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Shahzad Ali & Qurban Ali & Falk Melzer & Iahtasham Khan &Shamim Akhter & Heinrich Neubauer & Syed M. Jamal

Accepted: 2 July 2013 /Published online: 19 July 2013# Springer Science+Business Media Dordrecht 2013

Abstract Brucellosis is endemic in bovines in Pakistan. TheBrucella species and biovars involved, however, are un-known. The objectives of the present study were to isolateand characterize brucellae from seropositive milk samples,aborted fetuses, and vaginal swabs of cattle and buffaloeswhich had recently aborted. The seropositive milk samples,aborted fetuses, and vaginal swabs of cattle and buffaloeswere collected from the Potohar Plateau, Pakistan. Isolationof brucellae was done on modified Farrells serum dextrose

agar. Isolates were characterized by conventional biotypingmethods, while molecular typing was done by genus(B4/B5) and species-specific (Brucella abortus, Brucellamelitensis, Brucella ovis, and Brucella suis) polymerasechain reaction (PCR). A total of 30 isolates were recoveredfrom milk (n=5), aborted fetuses (n=13), and vaginal swabs(n=12). Most isolates were from cattle (56.7 %). All of themwere identified as B. abortus biovar 1 based on conventionalbiotyping methods and genus and species-specific PCR. Thispreliminary study provides the first report on the prevalenceof B. abortus biovar 1 in cattle and buffaloes in Pakistan.

Keywords Brucella abortus . Pakistan . Biochemical tests .

PCR

Introduction

Brucellosis is a widespread zoonotic disease affecting bothhumans and animals (Shahaza et al. 2009). Brucella abortusis usually considered as the causative agent of bovine bru-cellosis, but Brucella melitensis may also cause disease,especially in areas where bovines have close contact withsheep and goats (Ahmed et al. 2010). Apart from these twospecies, Brucella suis has also been reported in bovines (Taeet al. 2012).

Pakistan is bordered by Afghanistan and Iran in the west,India in the east, and China in the far northeast. In most of theneighboring countries of Pakistan, brucellosis outbreakshave been reported (Abubakar et al. 2012).

According to the World Health Organization, brucellosisis considered a neglected disease, particularly in Pakistan(World Health Organization (WHO) 2012). In Pakistan,23.9 % of the population lives below the poverty line. Cattleor buffaloes are mainly kept by the poor rural farmers as asubsistence farming (TheWorld Bank 2008). Brucellosis has anegative impact on animal health, pregnancy, and milk

S. Ali : S. AkhterPir Mehr Ali Shah Arid Agriculture University, Rawalpindi 46300,Pakistan

S. Akhtere-mail: sashraf1993@gmail.com

S. Ali : F. Melzer : I. Khan :H. NeubauerFriedrich-Loeffler-Institut, Bundesforschungsinstitut frTiergesundheit, Naumburger Strae 96a, 07743 Jena, Germany

F. Melzere-mail: Falk.Melzer@fli.bund.de

I. Khane-mail: drkhan_uaf@yahoo.com

H. Neubauere-mail: Heinrich.Neubauer@fli.bund.de

S. Ali (*) :Q. Ali : S. M. JamalNational Veterinary Laboratory, Park Road, Islamabad, Pakistane-mail: shahzaduaar772@yahoo.com

Q. Alie-mail: drqurban@yahoo.com

S. M. Jamale-mail: jamal115@yahoo.com

I. KhanUniversity of Veterinary and Animal Sciences, Lahore, Pakistan

S. M. JamalMinistry of National Food Security and Research, Islamabad,Pakistan

Trop Anim Health Prod (2014) 46:7378DOI 10.1007/s11250-013-0448-6

Isolation and identification of bovine Brucella isolatesfrom Pakistan by biochemical tests and PCR

production, and its zoonotic implications cannot be ignoreddue to lack of awareness and poor literacy rate of the dairyfarmers.

Pakistan is an agricultural country, and livestock sector isthe backbone of agriculture as it has 55.1 % stake in agricul-ture (Economic Survey 201112). Livestock is subsistencesector dominated by small holders to meet their needs ofmilk, food and cash income on daily basis. Livestock isconsidered as a more secure source of income in rural areas.The livestock population in Pakistan is assessed at 35.6million cattle, 31.7 million buffaloes, 28.1 million sheep,61.5 million goats, 1.0 million camels, 0.4 million horses,0.2 million mules and 4.7 million donkeys (EconomicSurvey 20112012). Apart from other diseases, bovine bru-cellosis is the major disease of livestock in Pakistan. Theprevalence of brucellosis in the country has been ascertainedusing only sero-surveillance (Hamidullah et al. 2009;Mukhtar 2010; Abubakar et al. 2010, 2012). However, noreport on isolation and characterization of prevalent Brucellaspecies and biovars based on biochemical tests and polymer-ase chain reaction (PCR) is available from Pakistan.Serological data may be misleading due to cross-reactivitybetween Brucella and other Gram-negative bacteria.Furthermore, serological methods cannot differentiate be-tween vaccinated and infected animals. Thus, the isolationof bacteria is considered as a gold standard for the diagnosisof brucellosis (Yu and Nielsen 2010). Moreover, molecularmethods like PCR assay enable safe identification of thebacteria at species level (Huber et al. 2009).

