world This study further highlights the usefulness of MLVA asan epidemiological tool for investigation of Brucella infections
1 Introduction
Thegenus Brucella has ten recognized species withmore than90 DNA homology [1 2] These species cause brucellosisthat is of economic and public health importance in terres-trial and aquatic animals and humans [1 3] Species of Bmelitensis B abortus some B suis biovars B canis B cetiand B inopinata are zoonotic and in humans the infectioncauses a debilitating disease with relapsing fever and flu-likesymptoms with multiple organ involvement [4ndash6] In cattleBrucella causes abortions placentitis orchitis mastitis andprenatal death [5 7]
Brucellosis in cattle is almost exclusively caused by Babortus [8] but B melitensis and B suis have been implicated
in some herds [9 10] making the vaccination of cattle usingvaccines targeting only B abortus less effective in preventingbrucellosis in cattle and transmission to humans [11 12]Brucella biovars and genotypes are known to be regionallyrestricted in their distribution [13] but the evolution ofinternational travel and trade and changing ecosystems haveled to introduction of new biovars and genotypes into regionsand hosts where they were not previously found [3] Astudy done in Uganda in 1958 isolated B abortus biovar 3from a human patient [14] and in the neighboring KenyaB melitensis biovar 1 and B abortus biovar 3 have beenisolated from cattle [9] Phylogenetic analysis of B melitensisisolates in Kenya showed a high degree of homology withisolates in Israel and the B abortus isolates closely resembled
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 720413 9 pageshttpdxdoiorg1011552015720413
2 BioMed Research International
that isolated in Uganda In a related study in Egypt Babortus B suis and B melitensis were isolated from cattleand all had a high level of phylogenetic variability withineach species although the isolates used in these studies werefew [15]The above findings indicate a complex epidemiologyof brucellosis in cattle in the region and call for refineddiagnostic methods beyond phenotypic typing
High resolution phenotypic and molecular approacheshave been developed for Brucella speciation biotypingand epidemiological trace-back [16 17] To date advancedmolecular technologies have not been widely used in lowincome countries where brucellosis is endemic in livestockand humans [7 18] Thus information on the prevailingBrucella species biovars and genotypesstrains in such areasof endemicity may shed new light on the epidemiology ofBrucella infection and the species and biovars circulatingBesides this generic scientific rationale for undertaking suchinvestigations increased understanding of the Brucella epi-demiology is critical for refining control of brucellosis inresource weak countries where the same measures as in highincome countries cannot be applied
In northern and eastern Uganda where this study wasperformed there was a considerable mixing of livestockspecies during years of insurgency in the 1990s presentingideal conditions for inter- and intraherd transmission ofdiseases such as brucellosis Indeed high herd and individualanimal seroprevalences of up to 27 and 75 respectivelywere recorded in a recent survey in the region [19] Thishigh prevalence may pose a severe threat to public health asprevious studies around the capital of Uganda suggest milkor milk products from cows as a major source of Brucellainfection in humans [20 21] The present study aimed atisolating and molecular-typing Brucella from cattle milk innorthern and eastern Uganda for better understanding of itsepidemiology
2 Materials and Methods
21 Study Design and Collection of Samples Milk sampleswere collected from 207 lactating cows in urban and peri-urban areas of Gulu and Soroti towns of northern andeastern Uganda from May 2011 to March 2012 for isolationof Brucella A total of 110 individual cow milk samples werecollected from 72 herds in Gulu and a total of 97 individualcow milk samples were collected from 33 herds in SorotiThese herds were part of the 166 herds whose animals hadbeen screened for brucellosis and were within a radius of15 Km in both Gulu and Soroti towns with the two townsbeing 200Km apart The cattle from which milk sampleswere taken had been screened for Brucella antibodies and intotal 17 of the 207 cows were seropositive In both townsthe seropositive herds from which the milk samples weretaken were near each other The numbers and selection ofhouseholds and animals included are described in detailpreviously [19] Midstreammilk samples were collected fromall quarters with 10ndash20mL collected from each teat intosterile 100mL falcon tubes The samples were transportedchilled to Makerere University College of Health Sciences
microbiology laboratory Kampala Uganda kept at 4∘C andcultured within three days
22 Sample Preparation Brucella Culturing and BiotypingThe milk was centrifuged at 3000timesg at 4∘C for 15 minutesand the pellet and supernatant were plated on both Farreland Centro de Investigacion y Tecnologıa Agroalimentaria(CITA) selective media supplemented with calf serum [22]Briefly Farrelrsquos medium was prepared from Brucellamediumbase (Oxoid UK) sterilized at 121∘C for 15 minutes andsupplemented with Brucella selective supplement (OxoidUK) according to the manufacturerrsquos instructions The CITAmedium was prepared according to De Miguel et al [22]Inoculated plates were incubated at 37∘C for 8 days in a5ndash10 carbon-dioxide incubator and read every 24 hoursfrom day three of incubation for colony growth Resultantcolonies were subcultured and biotyped based on theircolony morphology serum and carbon-dioxide requirementfor growth hydrogen sulphide production urease activityoxidase test and growth in presence of dye basic fuchsinand agglutination of anti-Brucella IgG monospecific seraA (Animal Health and Veterinary Laboratories AgenciesWeybridge UK) according to theOIETerrestrialManual [8]Representative colonies are stored at minus80∘C in 20 glycerolfor long-term storage
23 Genomic DNA Extraction and Real-Time PCR DetectionThe colonies that conformed to all the above phenotypiccharacteristics of Brucella were subjected to genomic DNAextraction using theNorgen bacterial genomicDNA isolationkit (Norgen Biotek Corp Ontario Canada) The extractedBrucella genomic DNAwas used to run a real-time multiplexPCR assay with oligonucleotide primers probes reactionmixture and PCR conditions according to Probert et al[23] Amplification and real-time fluorescence detectionweredone on the Rotor-Gene 3000 real-time PCR machine (Cor-bett Research-Corbett Life Sciences Mort Lake Australia)Three positive and two negative controls were included ineach runWhen the cycle threshold (CT) value of the sampleswas le40 samples were evaluated as positive This real-timemultiplex PCR was designed to detect Brucella at both thegenus and species levels for B abortus and B melitensis sinceit has the genus specific probe and the species specific probesfor the two Brucella species commonly infecting cattle
24 Brucella Species Confirmation Positive samples on real-time PCR were analysed further for Brucella speciation usingthe Bruce-ladder multiplex PCR assay kit (Ingenasa Spain)The oligonucleotide primers reaction mixture and PCRconditions were performed according to the manufacturersquosconditions in conformity with Lopez-Goni et al [16] How-ever amplification was done in a MyCycler thermal cycler(BioRad)
25 Characterization of Isolates by MLVA Genotyping Geno-typing was performed using the Multiple Locus VariableNumber Tandem Repeat Analysis (MLVA) using the 16-primer-pair assay [17 24ndash26] The oligonucleotide primer
BioMed Research International 3
Cycle5 10 15 20 25 30 35 40 45
Nor
m fl
uoro
035
030
025
020
015
010
005
000
Threshold
Figure 1 Triplex real-time PCR amplification pattern using theBrucella genus probe Fluorescence ratio is plotted against thenumber of PCR cycles to monitor amplification in real-time modeIsolates with weak Ct values (2918 and above) had Brucella-likephenotypic characteristics and were included in this assay
pairs incorporated in the Brucella MLVA 16 assay targetboth the conserved and highly discriminatory regions of theBrucella genome Each sample was run on three prescribedMLVA panels Panel 1 consisted of moderately polymorphicminisatellite primer pairs targeting the highly conservedgenomic regions of different Brucella species (bruce 06 bruce08 bruce 11 bruce 12 bruce 42 bruce 43 bruce 45 andbruce 55) Panel 2A (bruce 18 bruce 19 and bruce 21) andpanel 2B (bruce 04 bruce 07 bruce 09 bruce 16 and bruce30) consisted of microsatellite primer pairs targeting thediscriminatory genomic regions The PCR amplification andgenotyping were done according to le Fleche et al [17] withonly a modification in the total reaction volume to 30 120583LAt each run B suis reference strain REF 1330 (from Bruce-ladder kit) was included as shown in Figure 1
26 Gel-Electrophoresis Analysis of Panel 1 and Panel 2 LociAmplification Products Five microliters of the panel 1 andpanel 2 loci amplification products were loaded into 3 and2 agarose gel containing ethidium bromide (05 120583gmL)respectively to visualize the banding pattern in the samplesand positive controls underUV illuminationThe agarose gelwas run on 8Vcm current and a 100 base pair and 20 basepair ladders (BioRad) were included per run for panel 1 andpanel 2 respectively
27 Sequencing the VNTR Locus Amplicons In order toidentify repeat copy number variation among the isolatesin question the resulting PCR products were sequenced foreach VNTR locus at Macrogen Netherlands Sequenceswere viewed using BioEDIT version 7090 Sequencingwas performed in both directions using the M13-primersaccording to Applied Biosystems (ABI) Since each of theVNTR locus primers was tagged with M13 primer ([M13-Forward] 51015840-GTAAAACGACGGCCAGT-31015840 and [M13-Rev]51015840-GCGGATAACAATTTCACACAGG-31015840) this increaseseach VNTR locus PCR product size by 39 base pairs
Nor
m fl
uoro
045
040
035
030
025
020
015
010
005
000
Threshold
Cycle5 10 15 20 25 30 35 40 45
minus005
Figure 2 Triplex real-time PCR amplification pattern using the Bmelitensis probe Fluorescence ratio is plotted against the numberof PCR cycles to monitor amplification in real-time mode Only Bmelitensis (positive control) was picked by this probe
