Research Article Isolation and Molecular Characterization ... · Research Article Isolation and...

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)


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


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


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)


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


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


[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 Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


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


Cycle5 10 15 20 25 30 35 40 45

Nor

m fl

uoro

035

030

025

020

015

010

005

000

Threshold





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



3 Results





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


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




4 Discussion









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


B suis B melitensis

1682bp 1071bp587bp 587bp

1682bp1071bp587bp 587bp

1682bp1071bp

272bp


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




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




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cycle5 10 15 20 25 30 35 40 45

Nor

m fl

uoro

035

030

025

020

015

010

005

000

Threshold





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



3 Results





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


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




4 Discussion









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


B suis B melitensis

1682bp 1071bp587bp 587bp

1682bp1071bp587bp 587bp

1682bp1071bp

272bp


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




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




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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


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




4 Discussion









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


B suis B melitensis

1682bp 1071bp587bp 587bp

1682bp1071bp587bp 587bp

1682bp1071bp

272bp


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




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




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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




4 Discussion









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


B suis B melitensis

1682bp 1071bp587bp 587bp

1682bp1071bp587bp 587bp

1682bp1071bp

272bp


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




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




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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


B suis B melitensis

1682bp 1071bp587bp 587bp

1682bp1071bp587bp 587bp

1682bp1071bp

272bp


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




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




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



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




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




1) and RC


5 Conclusions



Ethical Approval




Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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