The aim of this study was to isolate brucellae from milk,aborted fetuses, and vaginal swabs of cattle and buffaloesand to characterize these isolates using the genus (B4/B5)and species-specific (AMOS) PCR, respectively, and to

ascertain biovar of Brucella species, employing biochemicaltests, growth in the presence of certain dyes, and agglutina-tion with specific sera.

Materials and methods

Sample collection

A total of 2,330 milk (1,168 cattle and 1,162 buffaloes)samples were randomly collected from different ecologicalregions of three districts located on the Potohar Plateau ofPakistan. These milk samples were initially screened usingBrucella milk ring test (MRT). Positive milk samples werecultured. In addition, 49 aborted fetuses and 95 vaginalswabs from cattle and buffaloes with a history of recentabortion were also investigated (Table 1).

Isolation of bacteria

Isolation of Brucella was conducted on modified Farrellsserum dextrose agar according to standard procedures(Farrell and Robinson 1972; Alton et al. 1988). ModifiedFarrells serum dextrose agar with 5 % horse serum, 1 %dextrose, and the following antibiotics (added to 1-l medi-um): cycloheximide (100 mg), bacitracin (25,000 IU), poly-myxin B sulfate (5,000 IU), vancomycin (20 mg), nalidixicacid (5 mg), and nystatin (100,000 IU) were used for primaryselective isolation of brucellae. Plates were inoculated withsample material and incubated aerobically and in the pres-ence of 510 % carbon dioxide at 37 C. These plates wereexamined 37 days post-inoculation for bacterial growth.

Table 1 List of samples from milk, aborted fetuses, and vaginal swabs collected from different sampling sites of the Potohar Plateau, Pakistan

Sampling sites Milka Aborted fetuses Vaginal swabs

Cattle Buffalo Cattle Buffalo Cattle Buffalo

ICTb

Chak Shahzad 30 12 8 7 28 11

Rawat 10 8 15 13 17 12

Rawalpindi

Kallar 9 7 4 1 5 8

Chountra 5 10 0 0 0 0

Kahuta 5 4 0 0 7 3

Attock

Kherimurat 8 13 1 0 3 1

Attock 10 6 0 0 0 0

Ahmadal 7 12 0 0 0 0

a Positive milk samples in MRT are given in table in front of each sampling siteb Islamabad Capital Territory

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Biovar typing and Brucella genus-specific (B4/B5)and species-specific AMOS PCR

Suspected colonies were subcultured for purity on serumdextrose agar. Identification of these isolates was doneaccording to standard procedures (Alton et al. 1988). Theisolates were initially examined for Gram and modifiedZiehlNeelsen (MZN) staining. Subsequent biochemicaltests for oxidase, catalase, urease production, hydrogen sul-fide production, carbon dioxide requirement, growth on me-dia containing basic fuchsin and thionin (20 g/ml), andagglutination by monospecific antisera (A, M, R; Anses,Paris) were carried out. DNA was extracted from coloniesusing the High Pure PCR Template Preparation Kit (RocheDiagnostics, Mannheim, Germany) according to manufac-turers instructions. Purity and concentrations of DNA wereassessed using the Nano-Drop ND-1000 UVVisSpectrophotometer (Nano-Drop Technologies, Wilmington,DE, USA), and preparations were stored at 20 C forsubsequent analysis.