28 Analysis of MLVA Sequence Data The MLVA PCRproductswere sequenced per loci and the forward and reversesequences were assembled into a contig in the Bionumericssoftware version 50 (Applied Maths Belgium) The M13primer tags were trimmed from the contig and the alleledesignation was determined by comparing the fragmentsize with the published allele numbering system (version36 httpmlvau-psudfr Brucella support website for MLVAtyping) The number of tandem repeats per loci was queriedin the Brucella MLVA 2012 public database (httpmlvau-psudfrmlvav4genotyping) accessed on February 21 2014for genotyping of our isolates The closest related knownstrains were determined based on the genetic distance (theminimumnumber of changes in the number of repeats of anylocus that converts one genotype to another)
3 Results
31 Biotyping Based on the biotyping (Table 1) B abortusbiovar 1 3 or 7 was isolated in 11 (53) out of 207 milksamples These 11 positive samples were all from seropositivecows (ie 11 of 17) The colonies being smooth eliminated Bcanis and B ovis which have rough colonies Production ofhydrogen sulfide eliminated B melitensis B ceti B microtiand B abortus biovars 5 and 6 and B suis except B suisbiovar 1 Ability to grow in absence of serum eliminated Babortus biovar 2 that generally requires serum for growthBrucella abortus biovar 2 B neotomae and B suis biovar 1were eliminated by their inability to grow in basic fuchsinAgglutination with anti-Brucella monospecific sera A elimi-nated B abortus biovars 4 and 9
32 Molecular Characterization
321 Brucella DNA Detection by Real-Time PCR DNA fromall the 11 isolates that was judged asB abortus by the biotypingwas detected as Brucella DNA by the Brucella genus probe inthe triplex real-time PCR (Figure 1) However the triplex real-time PCR was unable to detect the Brucella species involvedusing its B melitensis probe (Figure 2) and B abortus probe(Figure 3)
4 BioMed Research International
Table1Ph
enotypiccharacteris
ticso
fBrucella
sppiso
latedfro
mcattlem
ilkfro
mno
rthern
andeaste
rnUgand
ain2011-2012
SampleID
Serological
result
Colon
ymorph
ology
Serum
requ
irement
CO2
requ
irement
Oxidase
test
Ureasea
ctivity
Agglutin
ationwith
mon
ospecific
sera
AH
2Sprod
uctio
nGrowth
inbasic
fuchsin
Biovar
S40601
+S
minusminus
++
++
+1o
r3S40307
+S
minusminus
++
++
+1o
r3S0303
+S
minusminus
++
++
+1o
r3S3702
+S
minusminus
++
++
+1o
r3S40602
+S
minusminus
++
++
+1o
r3G4101
+S
minusminus
++
++
+1o
r3G8614
+S
minusminus
++
++
+1o
r3S40312
+S
minusminus
++
++
+1o
r3S40309
+S
minusminus
++
++
+1o
r3S010
8+
Sminus
minus+
++
++
1or3
S0210
+S
minusminus
++
++
+1o
r3Ssm
ooth
colonies
BioMed Research International 5
5 10 15 20 25 30 35
Cycle40 45
000
005Threshold010
015
020
025
030
035
040
045
050
Nor
m fl
uoro
055
060
065
070
Figure 3 Triplex real-time PCR amplification pattern using the Babortus probe Fluorescence ratio is plotted against the number ofPCR cycles to monitor amplification in real-time mode Only Babortus (positive control) was picked with a strong Ct value by thisprobe
322 Brucella Species Confirmation by Bruce-Ladder DNAfrom all the 11 isolates that were confirmed as belonging to thegenus Brucella in the triplex real-time PCRwas detected as Babortus DNA by the Bruce-ladder multiplex PCR (Figure 4)B abortus gives two bands of 1682 bp and 587 bp on Bruce-ladder PCR
323 MLVA Genotyping The MLVA-16 assay revealed thatnone of the 11 isolates did match any of the Brucella isolatesin the Brucella MLVA 2012 database but closely resembledthe former B abortus biovar 7 strain 07-994-2411 from Kenya(Table 2) All isolates obtained were monomorphic at all locias shown in Table 2 and Figure 5We designated these isolatesas UG Ba-m because they were isolated from cattle in Ugandaand had a B abortus profile on most biotyping assays butresembled both B melitensis and B abortus at genotypingBoth of the UG Ba-m isolates and the B abortus strain 07-994-2411 showed close resemblance to a human B melitensisbiovar 1 (strain BCCN87-92) strain isolated fromUSAWhencompared on MLVA-8 panel 1 loci the genetic distance wasonly zero between UG Ba-m isolates and B abortus strain07-994-2411 and one was between UG Ba-m isolates and Bmelitensis biovar 1 strain BCCN87-92
4 Discussion
Here we present for the first time phenotypic and molecularcharacterization of Brucella isolates from cattle milk inUganda These results contribute to better understanding ofgeographical transmission patterns of Brucella in cattle inUganda and are important if specific control measures areto be implemented in the future In Uganda most of themilk is marketed unprocessed through the informal milkmarketing linkages thus acting as a potential source ofhuman brucellosis infections
All UG Ba-m isolates were from Brucella seropositive cat-tle conforming to the well-known fact that Brucella infectedlactating female animals shed the bacteria in their milk sincethe organism relocates to the udder from the pregnant uterusupon delivery [27] This has public health implications in the
region since most of the milk is consumed unpasteurizedOne third of the seropositive cattle were not shedding theBrucella in the milk suggesting that these animals eitherhad cleared the infection or were chronically sick thus notshedding the bacteria as shown in a study by Capparelli etal [28] All UG Ba-m isolates being from seropositive cattlesuggest an active infection Notably Brucellawas not isolatedfrom milk from any of the seronegative cows This suggeststhat seroconversion precedes shedding of the Brucella inmilk and thus serological tests can be sufficient in predictingpossible shedders
The phenotypic characteristics of all UG Ba-m isolatesmatched those of B abortus biovars 1 3 or the formerbiovar 7 All isolates being B abortus conform to the well-known fact that B abortus is the predominant species incattle [14] Furthermore all 11 UG Ba-m isolates having thesame phenotypic profile suggest that they belong to the samebiovar attesting to the suggestion of regional predominance ofcertain Brucella biovars in Africa for instance predominanceof B abortus biovar 6 in nomadic cattle in Western Sudan[29] B abortus biovar 3 and B melitensis biovar 1 in cattlein Kenya [9] and B melitensis biovar 3 in ruminants in Egypt[15] However the numbers of isolates in these studies weretoo few to make a solid basis for generalization
Detection of all the 11 UG Ba-m isolates as Brucella bythe Brucella genus specific probe in the triplex real-time PCRwith strong signals confirmed them as Brucella The inabilityof the B melitensis probe in the triplex real-time PCR todetect the isolates as B melitensis suggested that they are notB melitensis The inability of the B abortus species specificprobe in the triplex real-time PCR to detect the isolates asB abortus suggested that they are not B abortus biovars 1 23 4 5 6 and 9 but could be the former B abortus biovar 7since the primers used were not targeting B abortus biovar 7[23 30]
All the 11 UG Ba-m isolates were confirmed as B abortusby the Bruce-ladder multiplex PCR The inability of thetriplex real-time PCR to detect these isolates at its B abortusspecies specific probe contrary to the Bruce-ladder multiplexPCR suggests that these isolates belong to the former Brucellaabortus biovar 7 This suggestion is based on the fact thatthe triplex real-time PCR was not designed to detect theformer Brucella abortus biovar 7 with its B abortus speciesspecific probe (detecting only B abortus biovars 1 2 34 5 6 and 9) and the Bruce-ladder multiplex PCR wasdesigned to detect all the B abortus biovars including theformer B abortus biovar 7 [16 23 30] This suggests that adiphasic PCR protocol involving the triplex real-time PCRby Probert et al [23] and the Bruce-ladder multiplex PCRby Lopez-Goni et al [16] could be used to replace the riskyprocedure of identifying the former B abortus biovar 7 usingthe conventional biotypingmethods by detecting particularlyits agglutination with monospecific anti-sera A andM and itsgrowth in both basic fuchsin and thionin dyes This protocolneeds however to be tested on all the former B abortusbiovar 7 isolates
The evidence adduced using a combination of phenotypicand molecular approaches designated all UG Ba-m isolatesas atypical B abortus without a biovar designation All the
6 BioMed Research International
DN
A m
arke
rS4
060
1M
FE 4
10M
FE 1
15G
410
1S4
060
2S3
702
S03
03S4
030
7G
861
4S4
031
2S4
030
9S0
108
S03
04B
Suis
B su
is
RB51
Rev1
N
eg ct
rlD
NA
mar
ker
30001000 500
The different species should givethe following pattern of bandsB abortus B ovis
B suis B melitensis
1682bp 1071bp587bp 587bp
1682bp1071bp587bp 587bp
1682bp1071bp
272bp
Figure 4 Bruce-ladder multiplex PCR agarose gel picture used to confirm the Brucella species isolated Extreme left and right lanes are for100 bp molecular weight marker from left to right lanes 2 5ndash13 are for DNA from isolates detected as Brucella with strong Ct values lanes 34 and 14 are for DNA from Brucella-like isolates with weak Ct values lanes 15 and 19 are for B suis positive control DNA lane 16 is for RB 51positive control DNA lane 17 is for Rev 1 positive control DNA and lane 18 is for PCR grade water (negative control)
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
Panel 1 Panel 2
Bruce 06 (134bP)
Bruce 11 (63bP)
Bruce 42 (125 bP)
Bruce 45 (18bP)
Bruce 08 (18bP)
Bruce 12 (15bP)
Bruce 43 (24bP)
Bruce 55 (40bP)
Bruce 04 (8bP)
Bruce 09 (8bP)
Bruce 18 (8bP)
Bruce 21 (8bP)
Bruce 07 (8bP)
Bruce 16 (8bP)
Bruce 19 (6bP)
Bruce 30 (8bP)
40bp
40bp
40bp
40bp
40bp
40bp
40bp
40bp
Figure 5 MLVA amplification pattern of isolates in this study and a B suis as control Lanes 1 8 and 15 show DNA marker lane 2 in eachgel shows pattern for B suis and lanes 3ndash7 and 9ndash14 show the amplification pattern of isolates 1ndash11 (G4101 S40602 S3702 S0303 S40307S40601 G8614 S40312 S4039 S0908 and S0210)
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Signal TransductionJournal of
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BioMed Research International
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Microbiology
2 BioMed Research International
that isolated in Uganda In a related study in Egypt Babortus B suis and B melitensis were isolated from cattleand all had a high level of phylogenetic variability withineach species although the isolates used in these studies werefew [15]The above findings indicate a complex epidemiologyof brucellosis in cattle in the region and call for refineddiagnostic methods beyond phenotypic typing
High resolution phenotypic and molecular approacheshave been developed for Brucella speciation biotypingand epidemiological trace-back [16 17] To date advancedmolecular technologies have not been widely used in lowincome countries where brucellosis is endemic in livestockand humans [7 18] Thus information on the prevailingBrucella species biovars and genotypesstrains in such areasof endemicity may shed new light on the epidemiology ofBrucella infection and the species and biovars circulatingBesides this generic scientific rationale for undertaking suchinvestigations increased understanding of the Brucella epi-demiology is critical for refining control of brucellosis inresource weak countries where the same measures as in highincome countries cannot be applied
In northern and eastern Uganda where this study wasperformed there was a considerable mixing of livestockspecies during years of insurgency in the 1990s presentingideal conditions for inter- and intraherd transmission ofdiseases such as brucellosis Indeed high herd and individualanimal seroprevalences of up to 27 and 75 respectivelywere recorded in a recent survey in the region [19] Thishigh prevalence may pose a severe threat to public health asprevious studies around the capital of Uganda suggest milkor milk products from cows as a major source of Brucellainfection in humans [20 21] The present study aimed atisolating and molecular-typing Brucella from cattle milk innorthern and eastern Uganda for better understanding of itsepidemiology
2 Materials and Methods
21 Study Design and Collection of Samples Milk sampleswere collected from 207 lactating cows in urban and peri-urban areas of Gulu and Soroti towns of northern andeastern Uganda from May 2011 to March 2012 for isolationof Brucella A total of 110 individual cow milk samples werecollected from 72 herds in Gulu and a total of 97 individualcow milk samples were collected from 33 herds in SorotiThese herds were part of the 166 herds whose animals hadbeen screened for brucellosis and were within a radius of15 Km in both Gulu and Soroti towns with the two townsbeing 200Km apart The cattle from which milk sampleswere taken had been screened for Brucella antibodies and intotal 17 of the 207 cows were seropositive In both townsthe seropositive herds from which the milk samples weretaken were near each other The numbers and selection ofhouseholds and animals included are described in detailpreviously [19] Midstreammilk samples were collected fromall quarters with 10ndash20mL collected from each teat intosterile 100mL falcon tubes The samples were transportedchilled to Makerere University College of Health Sciences
microbiology laboratory Kampala Uganda kept at 4∘C andcultured within three days
22 Sample Preparation Brucella Culturing and BiotypingThe milk was centrifuged at 3000timesg at 4∘C for 15 minutesand the pellet and supernatant were plated on both Farreland Centro de Investigacion y Tecnologıa Agroalimentaria(CITA) selective media supplemented with calf serum [22]Briefly Farrelrsquos medium was prepared from Brucellamediumbase (Oxoid UK) sterilized at 121∘C for 15 minutes andsupplemented with Brucella selective supplement (OxoidUK) according to the manufacturerrsquos instructions The CITAmedium was prepared according to De Miguel et al [22]Inoculated plates were incubated at 37∘C for 8 days in a5ndash10 carbon-dioxide incubator and read every 24 hoursfrom day three of incubation for colony growth Resultantcolonies were subcultured and biotyped based on theircolony morphology serum and carbon-dioxide requirementfor growth hydrogen sulphide production urease activityoxidase test and growth in presence of dye basic fuchsinand agglutination of anti-Brucella IgG monospecific seraA (Animal Health and Veterinary Laboratories AgenciesWeybridge UK) according to theOIETerrestrialManual [8]Representative colonies are stored at minus80∘C in 20 glycerolfor long-term storage
23 Genomic DNA Extraction and Real-Time PCR DetectionThe colonies that conformed to all the above phenotypiccharacteristics of Brucella were subjected to genomic DNAextraction using theNorgen bacterial genomicDNA isolationkit (Norgen Biotek Corp Ontario Canada) The extractedBrucella genomic DNAwas used to run a real-time multiplexPCR assay with oligonucleotide primers probes reactionmixture and PCR conditions according to Probert et al[23] Amplification and real-time fluorescence detectionweredone on the Rotor-Gene 3000 real-time PCR machine (Cor-bett Research-Corbett Life Sciences Mort Lake Australia)Three positive and two negative controls were included ineach runWhen the cycle threshold (CT) value of the sampleswas le40 samples were evaluated as positive This real-timemultiplex PCR was designed to detect Brucella at both thegenus and species levels for B abortus and B melitensis sinceit has the genus specific probe and the species specific probesfor the two Brucella species commonly infecting cattle
24 Brucella Species Confirmation Positive samples on real-time PCR were analysed further for Brucella speciation usingthe Bruce-ladder multiplex PCR assay kit (Ingenasa Spain)The oligonucleotide primers reaction mixture and PCRconditions were performed according to the manufacturersquosconditions in conformity with Lopez-Goni et al [16] How-ever amplification was done in a MyCycler thermal cycler(BioRad)
25 Characterization of Isolates by MLVA Genotyping Geno-typing was performed using the Multiple Locus VariableNumber Tandem Repeat Analysis (MLVA) using the 16-primer-pair assay [17 24ndash26] The oligonucleotide primer
BioMed Research International 3
Cycle5 10 15 20 25 30 35 40 45
Nor
m fl
uoro
035
030
025
020
015
010
005
000
Threshold
Figure 1 Triplex real-time PCR amplification pattern using theBrucella genus probe Fluorescence ratio is plotted against thenumber of PCR cycles to monitor amplification in real-time modeIsolates with weak Ct values (2918 and above) had Brucella-likephenotypic characteristics and were included in this assay
pairs incorporated in the Brucella MLVA 16 assay targetboth the conserved and highly discriminatory regions of theBrucella genome Each sample was run on three prescribedMLVA panels Panel 1 consisted of moderately polymorphicminisatellite primer pairs targeting the highly conservedgenomic regions of different Brucella species (bruce 06 bruce08 bruce 11 bruce 12 bruce 42 bruce 43 bruce 45 andbruce 55) Panel 2A (bruce 18 bruce 19 and bruce 21) andpanel 2B (bruce 04 bruce 07 bruce 09 bruce 16 and bruce30) consisted of microsatellite primer pairs targeting thediscriminatory genomic regions The PCR amplification andgenotyping were done according to le Fleche et al [17] withonly a modification in the total reaction volume to 30 120583LAt each run B suis reference strain REF 1330 (from Bruce-ladder kit) was included as shown in Figure 1
26 Gel-Electrophoresis Analysis of Panel 1 and Panel 2 LociAmplification Products Five microliters of the panel 1 andpanel 2 loci amplification products were loaded into 3 and2 agarose gel containing ethidium bromide (05 120583gmL)respectively to visualize the banding pattern in the samplesand positive controls underUV illuminationThe agarose gelwas run on 8Vcm current and a 100 base pair and 20 basepair ladders (BioRad) were included per run for panel 1 andpanel 2 respectively
27 Sequencing the VNTR Locus Amplicons In order toidentify repeat copy number variation among the isolatesin question the resulting PCR products were sequenced foreach VNTR locus at Macrogen Netherlands Sequenceswere viewed using BioEDIT version 7090 Sequencingwas performed in both directions using the M13-primersaccording to Applied Biosystems (ABI) Since each of theVNTR locus primers was tagged with M13 primer ([M13-Forward] 51015840-GTAAAACGACGGCCAGT-31015840 and [M13-Rev]51015840-GCGGATAACAATTTCACACAGG-31015840) this increaseseach VNTR locus PCR product size by 39 base pairs
Nor
m fl
uoro
045
040
035
030
025
020
015
010
005
000
Threshold
Cycle5 10 15 20 25 30 35 40 45
minus005
Figure 2 Triplex real-time PCR amplification pattern using the Bmelitensis probe Fluorescence ratio is plotted against the numberof PCR cycles to monitor amplification in real-time mode Only Bmelitensis (positive control) was picked by this probe
28 Analysis of MLVA Sequence Data The MLVA PCRproductswere sequenced per loci and the forward and reversesequences were assembled into a contig in the Bionumericssoftware version 50 (Applied Maths Belgium) The M13primer tags were trimmed from the contig and the alleledesignation was determined by comparing the fragmentsize with the published allele numbering system (version36 httpmlvau-psudfr Brucella support website for MLVAtyping) The number of tandem repeats per loci was queriedin the Brucella MLVA 2012 public database (httpmlvau-psudfrmlvav4genotyping) accessed on February 21 2014for genotyping of our isolates The closest related knownstrains were determined based on the genetic distance (theminimumnumber of changes in the number of repeats of anylocus that converts one genotype to another)
3 Results
31 Biotyping Based on the biotyping (Table 1) B abortusbiovar 1 3 or 7 was isolated in 11 (53) out of 207 milksamples These 11 positive samples were all from seropositivecows (ie 11 of 17) The colonies being smooth eliminated Bcanis and B ovis which have rough colonies Production ofhydrogen sulfide eliminated B melitensis B ceti B microtiand B abortus biovars 5 and 6 and B suis except B suisbiovar 1 Ability to grow in absence of serum eliminated Babortus biovar 2 that generally requires serum for growthBrucella abortus biovar 2 B neotomae and B suis biovar 1were eliminated by their inability to grow in basic fuchsinAgglutination with anti-Brucella monospecific sera A elimi-nated B abortus biovars 4 and 9
32 Molecular Characterization
321 Brucella DNA Detection by Real-Time PCR DNA fromall the 11 isolates that was judged asB abortus by the biotypingwas detected as Brucella DNA by the Brucella genus probe inthe triplex real-time PCR (Figure 1) However the triplex real-time PCR was unable to detect the Brucella species involvedusing its B melitensis probe (Figure 2) and B abortus probe(Figure 3)
4 BioMed Research International
Table1Ph
enotypiccharacteris
ticso
fBrucella
sppiso
latedfro
mcattlem
ilkfro
mno
rthern
andeaste
rnUgand
ain2011-2012
SampleID
Serological
result
Colon
ymorph
ology
Serum
requ
irement
CO2
requ
irement
Oxidase
test
Ureasea
ctivity
Agglutin
ationwith
mon
ospecific
sera
AH
2Sprod
uctio
nGrowth
inbasic
fuchsin
Biovar
S40601