The extracted DNA preparations were screened withgenus-specific Brucella PCR using B4/B5 primers(Table 2). Briefly, PCR was done in a 25 l reaction mixcontaining 18.3 l HPLC water, 2.5 l PCR puffer (10),1 l deoxyribonucleotide triphosphate (dNTP) (10 mM),1 l each primer (10 pmol/l), 0.2 l (5 U/l) Taq polymer-ase, and 1 l of DNA template. Amplification was done withinitial denaturation for 5 min at 93 C, followed by 35 cyclesfor denaturation for 1 min at 90 C, annealing for 1 min at60 C, elongation for 1 min at 72 C, and final elongation for5 min at 72 C. Positive samples were subjected to AMOS

PCR for species identification (Bricker and Halling 1994).Primers are given in Table 3. AMOS PCR was performed in25 l reaction volume having 19.3 l of HPLC water, 2.5 lof PCR puffer (10), 1 l dNTP mix (10 mM), 1 l primermix (10 pmol/l), 0.2 l Taq polymerase (5 U/l), and 1 lof DNA template. Amplification was carried out with initialdenaturation for 5 min at 95 C, followed by 30 cycles(denaturation for 1 min at 95 C, annealing for 2 min at58 C, elongation for 2 min at 72 C, and final elongationfor minutes at 72 C). The PCR product was run on a 1.5 %agarose gel along with DNA ladder for 90 min at 105 V,stained with ethidium bromide (1 mg/ml) and visualizedunder UV light using a gel documentation system (Syngene,UK).

Results

Out of 2,330 milk samples, 156 (6.7 %) were found positivein MRT. Among these MRT positive samples, 84 (53.8 %)were from cattle, and 72 (46.2 %) were from buffaloes.Thirty Brucella isolates were recovered from these samples,including 5 from milk, 13 from aborted fetuses, and 12 fromvaginal swabs (Table 4).

All the 30 isolates were Gram-negative andMZN-positivecoccobacilli. They were positive in the Brucella genus-specific (B4/B5) PCR and identified as B. abortus byAMOS PCR (Fig. 1). Biochemical tests showed that theseisolates were positive for oxidase and catalase production,but variation was seen in the strength of reaction; someisolates showed strong reaction, and others, weak reaction.

Table 2 PCR type along with primers type, sequence (5 to 3), target gene, and product length (in base pair) used for Brucella genus-specific (B4/B5) PCR

PCR type Primers type Sequence (5 to 3) Target gene Product length (bp)

Brucella Forward TGG CTC GGT TGC CAATAT CAA bcsp31 223

genus Reverse CGC GCT TGC CTT TCA GGT CTG

Table 3 List of primers along with sequence (5 to 3), target gene, and product length (in base pair) used for AMOS PCR

PCR type Primer type Sequence (5 to 3) Target gene Product length (bp)

AMOS BA (F) GAC GAA CGG AAT TTT TCC AAT CCC IS711 498

BA (R) TGC CGATCA CTT AAG GGC CTT CAT

BM (F) AAATCG CGT CCT TGC TGG TCT GA IS711 731

BM (R) TGC CGATCA CTT AAG GGC CTT CAT

BO (F) CGG GTT CTG GCA CCATCG TCG IS711 976

BO (R) TGC CGATCA CTT AAG GGC CTT CAT

BS (F) GCG CGG TTT TCT GAA GGT TCA GG IS711 285

BS (R) TGC CGATCA CTT AAG GGC CTT CAT

BA B. abortus, BM B. melitensis, BO Brucella ovis, BS B. suis, F forward, R reverse

Trop Anim Health Prod (2014) 46:7378 75

The isolates were positive for urease and hydrogen sulfideproduction. The isolates grew on media containing basicfuchsin but failed to grow on thionin media. Most colonies(83.3 %) required carbon dioxide for growth, while fewisolates (16.7 %) grew aerobically. All the isolates showedagglutination with Brucella-monospecific antiserum A,while no agglutination was observed with antisera M and R(Table 4). On the basis of these tests, the isolates wereconfirmed to be B. abortus biovar 1.

Discussion

Seroprevalence studies in animals show that brucellosis isendemic in Pakistan (Hamidullah et al. 2009; Munir et al.2011; Shafee et al. 2011). However, the Brucella species andtheir biovars endemic in Pakistan are unknown. The presentstudy provides the first evidence for the presence of B.abortus biovar 1 in cattle and buffaloes on the basis ofspecies-specific PCR and biochemical tests.

Isolation of Brucella is cumbersome, labor-intensive, andtime-consuming but can be achieved even with conventionaltechniques using the candle jar method to obtainmicroaerophilic conditions (Shareef 2006). Hence, up toour knowledge, no Pakistani investigation was made in thepast to culture brucellae from animals. Applying the bestpossible culture conditions including 510 % CO2 atmo-sphere, these authors cultured 43.3 % of brucellae fromaborted fetuses and 40 % from vaginal excretions/swabs.These findings are in accordance to the previous reports fromSri Lanka and Kenya (Priyantha 2011; Muendo et al. 2012).Ocholi et al. (2004) isolated brucellae from aborted fetuses,hygroma fluids, milk, and vaginal swabs obtained fromaborting cattle, sheep, goats, pigs, and horses in Nigeria.They reported a rate of isolation from milk to be 7.2 %.