+S
minusminus
++
++
+1o
r3S40307
+S
minusminus
++
++
+1o
r3S0303
+S
minusminus
++
++
+1o
r3S3702
+S
minusminus
++
++
+1o
r3S40602
+S
minusminus
++
++
+1o
r3G4101
+S
minusminus
++
++
+1o
r3G8614
+S
minusminus
++
++
+1o
r3S40312
+S
minusminus
++
++
+1o
r3S40309
+S
minusminus
++
++
+1o
r3S010
8+
Sminus
minus+
++
++
1or3
S0210
+S
minusminus
++
++
+1o
r3Ssm
ooth
colonies
BioMed Research International 5
5 10 15 20 25 30 35
Cycle40 45
000
005Threshold010
015
020
025
030
035
040
045
050
Nor
m fl
uoro
055
060
065
070
Figure 3 Triplex real-time PCR amplification pattern using the Babortus probe Fluorescence ratio is plotted against the number ofPCR cycles to monitor amplification in real-time mode Only Babortus (positive control) was picked with a strong Ct value by thisprobe
322 Brucella Species Confirmation by Bruce-Ladder DNAfrom all the 11 isolates that were confirmed as belonging to thegenus Brucella in the triplex real-time PCRwas detected as Babortus DNA by the Bruce-ladder multiplex PCR (Figure 4)B abortus gives two bands of 1682 bp and 587 bp on Bruce-ladder PCR
323 MLVA Genotyping The MLVA-16 assay revealed thatnone of the 11 isolates did match any of the Brucella isolatesin the Brucella MLVA 2012 database but closely resembledthe former B abortus biovar 7 strain 07-994-2411 from Kenya(Table 2) All isolates obtained were monomorphic at all locias shown in Table 2 and Figure 5We designated these isolatesas UG Ba-m because they were isolated from cattle in Ugandaand had a B abortus profile on most biotyping assays butresembled both B melitensis and B abortus at genotypingBoth of the UG Ba-m isolates and the B abortus strain 07-994-2411 showed close resemblance to a human B melitensisbiovar 1 (strain BCCN87-92) strain isolated fromUSAWhencompared on MLVA-8 panel 1 loci the genetic distance wasonly zero between UG Ba-m isolates and B abortus strain07-994-2411 and one was between UG Ba-m isolates and Bmelitensis biovar 1 strain BCCN87-92
4 Discussion
Here we present for the first time phenotypic and molecularcharacterization of Brucella isolates from cattle milk inUganda These results contribute to better understanding ofgeographical transmission patterns of Brucella in cattle inUganda and are important if specific control measures areto be implemented in the future In Uganda most of themilk is marketed unprocessed through the informal milkmarketing linkages thus acting as a potential source ofhuman brucellosis infections
All UG Ba-m isolates were from Brucella seropositive cat-tle conforming to the well-known fact that Brucella infectedlactating female animals shed the bacteria in their milk sincethe organism relocates to the udder from the pregnant uterusupon delivery [27] This has public health implications in the
region since most of the milk is consumed unpasteurizedOne third of the seropositive cattle were not shedding theBrucella in the milk suggesting that these animals eitherhad cleared the infection or were chronically sick thus notshedding the bacteria as shown in a study by Capparelli etal [28] All UG Ba-m isolates being from seropositive cattlesuggest an active infection Notably Brucellawas not isolatedfrom milk from any of the seronegative cows This suggeststhat seroconversion precedes shedding of the Brucella inmilk and thus serological tests can be sufficient in predictingpossible shedders
The phenotypic characteristics of all UG Ba-m isolatesmatched those of B abortus biovars 1 3 or the formerbiovar 7 All isolates being B abortus conform to the well-known fact that B abortus is the predominant species incattle [14] Furthermore all 11 UG Ba-m isolates having thesame phenotypic profile suggest that they belong to the samebiovar attesting to the suggestion of regional predominance ofcertain Brucella biovars in Africa for instance predominanceof B abortus biovar 6 in nomadic cattle in Western Sudan[29] B abortus biovar 3 and B melitensis biovar 1 in cattlein Kenya [9] and B melitensis biovar 3 in ruminants in Egypt[15] However the numbers of isolates in these studies weretoo few to make a solid basis for generalization
Detection of all the 11 UG Ba-m isolates as Brucella bythe Brucella genus specific probe in the triplex real-time PCRwith strong signals confirmed them as Brucella The inabilityof the B melitensis probe in the triplex real-time PCR todetect the isolates as B melitensis suggested that they are notB melitensis The inability of the B abortus species specificprobe in the triplex real-time PCR to detect the isolates asB abortus suggested that they are not B abortus biovars 1 23 4 5 6 and 9 but could be the former B abortus biovar 7since the primers used were not targeting B abortus biovar 7[23 30]
All the 11 UG Ba-m isolates were confirmed as B abortusby the Bruce-ladder multiplex PCR The inability of thetriplex real-time PCR to detect these isolates at its B abortusspecies specific probe contrary to the Bruce-ladder multiplexPCR suggests that these isolates belong to the former Brucellaabortus biovar 7 This suggestion is based on the fact thatthe triplex real-time PCR was not designed to detect theformer Brucella abortus biovar 7 with its B abortus speciesspecific probe (detecting only B abortus biovars 1 2 34 5 6 and 9) and the Bruce-ladder multiplex PCR wasdesigned to detect all the B abortus biovars including theformer B abortus biovar 7 [16 23 30] This suggests that adiphasic PCR protocol involving the triplex real-time PCRby Probert et al [23] and the Bruce-ladder multiplex PCRby Lopez-Goni et al [16] could be used to replace the riskyprocedure of identifying the former B abortus biovar 7 usingthe conventional biotypingmethods by detecting particularlyits agglutination with monospecific anti-sera A andM and itsgrowth in both basic fuchsin and thionin dyes This protocolneeds however to be tested on all the former B abortusbiovar 7 isolates
The evidence adduced using a combination of phenotypicand molecular approaches designated all UG Ba-m isolatesas atypical B abortus without a biovar designation All the
6 BioMed Research International
DN
A m
arke
rS4
060
1M
FE 4
10M
FE 1
15G
410
1S4
060
2S3
702
S03
03S4
030
7G
861
4S4
031
2S4
030
9S0
108
S03
04B
Suis
B su
is
RB51
Rev1
N
eg ct
rlD
NA
mar
ker
30001000 500
The different species should givethe following pattern of bandsB abortus B ovis
B suis B melitensis
1682bp 1071bp587bp 587bp
1682bp1071bp587bp 587bp
1682bp1071bp
272bp
Figure 4 Bruce-ladder multiplex PCR agarose gel picture used to confirm the Brucella species isolated Extreme left and right lanes are for100 bp molecular weight marker from left to right lanes 2 5ndash13 are for DNA from isolates detected as Brucella with strong Ct values lanes 34 and 14 are for DNA from Brucella-like isolates with weak Ct values lanes 15 and 19 are for B suis positive control DNA lane 16 is for RB 51positive control DNA lane 17 is for Rev 1 positive control DNA and lane 18 is for PCR grade water (negative control)
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
Panel 1 Panel 2
Bruce 06 (134bP)
Bruce 11 (63bP)
Bruce 42 (125 bP)
Bruce 45 (18bP)
Bruce 08 (18bP)
Bruce 12 (15bP)
Bruce 43 (24bP)
Bruce 55 (40bP)
Bruce 04 (8bP)
Bruce 09 (8bP)
Bruce 18 (8bP)
Bruce 21 (8bP)
Bruce 07 (8bP)
Bruce 16 (8bP)
Bruce 19 (6bP)
Bruce 30 (8bP)
40bp
40bp
40bp
40bp
40bp
40bp
40bp
40bp
Figure 5 MLVA amplification pattern of isolates in this study and a B suis as control Lanes 1 8 and 15 show DNA marker lane 2 in eachgel shows pattern for B suis and lanes 3ndash7 and 9ndash14 show the amplification pattern of isolates 1ndash11 (G4101 S40602 S3702 S0303 S40307S40601 G8614 S40312 S4039 S0908 and S0210)
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
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Signal TransductionJournal of
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BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
Cycle5 10 15 20 25 30 35 40 45
Nor
m fl
uoro
035
030
025
020
015
010
005
000
Threshold
Figure 1 Triplex real-time PCR amplification pattern using theBrucella genus probe Fluorescence ratio is plotted against thenumber of PCR cycles to monitor amplification in real-time modeIsolates with weak Ct values (2918 and above) had Brucella-likephenotypic characteristics and were included in this assay
pairs incorporated in the Brucella MLVA 16 assay targetboth the conserved and highly discriminatory regions of theBrucella genome Each sample was run on three prescribedMLVA panels Panel 1 consisted of moderately polymorphicminisatellite primer pairs targeting the highly conservedgenomic regions of different Brucella species (bruce 06 bruce08 bruce 11 bruce 12 bruce 42 bruce 43 bruce 45 andbruce 55) Panel 2A (bruce 18 bruce 19 and bruce 21) andpanel 2B (bruce 04 bruce 07 bruce 09 bruce 16 and bruce30) consisted of microsatellite primer pairs targeting thediscriminatory genomic regions The PCR amplification andgenotyping were done according to le Fleche et al [17] withonly a modification in the total reaction volume to 30 120583LAt each run B suis reference strain REF 1330 (from Bruce-ladder kit) was included as shown in Figure 1
26 Gel-Electrophoresis Analysis of Panel 1 and Panel 2 LociAmplification Products Five microliters of the panel 1 andpanel 2 loci amplification products were loaded into 3 and2 agarose gel containing ethidium bromide (05 120583gmL)respectively to visualize the banding pattern in the samplesand positive controls underUV illuminationThe agarose gelwas run on 8Vcm current and a 100 base pair and 20 basepair ladders (BioRad) were included per run for panel 1 andpanel 2 respectively
27 Sequencing the VNTR Locus Amplicons In order toidentify repeat copy number variation among the isolatesin question the resulting PCR products were sequenced foreach VNTR locus at Macrogen Netherlands Sequenceswere viewed using BioEDIT version 7090 Sequencingwas performed in both directions using the M13-primersaccording to Applied Biosystems (ABI) Since each of theVNTR locus primers was tagged with M13 primer ([M13-Forward] 51015840-GTAAAACGACGGCCAGT-31015840 and [M13-Rev]51015840-GCGGATAACAATTTCACACAGG-31015840) this increaseseach VNTR locus PCR product size by 39 base pairs
Nor
m fl
uoro
045
040
035
030
025
020
015
010
005
000
Threshold
Cycle5 10 15 20 25 30 35 40 45
minus005
Figure 2 Triplex real-time PCR amplification pattern using the Bmelitensis probe Fluorescence ratio is plotted against the numberof PCR cycles to monitor amplification in real-time mode Only Bmelitensis (positive control) was picked by this probe
28 Analysis of MLVA Sequence Data The MLVA PCRproductswere sequenced per loci and the forward and reversesequences were assembled into a contig in the Bionumericssoftware version 50 (Applied Maths Belgium) The M13primer tags were trimmed from the contig and the alleledesignation was determined by comparing the fragmentsize with the published allele numbering system (version36 httpmlvau-psudfr Brucella support website for MLVAtyping) The number of tandem repeats per loci was queriedin the Brucella MLVA 2012 public database (httpmlvau-psudfrmlvav4genotyping) accessed on February 21 2014for genotyping of our isolates The closest related knownstrains were determined based on the genetic distance (theminimumnumber of changes in the number of repeats of anylocus that converts one genotype to another)
3 Results
31 Biotyping Based on the biotyping (Table 1) B abortusbiovar 1 3 or 7 was isolated in 11 (53) out of 207 milksamples These 11 positive samples were all from seropositivecows (ie 11 of 17) The colonies being smooth eliminated Bcanis and B ovis which have rough colonies Production ofhydrogen sulfide eliminated B melitensis B ceti B microtiand B abortus biovars 5 and 6 and B suis except B suisbiovar 1 Ability to grow in absence of serum eliminated Babortus biovar 2 that generally requires serum for growthBrucella abortus biovar 2 B neotomae and B suis biovar 1were eliminated by their inability to grow in basic fuchsinAgglutination with anti-Brucella monospecific sera A elimi-nated B abortus biovars 4 and 9
32 Molecular Characterization
321 Brucella DNA Detection by Real-Time PCR DNA fromall the 11 isolates that was judged asB abortus by the biotypingwas detected as Brucella DNA by the Brucella genus probe inthe triplex real-time PCR (Figure 1) However the triplex real-time PCR was unable to detect the Brucella species involvedusing its B melitensis probe (Figure 2) and B abortus probe(Figure 3)
4 BioMed Research International
Table1Ph
enotypiccharacteris
ticso
fBrucella
sppiso
latedfro
mcattlem
ilkfro
mno
rthern
andeaste
rnUgand
ain2011-2012
SampleID
Serological
result
Colon
ymorph
ology
Serum
requ
irement
CO2
requ
irement
Oxidase
test
Ureasea
ctivity
Agglutin
ationwith
mon
ospecific
sera
AH
2Sprod
uctio
nGrowth
inbasic
fuchsin
Biovar
S40601
+S
minusminus
++
++
+1o
r3S40307
+S
minusminus
++
++
+1o
r3S0303
+S
minusminus
++
++
+1o
r3S3702
+S
minusminus
++
++
+1o
r3S40602
+S
minusminus
++
++
+1o
r3G4101
+S
minusminus
++
++
+1o
r3G8614
+S
minusminus
++
++
+1o
r3S40312
+S
minusminus
++
++
+1o
r3S40309
+S
minusminus
++
++
+1o
r3S010
8+
Sminus
minus+
++
++
1or3
S0210
+S
minusminus
++
++
+1o
r3Ssm
ooth
colonies
BioMed Research International 5
5 10 15 20 25 30 35
Cycle40 45
000
005Threshold010
015
020
025
030
035
040
045
050
Nor
m fl
uoro
055
060
065
070
Figure 3 Triplex real-time PCR amplification pattern using the Babortus probe Fluorescence ratio is plotted against the number ofPCR cycles to monitor amplification in real-time mode Only Babortus (positive control) was picked with a strong Ct value by thisprobe
322 Brucella Species Confirmation by Bruce-Ladder DNAfrom all the 11 isolates that were confirmed as belonging to thegenus Brucella in the triplex real-time PCRwas detected as Babortus DNA by the Bruce-ladder multiplex PCR (Figure 4)B abortus gives two bands of 1682 bp and 587 bp on Bruce-ladder PCR
323 MLVA Genotyping The MLVA-16 assay revealed thatnone of the 11 isolates did match any of the Brucella isolatesin the Brucella MLVA 2012 database but closely resembledthe former B abortus biovar 7 strain 07-994-2411 from Kenya(Table 2) All isolates obtained were monomorphic at all locias shown in Table 2 and Figure 5We designated these isolatesas UG Ba-m because they were isolated from cattle in Ugandaand had a B abortus profile on most biotyping assays butresembled both B melitensis and B abortus at genotypingBoth of the UG Ba-m isolates and the B abortus strain 07-994-2411 showed close resemblance to a human B melitensisbiovar 1 (strain BCCN87-92) strain isolated fromUSAWhencompared on MLVA-8 panel 1 loci the genetic distance wasonly zero between UG Ba-m isolates and B abortus strain07-994-2411 and one was between UG Ba-m isolates and Bmelitensis biovar 1 strain BCCN87-92
4 Discussion
Here we present for the first time phenotypic and molecularcharacterization of Brucella isolates from cattle milk inUganda These results contribute to better understanding ofgeographical transmission patterns of Brucella in cattle inUganda and are important if specific control measures areto be implemented in the future In Uganda most of themilk is marketed unprocessed through the informal milkmarketing linkages thus acting as a potential source ofhuman brucellosis infections
All UG Ba-m isolates were from Brucella seropositive cat-tle conforming to the well-known fact that Brucella infectedlactating female animals shed the bacteria in their milk sincethe organism relocates to the udder from the pregnant uterusupon delivery [27] This has public health implications in the
region since most of the milk is consumed unpasteurizedOne third of the seropositive cattle were not shedding theBrucella in the milk suggesting that these animals eitherhad cleared the infection or were chronically sick thus notshedding the bacteria as shown in a study by Capparelli etal [28] All UG Ba-m isolates being from seropositive cattlesuggest an active infection Notably Brucellawas not isolatedfrom milk from any of the seronegative cows This suggeststhat seroconversion precedes shedding of the Brucella inmilk and thus serological tests can be sufficient in predictingpossible shedders
The phenotypic characteristics of all UG Ba-m isolatesmatched those of B abortus biovars 1 3 or the formerbiovar 7 All isolates being B abortus conform to the well-known fact that B abortus is the predominant species incattle [14] Furthermore all 11 UG Ba-m isolates having thesame phenotypic profile suggest that they belong to the samebiovar attesting to the suggestion of regional predominance ofcertain Brucella biovars in Africa for instance predominanceof B abortus biovar 6 in nomadic cattle in Western Sudan[29] B abortus biovar 3 and B melitensis biovar 1 in cattlein Kenya [9] and B melitensis biovar 3 in ruminants in Egypt[15] However the numbers of isolates in these studies weretoo few to make a solid basis for generalization
Detection of all the 11 UG Ba-m isolates as Brucella bythe Brucella genus specific probe in the triplex real-time PCRwith strong signals confirmed them as Brucella The inabilityof the B melitensis probe in the triplex real-time PCR todetect the isolates as B melitensis suggested that they are notB melitensis The inability of the B abortus species specificprobe in the triplex real-time PCR to detect the isolates asB abortus suggested that they are not B abortus biovars 1 23 4 5 6 and 9 but could be the former B abortus biovar 7since the primers used were not targeting B abortus biovar 7[23 30]
All the 11 UG Ba-m isolates were confirmed as B abortusby the Bruce-ladder multiplex PCR The inability of thetriplex real-time PCR to detect these isolates at its B abortusspecies specific probe contrary to the Bruce-ladder multiplexPCR suggests that these isolates belong to the former Brucellaabortus biovar 7 This suggestion is based on the fact thatthe triplex real-time PCR was not designed to detect theformer Brucella abortus biovar 7 with its B abortus speciesspecific probe (detecting only B abortus biovars 1 2 34 5 6 and 9) and the Bruce-ladder multiplex PCR wasdesigned to detect all the B abortus biovars including theformer B abortus biovar 7 [16 23 30] This suggests that adiphasic PCR protocol involving the triplex real-time PCRby Probert et al [23] and the Bruce-ladder multiplex PCRby Lopez-Goni et al [16] could be used to replace the riskyprocedure of identifying the former B abortus biovar 7 usingthe conventional biotypingmethods by detecting particularlyits agglutination with monospecific anti-sera A andM and itsgrowth in both basic fuchsin and thionin dyes This protocolneeds however to be tested on all the former B abortusbiovar 7 isolates
The evidence adduced using a combination of phenotypicand molecular approaches designated all UG Ba-m isolatesas atypical B abortus without a biovar designation All the
6 BioMed Research International
DN
A m
arke
rS4
060
1M
FE 4
10M
FE 1
15G
410
1S4
060
2S3
702
S03
03S4
030
7G
861
4S4
031
2S4
030
9S0
108
S03
04B
Suis
B su
is
RB51
Rev1
N
eg ct
rlD
NA
mar
ker
30001000 500
The different species should givethe following pattern of bandsB abortus B ovis
B suis B melitensis
1682bp 1071bp587bp 587bp
1682bp1071bp587bp 587bp
1682bp1071bp
272bp
Figure 4 Bruce-ladder multiplex PCR agarose gel picture used to confirm the Brucella species isolated Extreme left and right lanes are for100 bp molecular weight marker from left to right lanes 2 5ndash13 are for DNA from isolates detected as Brucella with strong Ct values lanes 34 and 14 are for DNA from Brucella-like isolates with weak Ct values lanes 15 and 19 are for B suis positive control DNA lane 16 is for RB 51positive control DNA lane 17 is for Rev 1 positive control DNA and lane 18 is for PCR grade water (negative control)
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
Panel 1 Panel 2
Bruce 06 (134bP)
Bruce 11 (63bP)
Bruce 42 (125 bP)
Bruce 45 (18bP)
Bruce 08 (18bP)
Bruce 12 (15bP)
Bruce 43 (24bP)
Bruce 55 (40bP)
Bruce 04 (8bP)
Bruce 09 (8bP)
Bruce 18 (8bP)
Bruce 21 (8bP)
Bruce 07 (8bP)
Bruce 16 (8bP)
Bruce 19 (6bP)
Bruce 30 (8bP)
40bp
40bp
40bp
40bp
40bp
40bp
40bp
40bp
Figure 5 MLVA amplification pattern of isolates in this study and a B suis as control Lanes 1 8 and 15 show DNA marker lane 2 in eachgel shows pattern for B suis and lanes 3ndash7 and 9ndash14 show the amplification pattern of isolates 1ndash11 (G4101 S40602 S3702 S0303 S40307S40601 G8614 S40312 S4039 S0908 and S0210)
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
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International Journal of
Microbiology
4 BioMed Research International
Table1Ph
enotypiccharacteris
ticso
fBrucella
sppiso
latedfro
mcattlem
ilkfro
mno
rthern
andeaste
rnUgand
ain2011-2012
SampleID
Serological
result
Colon
ymorph
ology
Serum
requ
irement
CO2
requ
irement
Oxidase
test
Ureasea
ctivity
Agglutin
ationwith
mon
ospecific
sera
AH
2Sprod
uctio
nGrowth
inbasic
fuchsin
Biovar
S40601
+S
minusminus
++
++
+1o
r3S40307
+S
minusminus
++
++
+1o
r3S0303
+S