Table 4 Source and characterization of B. abortus isolates of cattle and buffaloes from the Potohar Plateau, Pakistan

Isolate number Animal species Sample type District Oxidase Catalase Urease H2S CO2 Growthon media

Agglutination Species/biovar

THa BFb A M R

1 B AF Attock +c + + + + d + + B. abortus/1

2 C AF Attock + + + + + + + B. abortus/1

34 C AF Rawalpindi + + + + + + + B. abortus/1

58 C VS ICT + + + + + + + B. abortus/1

910 B VS ICT + + + + + + + B. abortus/1

11 C VS ICT + + + + + + + B. abortus/1

1218 B AF ICT + + + + + + + B. abortus/1

1920 C M ICT ++e ++ + ++ + + B. abortus/1

2122 B VS ICT + + + + + + + B. abortus/1

23 C VS ICT + + + + + + + B. abortus/1

2426 C AF ICT + + + + + + + B. abortus/1

27 C M ICT ++ ++ + ++ + + B. abortus/1

28 B AF ICT + + + + + + + B. abortus/1

2930 C M ICT ++ ++ + ++ + + B. abortus/1

B buffalo, C cattle, AF aborted fetus, VS vaginal swab, M milka Thionin (20 g/ml)b Basic fuchsin (20 g/ml)cWeak positive reactiond Negative reactione Strong positive reaction

Fig. 1 AMOS PCR for B. abortus. Lane M 100-bp marker; lanes 115B. abortus DNA in samples from Potohar Plateau, Pakistan; lane 16positive control B. melitensis 16 M (10 ng/l); lane 17 B. abortusBA544 (10 ng/l); lane 18 B. suis (10 ng/l); lane 19 non-templatecontrol

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The recovery of B. abortus from milk samples in the presentstudy was low, i.e., 3.2 %.

Shedding of Brucella in the milk of infected animals is animportant source of transmission of disease to humans if theraw milk is consumed. Hence, pasteurized or boiled milk isrecommended to be used. Further, dairy product preparedfrom Brucella-contaminated milk have also been implicatedfor human brucellosis (Tantillo et al. 2003). The isolation ofBrucella from milk samples may be improved if more thanone culture medium is used.

All isolates showed a phenotype typically for B. abortusbiovar 1 except requirements of carbon dioxide. However,variation in carbon dioxide requirement has been reportedpreviously (Ocholi et al. 2004). B. abortus isolates pos-itive for catalase, oxidase, and urease were recoveredfrom the semen of breeding bulls in the neighboringcountry India, but among these isolates, some were neg-ative for hydrogen sulfide production in contrast to ourisolates (Nagalingam et al. 2012). All the isolates of B.abortus showed agglutination reaction with monospecificantiserum A. None of them agglutinated against mono-specific antisera M and R. These are characteristics ofsmooth strains of Brucella.

Although, B. abortus is identified as the major cause ofbrucellosis in bovines in neighboring countries, i.e., Indiaand Iran (Kumar and Basanti 2004; Doosti and Dehkordi2011) and worldwide (Adesiyun et al. 2011; Priyantha 2011;Martinez-Herrera et al. 2012), B. melitensis and B. suis havealso been reported from cattle (Tae et al. 2012; World HealthOrganization (WHO) 2012). This may be due to rearing ofdifferent species of animals together.

B. abortus biovar 1 is the most prevalent biovar in coun-tries where bovine brucellosis is endemic. Characterizationof B. abortus from India revealed the presence of B. abortusbiovars 1, 2, and 4 in India (Kanani 2007). Results of thebiochemical tests and growth of Brucella in the presence ofdyes revealed that all the isolates belonged to B. abortusbiovar 1 in the present study. To the best of our knowledge,there is no report of isolation of B. abortus so far fromPakistan. In this study, B. abortus was isolated frombovines and characterized to be biovar 1. This appearsto be the first report of isolation of B. abortus fromPakistan. Because the present study is restricted to lim-ited geographical area, study in other areas is needed toexplore the presence of B. abortus in bovine populationin other parts of the country.

Acknowledgments SA is a recipient of Indigenous PhD Fellowshipand International Research Support Initiative Program from the HigherEducation Commission, Pakistan, at the Friedrich-Loeffler-Institut,Jena, Germany.

Conflict of interest The authors declare that they have no conflict ofinterest.

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Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCRAbstractIntroductionMaterials and methodsSample collectionIsolation of bacteriaBiovar typing and Brucella genus-specific (B4/B5) and species-specific AMOS PCR

ResultsDiscussionReferences