minusminus
++
++
+1o
r3S3702
+S
minusminus
++
++
+1o
r3S40602
+S
minusminus
++
++
+1o
r3G4101
+S
minusminus
++
++
+1o
r3G8614
+S
minusminus
++
++
+1o
r3S40312
+S
minusminus
++
++
+1o
r3S40309
+S
minusminus
++
++
+1o
r3S010
8+
Sminus
minus+
++
++
1or3
S0210
+S
minusminus
++
++
+1o
r3Ssm
ooth
colonies
BioMed Research International 5
5 10 15 20 25 30 35
Cycle40 45
000
005Threshold010
015
020
025
030
035
040
045
050
Nor
m fl
uoro
055
060
065
070
Figure 3 Triplex real-time PCR amplification pattern using the Babortus probe Fluorescence ratio is plotted against the number ofPCR cycles to monitor amplification in real-time mode Only Babortus (positive control) was picked with a strong Ct value by thisprobe
322 Brucella Species Confirmation by Bruce-Ladder DNAfrom all the 11 isolates that were confirmed as belonging to thegenus Brucella in the triplex real-time PCRwas detected as Babortus DNA by the Bruce-ladder multiplex PCR (Figure 4)B abortus gives two bands of 1682 bp and 587 bp on Bruce-ladder PCR
323 MLVA Genotyping The MLVA-16 assay revealed thatnone of the 11 isolates did match any of the Brucella isolatesin the Brucella MLVA 2012 database but closely resembledthe former B abortus biovar 7 strain 07-994-2411 from Kenya(Table 2) All isolates obtained were monomorphic at all locias shown in Table 2 and Figure 5We designated these isolatesas UG Ba-m because they were isolated from cattle in Ugandaand had a B abortus profile on most biotyping assays butresembled both B melitensis and B abortus at genotypingBoth of the UG Ba-m isolates and the B abortus strain 07-994-2411 showed close resemblance to a human B melitensisbiovar 1 (strain BCCN87-92) strain isolated fromUSAWhencompared on MLVA-8 panel 1 loci the genetic distance wasonly zero between UG Ba-m isolates and B abortus strain07-994-2411 and one was between UG Ba-m isolates and Bmelitensis biovar 1 strain BCCN87-92
4 Discussion
Here we present for the first time phenotypic and molecularcharacterization of Brucella isolates from cattle milk inUganda These results contribute to better understanding ofgeographical transmission patterns of Brucella in cattle inUganda and are important if specific control measures areto be implemented in the future In Uganda most of themilk is marketed unprocessed through the informal milkmarketing linkages thus acting as a potential source ofhuman brucellosis infections
All UG Ba-m isolates were from Brucella seropositive cat-tle conforming to the well-known fact that Brucella infectedlactating female animals shed the bacteria in their milk sincethe organism relocates to the udder from the pregnant uterusupon delivery [27] This has public health implications in the
region since most of the milk is consumed unpasteurizedOne third of the seropositive cattle were not shedding theBrucella in the milk suggesting that these animals eitherhad cleared the infection or were chronically sick thus notshedding the bacteria as shown in a study by Capparelli etal [28] All UG Ba-m isolates being from seropositive cattlesuggest an active infection Notably Brucellawas not isolatedfrom milk from any of the seronegative cows This suggeststhat seroconversion precedes shedding of the Brucella inmilk and thus serological tests can be sufficient in predictingpossible shedders
The phenotypic characteristics of all UG Ba-m isolatesmatched those of B abortus biovars 1 3 or the formerbiovar 7 All isolates being B abortus conform to the well-known fact that B abortus is the predominant species incattle [14] Furthermore all 11 UG Ba-m isolates having thesame phenotypic profile suggest that they belong to the samebiovar attesting to the suggestion of regional predominance ofcertain Brucella biovars in Africa for instance predominanceof B abortus biovar 6 in nomadic cattle in Western Sudan[29] B abortus biovar 3 and B melitensis biovar 1 in cattlein Kenya [9] and B melitensis biovar 3 in ruminants in Egypt[15] However the numbers of isolates in these studies weretoo few to make a solid basis for generalization
Detection of all the 11 UG Ba-m isolates as Brucella bythe Brucella genus specific probe in the triplex real-time PCRwith strong signals confirmed them as Brucella The inabilityof the B melitensis probe in the triplex real-time PCR todetect the isolates as B melitensis suggested that they are notB melitensis The inability of the B abortus species specificprobe in the triplex real-time PCR to detect the isolates asB abortus suggested that they are not B abortus biovars 1 23 4 5 6 and 9 but could be the former B abortus biovar 7since the primers used were not targeting B abortus biovar 7[23 30]
All the 11 UG Ba-m isolates were confirmed as B abortusby the Bruce-ladder multiplex PCR The inability of thetriplex real-time PCR to detect these isolates at its B abortusspecies specific probe contrary to the Bruce-ladder multiplexPCR suggests that these isolates belong to the former Brucellaabortus biovar 7 This suggestion is based on the fact thatthe triplex real-time PCR was not designed to detect theformer Brucella abortus biovar 7 with its B abortus speciesspecific probe (detecting only B abortus biovars 1 2 34 5 6 and 9) and the Bruce-ladder multiplex PCR wasdesigned to detect all the B abortus biovars including theformer B abortus biovar 7 [16 23 30] This suggests that adiphasic PCR protocol involving the triplex real-time PCRby Probert et al [23] and the Bruce-ladder multiplex PCRby Lopez-Goni et al [16] could be used to replace the riskyprocedure of identifying the former B abortus biovar 7 usingthe conventional biotypingmethods by detecting particularlyits agglutination with monospecific anti-sera A andM and itsgrowth in both basic fuchsin and thionin dyes This protocolneeds however to be tested on all the former B abortusbiovar 7 isolates
The evidence adduced using a combination of phenotypicand molecular approaches designated all UG Ba-m isolatesas atypical B abortus without a biovar designation All the
6 BioMed Research International
DN
A m
arke
rS4
060
1M
FE 4
10M
FE 1
15G
410
1S4
060
2S3
702
S03
03S4
030
7G
861
4S4
031
2S4
030
9S0
108
S03
04B
Suis
B su
is
RB51
Rev1
N
eg ct
rlD
NA
mar
ker
30001000 500
The different species should givethe following pattern of bandsB abortus B ovis
B suis B melitensis
1682bp 1071bp587bp 587bp
1682bp1071bp587bp 587bp
1682bp1071bp
272bp
Figure 4 Bruce-ladder multiplex PCR agarose gel picture used to confirm the Brucella species isolated Extreme left and right lanes are for100 bp molecular weight marker from left to right lanes 2 5ndash13 are for DNA from isolates detected as Brucella with strong Ct values lanes 34 and 14 are for DNA from Brucella-like isolates with weak Ct values lanes 15 and 19 are for B suis positive control DNA lane 16 is for RB 51positive control DNA lane 17 is for Rev 1 positive control DNA and lane 18 is for PCR grade water (negative control)
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
Panel 1 Panel 2
Bruce 06 (134bP)
Bruce 11 (63bP)
Bruce 42 (125 bP)
Bruce 45 (18bP)
Bruce 08 (18bP)
Bruce 12 (15bP)
Bruce 43 (24bP)
Bruce 55 (40bP)
Bruce 04 (8bP)
Bruce 09 (8bP)
Bruce 18 (8bP)
Bruce 21 (8bP)
Bruce 07 (8bP)
Bruce 16 (8bP)
Bruce 19 (6bP)
Bruce 30 (8bP)
40bp
40bp
40bp
40bp
40bp
40bp
40bp
40bp
Figure 5 MLVA amplification pattern of isolates in this study and a B suis as control Lanes 1 8 and 15 show DNA marker lane 2 in eachgel shows pattern for B suis and lanes 3ndash7 and 9ndash14 show the amplification pattern of isolates 1ndash11 (G4101 S40602 S3702 S0303 S40307S40601 G8614 S40312 S4039 S0908 and S0210)
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
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Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 5
5 10 15 20 25 30 35
Cycle40 45
000
005Threshold010
015
020
025
030
035
040
045
050
Nor
m fl
uoro
055
060
065
070
Figure 3 Triplex real-time PCR amplification pattern using the Babortus probe Fluorescence ratio is plotted against the number ofPCR cycles to monitor amplification in real-time mode Only Babortus (positive control) was picked with a strong Ct value by thisprobe
322 Brucella Species Confirmation by Bruce-Ladder DNAfrom all the 11 isolates that were confirmed as belonging to thegenus Brucella in the triplex real-time PCRwas detected as Babortus DNA by the Bruce-ladder multiplex PCR (Figure 4)B abortus gives two bands of 1682 bp and 587 bp on Bruce-ladder PCR
323 MLVA Genotyping The MLVA-16 assay revealed thatnone of the 11 isolates did match any of the Brucella isolatesin the Brucella MLVA 2012 database but closely resembledthe former B abortus biovar 7 strain 07-994-2411 from Kenya(Table 2) All isolates obtained were monomorphic at all locias shown in Table 2 and Figure 5We designated these isolatesas UG Ba-m because they were isolated from cattle in Ugandaand had a B abortus profile on most biotyping assays butresembled both B melitensis and B abortus at genotypingBoth of the UG Ba-m isolates and the B abortus strain 07-994-2411 showed close resemblance to a human B melitensisbiovar 1 (strain BCCN87-92) strain isolated fromUSAWhencompared on MLVA-8 panel 1 loci the genetic distance wasonly zero between UG Ba-m isolates and B abortus strain07-994-2411 and one was between UG Ba-m isolates and Bmelitensis biovar 1 strain BCCN87-92
4 Discussion
Here we present for the first time phenotypic and molecularcharacterization of Brucella isolates from cattle milk inUganda These results contribute to better understanding ofgeographical transmission patterns of Brucella in cattle inUganda and are important if specific control measures areto be implemented in the future In Uganda most of themilk is marketed unprocessed through the informal milkmarketing linkages thus acting as a potential source ofhuman brucellosis infections
All UG Ba-m isolates were from Brucella seropositive cat-tle conforming to the well-known fact that Brucella infectedlactating female animals shed the bacteria in their milk sincethe organism relocates to the udder from the pregnant uterusupon delivery [27] This has public health implications in the
region since most of the milk is consumed unpasteurizedOne third of the seropositive cattle were not shedding theBrucella in the milk suggesting that these animals eitherhad cleared the infection or were chronically sick thus notshedding the bacteria as shown in a study by Capparelli etal [28] All UG Ba-m isolates being from seropositive cattlesuggest an active infection Notably Brucellawas not isolatedfrom milk from any of the seronegative cows This suggeststhat seroconversion precedes shedding of the Brucella inmilk and thus serological tests can be sufficient in predictingpossible shedders
The phenotypic characteristics of all UG Ba-m isolatesmatched those of B abortus biovars 1 3 or the formerbiovar 7 All isolates being B abortus conform to the well-known fact that B abortus is the predominant species incattle [14] Furthermore all 11 UG Ba-m isolates having thesame phenotypic profile suggest that they belong to the samebiovar attesting to the suggestion of regional predominance ofcertain Brucella biovars in Africa for instance predominanceof B abortus biovar 6 in nomadic cattle in Western Sudan[29] B abortus biovar 3 and B melitensis biovar 1 in cattlein Kenya [9] and B melitensis biovar 3 in ruminants in Egypt[15] However the numbers of isolates in these studies weretoo few to make a solid basis for generalization
Detection of all the 11 UG Ba-m isolates as Brucella bythe Brucella genus specific probe in the triplex real-time PCRwith strong signals confirmed them as Brucella The inabilityof the B melitensis probe in the triplex real-time PCR todetect the isolates as B melitensis suggested that they are notB melitensis The inability of the B abortus species specificprobe in the triplex real-time PCR to detect the isolates asB abortus suggested that they are not B abortus biovars 1 23 4 5 6 and 9 but could be the former B abortus biovar 7since the primers used were not targeting B abortus biovar 7[23 30]
All the 11 UG Ba-m isolates were confirmed as B abortusby the Bruce-ladder multiplex PCR The inability of thetriplex real-time PCR to detect these isolates at its B abortusspecies specific probe contrary to the Bruce-ladder multiplexPCR suggests that these isolates belong to the former Brucellaabortus biovar 7 This suggestion is based on the fact thatthe triplex real-time PCR was not designed to detect theformer Brucella abortus biovar 7 with its B abortus speciesspecific probe (detecting only B abortus biovars 1 2 34 5 6 and 9) and the Bruce-ladder multiplex PCR wasdesigned to detect all the B abortus biovars including theformer B abortus biovar 7 [16 23 30] This suggests that adiphasic PCR protocol involving the triplex real-time PCRby Probert et al [23] and the Bruce-ladder multiplex PCRby Lopez-Goni et al [16] could be used to replace the riskyprocedure of identifying the former B abortus biovar 7 usingthe conventional biotypingmethods by detecting particularlyits agglutination with monospecific anti-sera A andM and itsgrowth in both basic fuchsin and thionin dyes This protocolneeds however to be tested on all the former B abortusbiovar 7 isolates
The evidence adduced using a combination of phenotypicand molecular approaches designated all UG Ba-m isolatesas atypical B abortus without a biovar designation All the
6 BioMed Research International
DN
A m
arke
rS4
060
1M
FE 4
10M
FE 1
15G
410
1S4
060
2S3
702
S03
03S4
030
7G
861
4S4
031
2S4
030
9S0
108
S03
04B
Suis
B su
is
RB51
Rev1
N
eg ct
rlD
NA
mar
ker
30001000 500
The different species should givethe following pattern of bandsB abortus B ovis
B suis B melitensis
1682bp 1071bp587bp 587bp
1682bp1071bp587bp 587bp
1682bp1071bp
272bp
Figure 4 Bruce-ladder multiplex PCR agarose gel picture used to confirm the Brucella species isolated Extreme left and right lanes are for100 bp molecular weight marker from left to right lanes 2 5ndash13 are for DNA from isolates detected as Brucella with strong Ct values lanes 34 and 14 are for DNA from Brucella-like isolates with weak Ct values lanes 15 and 19 are for B suis positive control DNA lane 16 is for RB 51positive control DNA lane 17 is for Rev 1 positive control DNA and lane 18 is for PCR grade water (negative control)
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
Panel 1 Panel 2
Bruce 06 (134bP)
Bruce 11 (63bP)
Bruce 42 (125 bP)
Bruce 45 (18bP)
Bruce 08 (18bP)
Bruce 12 (15bP)
Bruce 43 (24bP)
Bruce 55 (40bP)
Bruce 04 (8bP)
Bruce 09 (8bP)
Bruce 18 (8bP)
Bruce 21 (8bP)
Bruce 07 (8bP)
Bruce 16 (8bP)
Bruce 19 (6bP)
Bruce 30 (8bP)
40bp
40bp
40bp
40bp
40bp
40bp
40bp
40bp
Figure 5 MLVA amplification pattern of isolates in this study and a B suis as control Lanes 1 8 and 15 show DNA marker lane 2 in eachgel shows pattern for B suis and lanes 3ndash7 and 9ndash14 show the amplification pattern of isolates 1ndash11 (G4101 S40602 S3702 S0303 S40307S40601 G8614 S40312 S4039 S0908 and S0210)
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
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Signal TransductionJournal of
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
DN
A m
arke
rS4
060
1M
FE 4
10M
FE 1
15G
410
1S4
060
2S3
702
S03
03S4
030
7G
861
4S4
031
2S4
030
9S0
108
S03
04B
Suis
B su
is
RB51
Rev1
N
eg ct
rlD
NA
mar
ker
30001000 500
The different species should givethe following pattern of bandsB abortus B ovis
B suis B melitensis
1682bp 1071bp587bp 587bp
1682bp1071bp587bp 587bp
1682bp1071bp
272bp
Figure 4 Bruce-ladder multiplex PCR agarose gel picture used to confirm the Brucella species isolated Extreme left and right lanes are for100 bp molecular weight marker from left to right lanes 2 5ndash13 are for DNA from isolates detected as Brucella with strong Ct values lanes 34 and 14 are for DNA from Brucella-like isolates with weak Ct values lanes 15 and 19 are for B suis positive control DNA lane 16 is for RB 51positive control DNA lane 17 is for Rev 1 positive control DNA and lane 18 is for PCR grade water (negative control)
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
1 8 15 1 8 15
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
100 bp 100 bp
Panel 1 Panel 2
Bruce 06 (134bP)
Bruce 11 (63bP)
Bruce 42 (125 bP)
Bruce 45 (18bP)
Bruce 08 (18bP)
Bruce 12 (15bP)
Bruce 43 (24bP)
Bruce 55 (40bP)
Bruce 04 (8bP)
Bruce 09 (8bP)
Bruce 18 (8bP)
Bruce 21 (8bP)
Bruce 07 (8bP)
Bruce 16 (8bP)
Bruce 19 (6bP)
Bruce 30 (8bP)
40bp
40bp
40bp
40bp
40bp
40bp
40bp
40bp
Figure 5 MLVA amplification pattern of isolates in this study and a B suis as control Lanes 1 8 and 15 show DNA marker lane 2 in eachgel shows pattern for B suis and lanes 3ndash7 and 9ndash14 show the amplification pattern of isolates 1ndash11 (G4101 S40602 S3702 S0303 S40307S40601 G8614 S40312 S4039 S0908 and S0210)
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Virolog y
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Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
Table2Brucellasppiso
latesrecovered
from
cattlemilk
samples
inno
rthern
andeaste
rnUgand
a(2011-2
012)
andtheirM
LVA16
profi
lecomparedto
thatof
somekn
ownBrucellastrains
from
BrucellaMLV
Adatabase
andstr
ains
conformingto
Babortusformer
biovar
7
SampleIDstrains
Year
Region
Host
Specim
enNum
bero
frepeatsforthe
polymorph
icpanel1
andpanel2
Bruceloci
Species
Biovar
Genotype
Panel1
Panel2A
Panel2B
68
1112
4243
4555
1819
214
79
1630
G4101
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40602
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS3702
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0303
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40307
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40601
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mG8614
2011
Gulu
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40312
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS40309
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0908
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mS0210
2012
Soroti
Cattle
Milk
24
212
32
33
59
64
29
84
Babortus
ND
UGBa
-mlowast
REF1330
mdashUSA
Swine
mdash2
36
104
15
24
389
66
55
3B
suis
Biovar
133
(MLV
A-11)
a BCC
N87-92
1997
USA
Hum
anmdash
24
212
42
33
936
72
43
35
Bmelitensis
Biovar
1126(M
LVA-
11)
a REF
544
1942
England
Bovine
mdash4
54
122
23
35
428
35
34
5B
abortus
Biovar
183
(MLV
A-11)
a REF
86859
1959
England
Bovine
mdash4
54
122
13
36
428
34
33
5B
abortus
Biovar
280
(MLV
A-11)
a REF
Tulya
1958
Ugand
aHum
anmdash
35
411
22
33
840
86
53
115
Babortus
Biovar
364
(MLV
A-11)
a REF
292
1961
England
Bovine
mdash4
54
122
23
26
428
34
33
5B
abortus
Biovar
478
(MLV
A-11)
a REF
B3196
1959
England
Bovine
mdash3
53
122
22
37
428
67
33
3B
abortus
Biovar
567
(MLV
A-11)
a REF
870
1959
Africa
Bovine
mdash3
53
122
23
37
428
36
33
3B
abortus
Biovar
666
(MLV
A-11)
a REF
C68
1958
England
Bovine
mdash3
53
122
22
37
428
66
33
3B
abortus
Biovar
967
(MLV
A-11)
a BCC
NV
1(S19)
1943
USA
Bovine
mdash4
54
122
23
36
428
35
33
5B
abortus
Biovar
182
(MLV
A-11)
a BCC
NV
5(RB5
1)mdash
USA
Bovine
mdash4
54
122
33
36
428
37
33
5B
abortus
Biovar
179
(MLV
A-11)
a 13
2006-2007
Egypt
Cattle
Spleen
45
412
23
33
6mdash
83
73
35
Babortus
mdashmdash
a 42002ndash2007
Egypt
Cattle
mdash4
54
122
33
36
mdash8
39
33
5B
abortus
mdashmdash
a 6-K
EBa1
2009
Kenya
Cattle
Abortio
nmaterial
35
411
22
33
740
86
63
116
Babortus
Biovar
334
(MLV
A-8)
a 11-K
EBa2
2009
Kenya
Cattle
Vaginald
ischarge
35
411
22
33
740
86
53
125
Babortus
Biovar
334
(MLV
A-8)
a BCC
N93-26
1993
Sudan
Dromedary
mdash3
54
112
23
36
408
68
37
7B
abortus
Biovar
363
(MLV
A-11)
b 07-994-2411
1963
Kenya
Bovine
mdash2
42
123
23
35
226
52
67
5B
abortus
ND
ND
b 03-4923-239
2003
Turkey
Bovine
mdash4
53
122
23
16
218
67
63
3B
abortus
ND
ND
b 99-9971-135
1988
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
b 99-9971-159
1993
Mon
golia
Bovine
mdash4
55
122
22
26
218
56
43
3B
abortus
ND
ND
a Strains
downloadedfro
mBrucellaMLV
Adatabase
February
2014http
mlvau
-psudfrlowast
thereference
strainused
(from
Bruce-ladd
erkit)
NDnot
desig
nated
b strains
conformingto
Babortusformer
biovar
7
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
isolates were monomorphic at molecular analysis whichcould be due to the isolates being from a small geographicalregion of approximately 15 Km radius per region (data notshown here) and having been collected in a short timeframe making it possible for all the isolates to be from acommon source as animals mix in the grazing grounds Thegenetic monomorphism observed is partly congruent withthat observed in five B melitensis biovar 1 isolates obtainedfrom bovine milk in central Kenya by Muendo et al [9]although their finding was in a different Brucella species Ourresults are further supported by findings by Garin-Bastujiet al [31] who found similar monomorphism in isolates inMongolia isolated 5 years apart in the same region Thegenetic monomorphism exhibited at the minisatellite andmicrosatellite loci that are otherwise polymorphic even inhighly genetic homogenous species like Brucella suggests thatthere is one or very few circulating strains of Brucella in thisregion of Uganda attesting to the regional predominance ofBrucella biovars and strains
The closest known strain for the 11 UG Ba-m isolates wasa B abortus strain 07-994-2411 isolated from cattle in theneighboring Kenya in 1963 This strain has no biovar desig-nation but was formerly known as B abortus biovar 7 beforebiovar 7 was suspended from the Brucella nomenclature(International Committee on Systematic Bacteriology 1988)B abortus biovar 7 was suspended from Brucella nomencla-ture because the reference strain (6375) was thought to bea mixture of B abortus biovars 3 and 5 A recent study byGarin-Bastuji et al [31] proposed the reintroduction of Babortus biovar 7 in the approved list of bacterial names havingidentified B abortus strains from Turkey Mongolia andKenya that perfectly matched the former B abortus biovar7 characteristics B abortus biovar 7 can be differentiatedfrom other B abortus biovars by its ability to agglutinatewith anti-A and anti-B monospecific sera B abortus biovar7 has smooth colonies does not require carbon dioxide forgrowth produces hydrogen sulphide is oxidase and ureasepositive does not agglutinate with monospecific anti-sera Rgrows in the presence of dyes thionin and basic fuchsin ata concentration of 20120583gmL and is lysed by phages Tbilisi(Tb) Weybridge (Wb) Izatnagar 1 (LZ
1) and RC
A genetic difference in the UG Ba-m isolates at 5 loci outof 16 polymorphic loci examined compared to the knownclosest related strain (07-994-2411) from Kenya in a periodof half a century could be a result of mutations and provesthe ability of MLVA to differentiate strains from differentlocalities a finding congruent with that of Verger et al [2]The observed genetic similarity between the Kenyan strainand the 11 UG Ba-m isolates compared to other isolates fromdistant places could be due to the cross-border transmissionof Brucella in cattle that could have been facilitated by cattlerustling across the pastoral Karamoja subregion of Ugandaand Kenya over the past years
5 Conclusions
In conclusion our findings suggest B abortus without biovardesignation (atypical B abortus) as a cause of brucellosis
in cattle in northern and eastern Uganda The Ugandanisolates exhibited a single MLVA-16 pattern and show in turnhigh levels of genetic variation when compared with otherAfrican strains highlighting the usefulness of MLVA as anepidemiological tool for investigation of Brucella infectionsFurthermore the ability of a diphasic PCR protocol involvingthe triplex real-time PCR by Probert et al [23] and theBruce-ladder multiplex PCR by Lopez-Goni et al [16] todetect B abortus could be used to replace the procedureof identifying the former B abortus biovar 7 using theconventional biotyping methods
Ethical Approval
This study entailed collection of milk samples from farmersrsquocattle Ethical clearancewas obtained from the Ethical ReviewCommittee of the College of Veterinary Medicine AnimalResources and Biosecurity Makerere UniversityThe farmerswere informed of the study and their verbal consent wassought prior to commencement of data collection
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was conducted in part using funds from theSwedish International Development Cooperation Agency(Sida) Carnegie Corporation of New York Makerere Uni-versity and the SLU Global Food Security Research andEducation Program 2010ndash2013 A Swedish Government Ini-tiative Ministry of Foreign Affairs Sweden The authors aregrateful for the support and cooperation offered by the fieldveterinary staff and farmers inGulu and Soroti during samplecollectionThey thank Professors Ignacio Moriyon and AmiaZuniga Ripa of Instituto de Salud Tropical Universidad deNavarra Spain for their guidance and for providing part ofthe reagents used in this study
References
[1] J Godfroid H C Scholz T Barbier et al ldquoBrucellosis at theanimalecosystemhuman interface at the beginning of the 21stcenturyrdquo Preventive VeterinaryMedicine vol 102 no 2 pp 118ndash131 2011
[2] J-M Verger F Grimont P A D Grimont and M GrayonldquoBrucella a monospecific genus as shown by deoxyribonucleicacid hybridizationrdquo International Journal of Systematic Bacteri-ology vol 35 no 3 pp 292ndash295 1985
[3] G Pappas ldquoThe changing Brucella ecology novel reservoirsnew threatsrdquo International Journal of Antimicrobial Agents vol36 no 1 pp S8ndashS11 2010
[4] D Bruce ldquoNote on the discovery of a micro-organism in Maltafeverrdquo Practitioner vol 39 pp 161ndash170 1887
[5] M P Franco M Mulder R H Gilman and H L SmitsldquoHuman brucellosisrdquo The Lancet Infectious Diseases vol 7 no12 pp 775ndash786 2007
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 9
[6] H C Scholz K Nockler C G Llner et al ldquoBrucella inopinatasp nov isolated from a breast implant infectionrdquo InternationalJournal of Systematic and EvolutionaryMicrobiology vol 60 no4 pp 801ndash808 2010
[7] J J McDermott and S M Arimi ldquoBrucellosis in sub-SaharanAfrica epidemiology control and impactrdquo Veterinary Microbi-ology vol 90 no 1ndash4 pp 111ndash134 2002
[8] World Organisation for Animal Health and OIE ldquoBovinebrucellosisrdquo in OIE Terrestrial Manual chapter 243 2009
[9] E N Muendo P M Mbatha J Macharia et al ldquoInfection ofcattle in Kenya with Brucella abortus biovar 3 and Brucellamelitensis biovar 1 genotypesrdquo Tropical Animal Health andProduction vol 44 no 1 pp 17ndash20 2012
[10] D R Ewalt J B Payeur J C Rhyan and P L Geer ldquoBrucellasuis biovar 1 in naturally infected cattle a bacteriological sero-logical and histological studyrdquo Journal of Veterinary DiagnosticInvestigation vol 9 no 4 pp 417ndash420 1997
[11] G G Schurig N Sriranganathan andM J Corbel ldquoBrucellosisvaccines past present and futurerdquoVeterinaryMicrobiology vol90 no 1ndash4 pp 479ndash496 2002
[12] I Moriyon M J Grillo D Monreal et al ldquoRough vaccinesin animal brucellosis structural and genetic basis and presentstatusrdquo Veterinary Research vol 35 no 1 pp 1ndash38 2004
[13] S Valdezate A Navarro P Villalon G Carrasco and JA Saez-Nieto ldquoEpidemiological and phylogenetic analysisof Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing hypervariableoctameric oligonucleotide fingerprinting and rpoB typingrdquoJournal of Clinical Microbiology vol 48 no 8 pp 2734ndash27402010
[14] M E Meyer and W J B Morgan ldquoDesignation of neotypestrains and of biotype reference strains for species of the genusBrucella Meyer and Shawrdquo International Journal of SystematicBacteriology vol 23 no 2 pp 135ndash141 1973
[15] A M S Menshawy M Perez-Sancho T Garcia-Seco et alldquoAssessment of genetic diversity of zoonotic Brucella sppRecovered from livestock in Egypt using multiple locus VNTRanalysisrdquo BioMed Research International vol 2014 Article ID353876 7 pages 2014
[16] I Lopez-Goni D Garcıa-Yoldi C M Marın et al ldquoEvaluationof a multiplex PCR assay (Bruce-ladder) for molecular typingof all Brucella species including the vaccine strainsrdquo Journal ofClinical Microbiology vol 46 no 10 pp 3484ndash3487 2008
[17] P le Fleche I Jacques M Grayon et al ldquoEvaluation andselection of tandem repeat loci for a Brucella MLVA typingassayrdquo BMCMicrobiology vol 6 article 9 2006
[18] G Pappas P Papadimitriou N Akritidis L Christou and E VTsianos ldquoThe new globalmap of human brucellosisrdquoTheLancetInfectious Diseases vol 6 no 2 pp 91ndash99 2006
[19] R D Mugizi S Boqvist and G W Nasinyama ldquoPrevalence ofand factors associated with Brucella sero-positivity in cattle inurban and peri-urban farming systems in eastern and northernUgandardquo Journal of Veterinary and Medical Science In press
[20] K Makita E M Fevre C Waiswa et al ldquoHuman Brucellosis inurban and peri-urban areas of Kampala Ugandardquo Annals of theNew York Academy of Sciences vol 1149 pp 309ndash311 2008
[21] K Makita E M Fevre C Waiswa M C Eisler and S CWelburn ldquoHow human brucellosis incidence in urban Kampalacan be reduced most efficiently A stochastic risk assessment ofinformally-marketed milkrdquo PLoS ONE vol 5 no 12 Article IDe14188 2010
[22] M J De Miguel C M Marın P M Munoz L Dieste M JGrillo and J M Blasco ldquoDevelopment of a selective culturemedium for primary isolation of the main Brucella SpeciesrdquoJournal of Clinical Microbiology vol 49 no 4 pp 1458ndash14632011
[23] W S Probert K N Schrader N Y Khuong S L Bystrom andM H Graves ldquoReal-time multiplex PCR assay for detection ofBrucella spp B abortus and B melitensisrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1290ndash1293 2004
[24] G Borriello S Peletto M G Lucibelli P L Acutis DErcolini andG Galiero ldquoLink between geographical origin andoccurrence of Brucella abortus biovars in cow and water buffaloherdsrdquo Applied and Environmental Microbiology vol 79 no 3pp 1039ndash1043 2013
[25] S Al Dahouk P L Fleche K Nockler et al ldquoEvaluationof Brucella MLVA typing for human brucellosisrdquo Journal ofMicrobiological Methods vol 69 no 1 pp 137ndash145 2007
[26] A M Whatmore S J Shankster L L Perrett et al ldquoIden-tification and characterization of variable-number tandem-repeat markers for typing of Brucella spprdquo Journal of ClinicalMicrobiology vol 44 no 6 pp 1982ndash1993 2006
[27] M Tittarelli M di Ventura F de Massis et al ldquoThe persistenceof Brucella melitensis in experimentally infected ewes throughthree reproductive cyclesrdquo Journal of Veterinary Medicine SeriesB Infectious Diseases and Veterinary Public Health vol 52 no9 pp 403ndash409 2005
[28] R Capparelli M Parlato M Iannaccone et al ldquoHeterogeneousshedding of Brucella abortus inmilk and its effect on the controlof animal brucellosisrdquo Journal of Applied Microbiology vol 106no 6 pp 2041ndash2047 2009
[29] M T Musa K L Jahans and M E Fadalla ldquoBrucella biovarsisolated from nomadic cattle in the southern Darfur Provinceof Western Sudanrdquo Journal of Comparative Pathology vol 102no 1 pp 49ndash54 1990
[30] R Redkar S Rose B Bricker and V Delvecchio ldquoReal-timedetection of Brucella abortus Brucella melitensis and Brucellasuisrdquo Molecular and Cellular Probes vol 15 no 1 pp 43ndash522001
[31] B Garin-Bastuji V Mick G Le Carrou et al ldquoExamination oftaxonomic uncertainties surrounding Brucella abortus bv 7 byphenotypic and molecular approachesrdquo Applied and Environ-mental Microbiology vol 80 no 5 pp 1570ndash1579 2014
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology