Detection ofMycobacteriumbovis in Organs of Slaughtered Cattle by DNA-Based Polymerase Chain Reaction and Ziehl-Neelsen Techniques in Bauchi State, Nigeria

Research ArticleDetection of Mycobacterium bovis in Organs ofSlaughtered Cattle by DNA-Based Polymerase Chain Reactionand Ziehl-Neelsen Techniques in Bauchi State Nigeria

A S Sarsquoidu1 E C Okolocha1 A A Dzikwi1 J K P Kwaga1 A A Gamawa2

A Usman3 S A Maigari4 and S Ibrahim5

1Department of Veterinary Public Health and Preventive Medicine Ahmadu Bello University PMB 1013Zaria 2222 Kaduna State Nigeria2Area Veterinary Clinic (Kofar Ran) Ministry of Animal Resources and Nomadic Resettlement Bauchi State Nigeria3TB-Laboratory Department of Medicine Faculty of Veterinary Medicine Ahmadu Bello University PMB 1013Zaria 2222 Kaduna State Nigeria4Department of Medicine Faculty of Veterinary Medicine Ahmadu Bello University PMB 1013 Zaria 2222 Kaduna State Nigeria5University of Maiduguri Teaching Hospital PMB 1069 Maiduguri Borno State Nigeria

Correspondence should be addressed to A S Sarsquoidu adamudvm13gmailcom

Received 16 August 2014 Accepted 11 November 2014

Academic Editor Carlos Gonzalez-Rey

Copyright copy 2015 A S Sarsquoidu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Bovine tuberculosis is a chronic infectious and contagious zoonotic disease of domestic animals wild animals and humans Italso poses a public health threat and economic losses This study was aimed at determining the prevalence of bovine tuberculosisin slaughtered cattle based on PM meat inspection Ziehl-Neelsen staining and PCR techniques in Bauchi State Nigeria AProspective study was conducted on 800 cattle slaughtered in the three Zonal abattoirs of Bauchi State Nigeria One hundredand twenty (15) tissues from different organs had suspected bTB lesions at PM Out of the samples examined 35 (292) wereAFB positive by ZN and 10 (83) were confirmed positive for M bovis by PCR with an overall prevalence of 2916 and 833respectively Female had a higher prevalence rate than male cattle at 1666 and 125 by ZN and 500 and 333 by PCRrespectively (119875 gt 005 1205942 = 0218) However there was a statistically significant association (119875 lt 005 1205942 = 7002) betweendetection of bTB and the age of cattle ZN revealed that cattle aged 6 years and above had the highest number of positive bTB cases679 while cattle aged 3ndash5 years had the lowest 1481 PCR technique revealed that the cattle aged 6 and above years also hadthe highest percentage positive M bovis cases of 2284 whereas cattle aged 3ndash5 years had the lowest and the overall prevalencerate of 833 The study found a high infection rate of bTB among cattle and majority of the lesions 542 were from lungs Theprevalence of bTB was higher in Bauchi metropolitan abattoir which supplies larger population of the state with beef

1 Introduction

Bovine tuberculosis (bTB) is a chronic infectious and con-tagious zoonotic disease of domestic animals wild animalsand humans [1] It also occurs in a wide range of mammalianspecies [2] It is characterized by the formation of granu-lomas in tissues especially in the lungs lymph nodes liverintestines and kidney [3] According to the World HealthOrganization report in 2013 tuberculosis still remains amajor health problemwith 9million new cases and 15milliondeaths annually worldwide [4]Themajority of these occur in

the developing nations In Nigeria there have been limitedstudies to determine the prevalencerelationship betweenbovine and human TB especially with the emerging cultureof eating improperly cooked beef and mutton along with thedrinking of unpasteurized fresh milk [5 6]

Bovine tuberculosis is caused by Mycobacterium bovis(bovine tubercle bacillus) which is a member of Mycobacte-rium tuberculosis complex [7 8] The aetiological agents ofmammalian tuberculosis classified as members of theMyco-bacterium tuberculosis complex (MTBC) include Mycobac-terium tuberculosis M bovis M microti M caprae M

Hindawi Publishing CorporationJournal of Veterinary MedicineVolume 2015 Article ID 921868 7 pageshttpdxdoiorg1011552015921868

2 Journal of Veterinary Medicine

africanum M canettii and M pinnipedii Mycobacteriumafricanum consists of a rather heterogeneous group of strainsisolated from humans in Africa [9] The organism may betransmitted by aerosol or droplets of exudates containingthe bacilli It can be transmitted by ingestion of feed andwater contaminated with urine fecal material or exudatesfrom diseased animals that contain the tubercle bacilli Thebovine tubercle bacillus is the cause of the bTB in cattle butstill is often used to denote bovine strains of the tuberclebacillus irrespective of the host In fact the bovine tuberclebacillus has one of the broadest host ranges of all knownpathogens The species has been reported in domesticatedand feral Bovidae Other species in which the disease hasbeen reported include goat sheep pig horse cat dog fennecfox bison buffalo badger wild and feral pig antelopecamel and man and nonhuman primates among others [10]Cattle movements particularly those from areas where bTBis reported are the best predictor of disease occurrence [11]

Bovine TB in cattle is initially asymptomatic and priorto testing farmers usually remain unaware of the outbreakduring this stage It can take as long as a few years before anysigns can be observed As the infection progresses in cattlebovine tuberculosis causes a slight fever and roughened haircoat and the lymph nodes usually swell or have abscesses andshow signs of dyspnoea with an associated cough The cowwill appear weak show signs of laboured breathing and havean increased breathing rate A loss of appetite will be noticedand the decrease in weight may be so significant that the cowactually becomes emaciated Tuberculosis (TB) in humansis characterized by prolonged coughing general body weak-ness wasting and sweating at night with slight fever [12]

In UK badgers have been culled since 1971 when atuberculous dead badger was found in an infected farmBovine TB control has proved difficult to achieve wherethere is a persistent reservoir of infection in wildlife [13]Consequently interpreting subsequent trends in incidence ofbTB which are complicated by multiple factors such as cattlemanagement practice changing specificity of tests changesto the cattle meat factory (abattoir) including pollutioncontrolregulations and increase in level of aflatoxins in feeds(known for their immunosuppressive qualities etc) [14 15]

Despite the availability of effective therapy tuberculosisremains a major public health problem worldwide [16 17]Factors such as inadequate health service infrastructuredecreased access to health care and limited human andfinancial resources have prevented adequate implementationof control measures against TB [18]

Recent advances in the treatment of human TB havepermitted effective management of cases on an ambula-tory basis [19] However in many developing countries anirregular drug taking practice and premature termination oftreatmentmainly by self-discharge are themain cause of poorperformance of control programmesThis study was aimed atdetermining the prevalence of bovine tuberculosis in slaugh-tered cattle based on postmortem (PM) meat inspectionZiehl-Neelsen staining (ZN) and polymerase chain reaction(PCR) techniques in abattoirs of Bauchi State Nigeria

2 Materials and Methods

21 Study Area Bauchi State occupies a total land area of49119 Km2 representing about 53 of Nigeriarsquos total landmass and is located between latitudes 9∘31015840 and 12∘31015840 north ofthe equator and longitudes 8∘501015840 and 11∘ east of theGreenwichMeridean [20] The state is bordered by Kano and Jigawato the north Taraba and Plateau to the south Gombe andYobe to the east and Kaduna to the west The state is highlypopulated with cattle mainly owned by Fulani herdsmenThe cattle population is estimated at 1789000 about 13 ofthe Nigerian cattle population of 13900000 [21] The Statehas a human population of 4653066 ranked 11th of the 36states density of 95 km2 (250sqmi) and per capita incomeof $983 [22] Bauchi State has a total of 55 tribal groups inwhich Hausa Fulani and Kanuri are the main tribes Thestudy was carried out in Bauchi Katagum and Misau LocalGovernment Areas (out of the 20 LGAs) each representingthe three senatorial zones as Bauchi South Bauchi Northand Bauchi Central with populations of 493810 295970 and263487 respectively

22 Study Design The three major slaughter houses namelyBauchi Misau and Katagum (Azare) in each of the threesenatorial zones of Bauchi State were selected for the studyfor sampling purposes based on their daily slaughter capacityand socioeconomic impact of the abattoir Accordingly out ofthe 800 head of cattle examined (sample size) a proportionatedistribution was employed given 50 (400) to Bauchi abat-toir and 25 (200) each to Katagum and Misau slaughterhouses This was based on their daily slaughter capacityof 40ndash60 20ndash30 and 20ndash25 head of cattle from Bauchiabattoir Katagum and Misau slaughter houses respectivelyThe selection of the cattle sampled at each of the abattoirs wasstrictly on the basis of the age of the cattle (meant for slaughterand three years above) following then PM observation oftypical granulomatous bTB lesions in any of the visceralorgans before the cattle is considered a case and sample

23 Source of Animals for Slaughter Most of the animalsbrought for slaughter were mainly sourced from MaiduguriChad and Niger

24 Sampling Procedure and Transportation Identification ofcases was based on presence of typical tubercle yellowishgranulomatous and caseous lesions in the lungs lymphnodes kidneys intestines and liver [23] Aging of cattle wascarried out at the abattoir after slaughter as described by[24] using the time of appearance and the degree of wear onthe temporary and permanent teeth Additional data takenincluded sex and breed of each animal sampled

A total of 800 head of slaughtered cattle were examined atpostmortem for TB lesions Using a convenience samplinga total of 400 head of cattle from Bauchi 200 head of cattleeach from Misau and Katagum LGA slaughter houses wereexaminedOne hundred and twenty (120) tissue samples werecollected from cattle with suspected TB lesions into sterilescrew-capped containers (with 9 normal saline solution tokeep tissuesmoist) and transported inside a cooler containing

Journal of Veterinary Medicine 3

ice pack to the Veterinary Public Health Bacterial ZoonosesLaboratory Ahmadu Bello University Zaria and stored atminus20∘C until analysis was carried out Stored samples wereprocessed (crushed and analysed) for microscopic examina-tion (modified Ziehl-Neelsen staining technique) and onlyAFB positive samples were taken to the DNA Labs Kadunaand used for conventional PCR amplification protocols spe-cific forM bovis as described by [25]

3 Microscopic Examination

31 Acid FastnessZiehl-Neelsen Staining Technique Ziehl-Neelsen staining (ldquoHotrdquo ZN technique) was carried out on thetissues stored at minus20∘C after thawing them to room tempera-ture using standard protocol for mycobacterial identificationof [26] to detect acid fast bacilli from granulomatous tissuesamples collected during the meat inspection

Direct smears were prepared from tissues presentingtuberculous lesions using Ziehl-Neelsen stainingacid fasttechnique The collected samples were crushed using a pestlein a clean mortar and homogenized using a stomacher Thecrushed tissues were placed on a new clean and labelledgrease-free glass slide and spread with a sterile pipette tipto make a smear (1 cm times 2 cm) The smeared slides wereair-dried and heat-fixed by passing them gently over theflame (Bunsen burner) with the specimen side up Thisfixed the specimen to the slide and preserved the bacterialmorphology

32 Protocol for Hot Ziehl-Neelsen Direct smears were pre-pared The crushed tissues were placed on a grease-freeglass slide to make a smear (1 cm times 2 cm) The smearswere then stained with carbol fuchsin and allowed to standfor 5 minutes The stained smear was washed with tapwater (to remove excess stain from the slide) The slide wasdecolourized with 3 acid alcohol After decolourization theslide was counterstained with methylene blue for 1 minuteAdditional rinsing was done with water to remove excesscolour and the slide was allowed to air-dry Finally the slidewas viewed in an optical microscope (times100mdashan oil emersionobjective) Results were that acid fast bacilli appeared red Ared straight or slightly curved rod occurring singly or in acluster indicates the presence of tubercle bacilli

33 Genomic DNA Preparation from Tissues The tissuesamples positive byZNwere collected into a 15mLmicrofugetube containing lysis buffer stored at room temperature andtransported on ice pack to DNA Labs Kaduna About 1 g ofthe tissue sample was homogenized with pestle and mortarfor chromosomalDNAextraction using a phenol-chloroformtechnique as described by [25 27] The supernatant wasdiscarded the suspension was used then cooled at 4∘Cneutralizedwith 3 volumes of 01mTris-HCL (PH 74) bufferand centrifuged (5000timesg 5min) to get rid of the tissuemembrane and possible contaminants The pellet was driedand redissolved in 20120583L of 1x Tris-Borate (1times TBE) bufferandDNAwas precipitatedwith ethanol collected by centrifu-gation and dissolved in 50120583L of distilled water 5120583L of the

Table 1 Species-specific primers for Mycobacterium bovis used forthe study

Primerdirection PCR primer sequence Amplicon size

(bp)Forward (51015840CCCGCTGATGCAAGTGCC31015840) 285 bpM bovisReverse (51015840CCCGCACATCCCAACACC31015840)These primer oligonucleotide sequences were used to amplify a fragment ofthe oxyR gene [29]

extracted DNA was run on 10 agarose gel and spectropho-tometer to confirm the presence of DNA The remainingDNA samples were stored at minus20∘C until further use

Table 1 showed the forward and reverse oligonucleotideprimer sequences that were complimentary to the DNA tem-plate extracts at the 31015840 ends in the nucleotide sequences of theMycobacterium bovis genome The primers were synthesizedbased on theG+Ccontents of theMycobacteriumbovis strainsavailable at the gene bank of the Bioneer USA [28]

34 PCR Amplification Protocols Ten (10) microlitres sus-pended DNA was used as a template for PCR amplificationunder standard conditions as described by [29] A com-mercial ldquoHot-Statrdquo PCR Premix (Bioneer USA) a mixtureprepared in a lyophilized format containing Taq DNA poly-merase reaction buffer dNTPs (dATP dGTP dCTP dTTP)and MgCl

2were used All amplification reactions were

performed using a Perkin ElmerThermocycler (Perkin ElmerCetus) programmed for 40 amplification cycles [28 30] Thereaction was performed in a final volume of 50120583L containing10 120583L of DNA template 1x TBE (1x Tris-Borate) reactionbuffer (containing 10mM Tris-HCl (pH 83) 50mM KCl13mMMgCl

2 and 0001 of gelatin) 25U Taq polymerase

02mM of each deoxynucleoside triphosphate and 75 pmolof each primer (Table 1) DNA from M bovis ATCC 19210and BCG Pasteur 27291 and sterile nuclease-free water wereused as positive and negative PCR controls respectivelyAfter an initial denaturation step (at 94∘C for 5min) 40amplification cycles were performed as follows denaturationat 94∘C for 1min annealing at 58∘C for 30 sec and extensionat 72∘C for 30 sec with an increment of 1 sec per cycle for thedenaturation and extension segment A final extension wasperformed at 72∘C for 15min

35 Gel Electrophoresis After amplification 20120583L of thePCR product was loaded with gel loading buffer in a 1agarose gel (Swekem FMC Bioproducts Rockland MaineUSA) containing 05 pugmL ethidium bromide The gel wasalso loaded with the 100 base pair (bp) DNA molecularmarker (ladder) Gel electrophoresis was done at a 5 vcmfor 1 hour Finally after electrophoresis the DNA bands werevisualized (using UV light box or gel imaging system) andphotographed

36 Data Analysis Descriptive statistical tables and barcharts were constructed using Microsoft Excel 2010The datawere also analysed using statistical package for social sciences(SPSS) version 200 Chi-square (1205942) was used to determine


association between variables and M bovis infection Oddsratio (OR) and 95 confidence interval were calculatedto measure the strengths of associations between variablesand bTB (M bovis) Values of 119875 lt 005 were consideredsignificant

4 Results

Of the 800 samples examined at postmortem for bTB lesionsonly 15 (120) tissues from different organs were suspectedto have bTB lesions Out of the 120 tissues thirty-five 2916(35) were positive for bTB using Ziehl-Neelsen stainingtechnique (Figure 2) and gave a prevalence rate of 2916(Tables 3 and 4) Further confirmation using PCR technique(Figure 3) gave an overall prevalence of 833 (10) Theoccurrence of bTB lesions in the organs of slaughteredcattle in Bauchi State showed that the lungs had the highestnumber of bTB suspected tissues 5420 (65120) Figure 1showed a clear photograph of tuberculous lung lesionsobtained from affected cattle at PM Followed by the lymphnodes 2330 (28120) while the heart liver spleen intestinesand mammary glands made up the other 2250 (27120)suspected bTB tissues (Table 2) By ZN microscopic staining2923 (1965) of the lungswere positive for bTB (AFB)while2143 (628) of the lymphnodeswereAFBpositive followedby the heart the intestines the liver and the spleen with thefew AFB positive and mammary gland was AFB negativeMore so by PCR the lungs also had significantly highernumber of positive results forM bovis 77 (565) followedby the lymph nodes 714 (228) and then the intestines andthe liver had the least number of positives for bTB (M bovis)while the spleen heart andmammary glandwere all negativefor bTB (Table 2)

There was a statistically significant (119875 lt 005 1205942 = 4623OR = 3680) association between detection of the bTB inthe tissues using ZN and the age of cattle (Table 2) Cattleaged 6 years and above had the highest percentage tissuepositive for bTB 3333 (3193) using ZN while cattle aged 3ndash5 years had 1481 (427) of their tissues positive for bTB anda prevalence rate of 2916 for the three age groupsThe PCRalso showed a statistically significant (119875 lt 005 OR = 3363)association between detection of bTB in the tissues and theage of the cattle Cattle aged six (6) years had a prevalencerate of 833 while cattle aged 3ndash5 years were negative forbTB using PCR method

There was no statistically significant association (119875 gt005 1205942 = 2017 OR = 180) between the presence of bTB inthe tissues sampled and the sex of the cattle using ZNmethodHowever the female cattle had higher prevalence and sex-specific rates than themale cattle sampled (Table 5)Themalecattle also had higher sex-specific rate of 10 (440) usingPCR technique than the female cattle 750 (680) Howeverfemale cattle had higher prevalence rate of 500 than malecattle with 333 and there was no statistically significantassociation between detection of bTB and sex using PCRmethod (1205942 = 0218 df = 1 OR = 137 95 CI onOR = 0364ndash5164) (Table 5)

Table 5 also summarises the overall prevalence and sex-specific rates using ZN and PCR tests Using ZN the

Table 2 Distribution of suspected bTB lesions in various organs ofslaughtered cattle in Bauchi state Nigeria

Organsexamined

Number of TBsuspected lesions

()

ZN staining( +ve) PCR ( +ve)

Lungs 65 (5420) 19 (2923) 5 (780)Lymph nodes 28 (2330) 6 (2143) 2 (714)Intestines 2 (170) 2 (100) 2 (100)Liver 8 (670) 2 (25) 1 (1250)Spleen 6 (500) 1 (1667) 0 (0)Heart 10 (830) 5 (50) 0 (0)Mam gland 1 (080) 0 (0) 0 (0)Total 120 35 10

prevalence and sex-specific rates of 125 (15120) and 3750(1540) respectively out of the male cattle sampled wereAFB positive while 1666 (20120) out of the female cattlepopulation 250 (2080) respectively were also AFB pos-itive given an overall prevalence of 2916 by ZN whereasusing PCR the prevalence and sex-specific rates of the cattlepopulation sampled indicated a rate of 833 (10120) out ofthe 40 head of male cattle sampled 100 (440) while outof the 80 head of female cattle sampled 750 (680) werepositive for bTB specifically withM bovisThe infection rateswere higher in females than in males By the confirmatoryPCR the overall prevalence rate ofM bovis (bTB) in BauchiState was 833

5 Discussion

The prevalence rates of bTB found in this present studybased on ZN and confirmatory PCR were 2916 and 833respectively This study also showed that PCR is a highlysensitive and specific (833 and 901 resp) test that can beadapted as a confirmatory test to conventional tests such asPM tuberculin skin test (TST) and ZN because of its abilityto rule out possible false positive results associated with thesetests However ZNhas the ability to detectmoreAFB positivesamples as fastidious organisms that may be present in thegranulomatous lesions obtained from slaughtered cattleThisagreed with the previous report by [31] using ZN methodalone The overall prevalence rate (833) of this study isrelatively high and may be related to the poor control mea-sures in the state (lack of test and slaughter policy absenceof control at borders inadequate quarantine measures andthe lack of effective preventive measures against bovine TB)and the influx of possibly infected cattle from neighbouringstates and countries (Cameroon Chad and Niger) Also theincrease in intensive farming practice where large herds arehoused together for long periods of time and poor hygieneare possible risk factors to the spread and endemicity natureof the disease in the state There are many studies on bTB inNigeria and other countries including a retrospective studyby [32] who reported a relatively lower prevalence of 172in Bauchi State as compared to our findings which may bedue to the improved diagnostic technique used in this study


(a) (b)

Figure 1 Photograph of the affected lungs showing massive granulomatous (tuberculous) lesions from slaughtered cattle during PM ((a)bilateral lobes and (b) unilateral lobe) See the blue arrows

(a) (b)

Figure 2 Photographs (a) and (b) showing the acid fast bacilli (AFB) organisms appearing reddish under amicroscope (times100 oil immersion)pointed by the arrows on the methylene blue background after the Ziehl-Neelsen (ZN) staining

Table 3 Distribution of bTB among cattle of different age groups in Bauchi State Nigeria

Age (years) Number sampled Number of ZN positive () Prevalence OR (95 CI)3ndash5 27 4 (1481) 333 16ndash8 41 16 (3902) 1333 3680 (1072ndash12630)lowast

9ndash11 52 15 (2885) 1250 2331 (0688ndash7892)Total 120 35 2916lowastSignificant at 95 CI 1205942 = 4623 df = 2

Table 4 Overall prevalence of bTB (M bovis) among various age groups of cattle in Bauchi State Nigeria

Age (years) Number sampled PCR-positive () Prevalence () OR 95 CI3ndash5 27 0 (000) 000 1lowastlowast

6ndash8 41 7 (1707) 583 3363lowast 0812ndash139309ndash11 52 3 (577) 250Total 120 10 8331205942 = 7002 lowast119875 lt 005 df = 2 lowastlowastreference value (1)


Table 5 The overall prevalence and sex-specific rates of bTB in Bauchi State Nigeria

SexlowastlowastTests Overall prevalence ()

ZN PCR ZNlowast PCRNumber sampled Positive () Positive ()

Males 40 15 (3750) 4 (1000) 1250 333Females 80 20 (2500) 6 (750) 1666 500Total 120 35 10 2916 833lowast1205942 = 1709 df = 1 OR = 453 119875 lt 005 and 95 CI = 212ndash9664lowastSignificant between testslowastlowast1205942= 0218 OR = 137

lowastlowastBetween sexes

Agarose gel electrophoresis of oxyR gene amplicon

M 1 2 3 4 5 6 7 8 9 10 11 M

M 12 13 14 15 16 17 18 19 20 21 22 M

Figure 3 A 2-panel agarose gel electrophoresis of PCR amplifica-tion of oxyR gene specific for M bovis Lane M 13 kb molecularweight markers (100 bp DNA ladder Bioneer labs USA) lanes 1 and2 are positive controls (BCG Pasteur strain and BCG vaccine) lane10 is negative control (nuclease free water) and lanes 3 5 6 8 17and 18 are positive samples diagnostic forM bovis (285 bp)

Moreover increase in free influx of possibly infected cattlefrom neighbouring endemic states especially Gombe Statewith a prevalence rate of 1227 as reported by [32] whodescribed Bauchi State as a population at risk could also be areason for the high prevalence reported in this study Also ahigh prevalence rate of the disease in the Northern Nigeriaand increase in livestock density and contact rates amongcattle of different sources could be another reason

The findings that 120 (15) bTB suspected lesions wereobserved in the 800 head of slaughtered cattle examined inBauchi State abattoirs had emphasized the importance ofPM meat inspection This agrees with the report of [33]as postmortem examination still remains the immediatediagnostic tool to be used in endemic slaughter houses

The distribution and occurrence of suspected bTB lesionsin different organs of slaughtered cattle in Bauchi Stateshowed 5420 (65120) in the lungs and 2330 (28120) inthe lymph nodes while the heart liver spleen intestines andmammary glandsmade up the least number of bTB positivesThis agreed with the retrospective study reported by [33] ongross bTB lesions in different organs in slaughtered cattle inMaiduguri Nigeria with 678 of the lungs and 139 lymphnodes with the least number in the other affected visceralorgans This also agreed with the report of [34]

6 Conclusion

The present study estimated the prevalence rate of bTB inBauchi State using PM ZN and PCR techniques at (1502916 and 833 resp) Bovine TB lesions found at PMwere not all due to M bovis alone as other MTBC and AFBorganisms may cause bTB-like lesions which were excludedby PCR

7 Recommendation

Proper PMmeat inspection should be practiced efficiently atthe abattoir and slaughter houses before taking beef to thepublic The emergence MDR and XDR-TB strains are also amajor concern in Nigeria Thus further molecular epidemi-ological studies with more improved techniques like MIRU-VNTR and spoligotyping should be carried out on isolatesfrom the state to look for other zoonotics likeM africanum

Conflict of Interests

The authors unanimously agreed and declared that ldquothereis not any conflict of interests among them regarding thepublication of this paperrdquo

Authorsrsquo Contribution

This work was carried out in collaboration between allauthors A S Sarsquoidu designed the study did the samplingrun the Ziehl-Neelsen staining technique and wrote the firstdraft of the paper E C Okolocha A A Dzikwi and J K PKwaga supervised the whole research and reviewed the paperA Usman managed the molecular aspect and provided theprimers and positive controls used in the PCR A A Gamawacontributed in the data collation andmanaged the data analy-ses of the study S AMaigari and S Ibrahim did the literaturesearches All authors read and approved the final paper

Acknowledgments

The authors are indebted to the Area Veterinary ClinicBauchi Metropolitan abattoir Azare and Katagum slaughterhouses managements other abattoir staff and Ministry ofAnimal Resources and Nomadic Resettlement Bauchi Statefor cooperation and assistance during the research fieldwork


References

[1] O M Radostits D Blood K Hinchey et al VeterinaryMedicine ATextbook of theDiseases of Cattle Horses Sheep Pigsand Goats Saunders 10th edition 2007

[2] LMOrsquoReilly andC J Daborn ldquoThe epidemiology ofMycobac-terium bovis infections in animals and man a reviewrdquo Tubercleand Lung Disease vol 76 no 1 pp 1ndash46 1995

[3] J E Shitaye W Tsegaye and I Pavlik ldquoBovine tuberculosisinfection in animal and human populations in Ethiopia areviewrdquo Veterinarni Medicina vol 52 no 8 pp 317ndash332 2007

[4] WHO ldquoWorld Health Organization global TB report (2014)Tuberculosis (TB)rdquo Fact sheet 104 WHO Geneva Switzerland2014 httpwwwwhointmediacentrefactsheetsfs104en

[5] J P Caffrey ldquoStatus of bovine tuberculosis eradication pro-grammes in Europerdquo Veterinary Microbiology vol 40 no 1-2pp 1ndash4 1994

[6] L M Shehu Survey of tuberculosis and tubercle bacilli in Fulaniherds ldquoNonordquo and some herdsmen in Zaria area Nigeria [MSthesis] Ahmadu Bello University Zaria Nigeria 1988

[7] C H Collins and J M Grange ldquoThe bovine tubercle bacillusrdquoJournal of Applied Bacteriology vol 55 no 1 pp 13ndash29 1983

[8] U Pfeiffer ldquoTuberculosis in animalsrdquo in Clinical Tuberculosis PD Deviewa Ed Armold London UK 3rd edition 2003

[9] C H Collins and J M Grange ldquoZoonotic implication ofMycobacterium bovis infectionrdquo International Veterinary Jour-nal vol 41 pp 363ndash366 1987

[10] J Francis Tuberculosis in Animals and Man Cassell LondonUK 1958

[11] M Gilbert A Mitchell D Bourn J Mawdsley R Clifton-Hadley and W Wint ldquoCattle movements and bovine tubercu-losis in Great Britainrdquo Nature vol 435 no 7041 pp 491ndash4962005

[12] C OThoen P A Lobue D A Enarson J B Kaneene and I Nde Kantor ldquoTuberculosis a re-emerging disease in animals andhumansrdquo Veterinaria Italiana vol 45 no 1 pp 135ndash181 2009

[13] R SMorris DU Pfeiffer andR Jackson ldquoThe epidemiology ofMycobacterium bovis infectionsrdquo Veterinary Microbiology vol40 no 1-2 pp 153ndash177 1994

[14] R S Clifton-Hadley J W Wilesmith M S Richards P Uptonand S Johnston ldquoThe occurrence of Mycobacterium bovis incattle in and around an area subject to extensive badger (Malesmales) controlrdquo Epidemiology and infection vol 114 no 1 pp179ndash193 1995

[15] FAWC ldquoFarm Animal Welfare Councilannual reportrdquo FAWCPBN3797 1997

[16] M C Raviglione D E Snider Jr and A Kochi ldquoGlobalepidemiology of tuberculosis morbidity and mortality of aworldwide epidemicrdquo Journal of the American Medical Associ-ation vol 273 no 3 pp 220ndash226 1995

[17] T Wada S Maeda A Hase and K Kobayashi ldquoEvaluation ofvariable numbers of tandem repeat as molecular epidemiologi-cal markers ofMycobacterium tuberculosis in Japanrdquo Journal ofMedical Microbiology vol 56 no 8 pp 1052ndash1057 2007

[18] D Maher J L C Van Gorkom P C F M Gondrie and MRaviglione ldquoCommunity contribution to tuberculosis care incountries with high tuberculosis prevalence past present andfuturerdquo International Journal of Tuberculosis and Lung Diseasevol 3 no 9 pp 762ndash768 1999

[19] D E Morisky C K Malotte P Choi et al ldquoA patient educationprogram to improve adherence rates with antituberculosis drug

regimensrdquo Health Education Quarterly vol 17 no 3 pp 253ndash267 1990

[20] Bauchi State Diary ABUICT-Centre database 2009 httpwwwnigeriagalleriacomnigeriastatenigeriabauchistate

[21] FAO ldquoCorporate documentary repositoryrdquo in Nigerian CattlePopulation FAO Rome Italy 2010

[22] Census 2006 Nigeria 2006 httpwwwnigeriamasterwebcomNigeria06CensusFigshtml

[23] L Corner L Melville K McCubbin et al ldquoEfficiency of inspec-tion procedures for the detection of tuberculous lesions incattlerdquoAustralian Veterinary Journal vol 67 no 11 pp 389ndash3921990

[24] T Ron B Ben K Bill and C Ken ldquoMethods of determiningage in cattlerdquo Cattle Producerrsquos Library CL712 2003

[25] P del Portillo L A Murillo and M E Patarroyo ldquoAmplifi-cation of a species-specific DNA fragment of Mycobacteriumtuberculosis and its possible use in diagnosisrdquo Journal of ClinicalMicrobiology vol 29 no 10 pp 2163ndash2168 1991

[26] R R Kazwala C J Daborn J M Sharp D M Kambarage SF H Jiwa and N A Mbembati ldquoIsolation of Mycobacteriumbovis fromhuman cases of cervical adenitis in Tanzania a causefor concernrdquo International Journal of Tuberculosis and LungDisease vol 5 no 1 pp 87ndash91 2001

[27] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[28] J G Rodriguez G AMejia P Del PortilloM E Patarroyo andL A Murillo ldquoSpecies-specific identification ofMycobacteriumbovis by PCRrdquoMicrobiology vol 141 no 9 pp 2131ndash2138 1995

[29] R E Romero D L Garzon G A Mejıa W Monroy ME Patarroyo and L A Murillo ldquoIdentification of Mycobac-terium bovis in bovine clinical samples by PCR species-specificprimersrdquo Canadian Journal of Veterinary Research vol 63 no2 pp 101ndash106 1999

[30] TMamiatis E F Fritsch and J SambrookMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory New YorkNY USA 1982

[31] I A Abubakar Molecular epidemiology of human and bovinetuberculosis in the Federal Capital Territory and Kaduna state[PhD thesis] Plymouth University Plymouth UK 2007

[32] M M Aliyu A J Adamu and Y A Bilyaminu ldquoCurrentprevalence of tuberculous lesions among slaughtered cattle innortheastern state of Nigeriardquo Revue Elevage Veterinaire paysdes Tropicaux vol 62 no 1 pp 13ndash16 2009

[33] U B Abubakar S A Shehu and F U Mohammed ldquoRetro-spective study of tuberculosis in slaughtered cattle atMaiduguriabattoir NigeriardquoVeterinary Research vol 4 no 1 pp 1ndash4 2011

[34] I O Igbokwe I Y Madaki S Danburam J A Ameh M MAliyu and C O Nwosu ldquoPrevalence of pulmonary tubercu-losis lesions in cattle slaughtered in abattoirs in NortheasternNigeriardquo Revue drsquoElevage et de Medecine Veterinaire des PaysTropicaux vol 54 pp 191ndash195 2001

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Veterinary Medicine International



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Case Reports in Veterinary Medicine


africanum M canettii and M pinnipedii Mycobacteriumafricanum consists of a rather heterogeneous group of strainsisolated from humans in Africa [9] The organism may betransmitted by aerosol or droplets of exudates containingthe bacilli It can be transmitted by ingestion of feed andwater contaminated with urine fecal material or exudatesfrom diseased animals that contain the tubercle bacilli Thebovine tubercle bacillus is the cause of the bTB in cattle butstill is often used to denote bovine strains of the tuberclebacillus irrespective of the host In fact the bovine tuberclebacillus has one of the broadest host ranges of all knownpathogens The species has been reported in domesticatedand feral Bovidae Other species in which the disease hasbeen reported include goat sheep pig horse cat dog fennecfox bison buffalo badger wild and feral pig antelopecamel and man and nonhuman primates among others [10]Cattle movements particularly those from areas where bTBis reported are the best predictor of disease occurrence [11]

Bovine TB in cattle is initially asymptomatic and priorto testing farmers usually remain unaware of the outbreakduring this stage It can take as long as a few years before anysigns can be observed As the infection progresses in cattlebovine tuberculosis causes a slight fever and roughened haircoat and the lymph nodes usually swell or have abscesses andshow signs of dyspnoea with an associated cough The cowwill appear weak show signs of laboured breathing and havean increased breathing rate A loss of appetite will be noticedand the decrease in weight may be so significant that the cowactually becomes emaciated Tuberculosis (TB) in humansis characterized by prolonged coughing general body weak-ness wasting and sweating at night with slight fever [12]

In UK badgers have been culled since 1971 when atuberculous dead badger was found in an infected farmBovine TB control has proved difficult to achieve wherethere is a persistent reservoir of infection in wildlife [13]Consequently interpreting subsequent trends in incidence ofbTB which are complicated by multiple factors such as cattlemanagement practice changing specificity of tests changesto the cattle meat factory (abattoir) including pollutioncontrolregulations and increase in level of aflatoxins in feeds(known for their immunosuppressive qualities etc) [14 15]

Despite the availability of effective therapy tuberculosisremains a major public health problem worldwide [16 17]Factors such as inadequate health service infrastructuredecreased access to health care and limited human andfinancial resources have prevented adequate implementationof control measures against TB [18]

Recent advances in the treatment of human TB havepermitted effective management of cases on an ambula-tory basis [19] However in many developing countries anirregular drug taking practice and premature termination oftreatmentmainly by self-discharge are themain cause of poorperformance of control programmesThis study was aimed atdetermining the prevalence of bovine tuberculosis in slaugh-tered cattle based on postmortem (PM) meat inspectionZiehl-Neelsen staining (ZN) and polymerase chain reaction(PCR) techniques in abattoirs of Bauchi State Nigeria

2 Materials and Methods

21 Study Area Bauchi State occupies a total land area of49119 Km2 representing about 53 of Nigeriarsquos total landmass and is located between latitudes 9∘31015840 and 12∘31015840 north ofthe equator and longitudes 8∘501015840 and 11∘ east of theGreenwichMeridean [20] The state is bordered by Kano and Jigawato the north Taraba and Plateau to the south Gombe andYobe to the east and Kaduna to the west The state is highlypopulated with cattle mainly owned by Fulani herdsmenThe cattle population is estimated at 1789000 about 13 ofthe Nigerian cattle population of 13900000 [21] The Statehas a human population of 4653066 ranked 11th of the 36states density of 95 km2 (250sqmi) and per capita incomeof $983 [22] Bauchi State has a total of 55 tribal groups inwhich Hausa Fulani and Kanuri are the main tribes Thestudy was carried out in Bauchi Katagum and Misau LocalGovernment Areas (out of the 20 LGAs) each representingthe three senatorial zones as Bauchi South Bauchi Northand Bauchi Central with populations of 493810 295970 and263487 respectively

22 Study Design The three major slaughter houses namelyBauchi Misau and Katagum (Azare) in each of the threesenatorial zones of Bauchi State were selected for the studyfor sampling purposes based on their daily slaughter capacityand socioeconomic impact of the abattoir Accordingly out ofthe 800 head of cattle examined (sample size) a proportionatedistribution was employed given 50 (400) to Bauchi abat-toir and 25 (200) each to Katagum and Misau slaughterhouses This was based on their daily slaughter capacityof 40ndash60 20ndash30 and 20ndash25 head of cattle from Bauchiabattoir Katagum and Misau slaughter houses respectivelyThe selection of the cattle sampled at each of the abattoirs wasstrictly on the basis of the age of the cattle (meant for slaughterand three years above) following then PM observation oftypical granulomatous bTB lesions in any of the visceralorgans before the cattle is considered a case and sample

23 Source of Animals for Slaughter Most of the animalsbrought for slaughter were mainly sourced from MaiduguriChad and Niger

24 Sampling Procedure and Transportation Identification ofcases was based on presence of typical tubercle yellowishgranulomatous and caseous lesions in the lungs lymphnodes kidneys intestines and liver [23] Aging of cattle wascarried out at the abattoir after slaughter as described by[24] using the time of appearance and the degree of wear onthe temporary and permanent teeth Additional data takenincluded sex and breed of each animal sampled

A total of 800 head of slaughtered cattle were examined atpostmortem for TB lesions Using a convenience samplinga total of 400 head of cattle from Bauchi 200 head of cattleeach from Misau and Katagum LGA slaughter houses wereexaminedOne hundred and twenty (120) tissue samples werecollected from cattle with suspected TB lesions into sterilescrew-capped containers (with 9 normal saline solution tokeep tissuesmoist) and transported inside a cooler containing






















4 Results






Organsexamined


()




5 Discussion



(a) (b)


(a) (b)















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M 12 13 14 15 16 17 18 19 20 21 22 M





6 Conclusion


7 Recommendation






Acknowledgments



References











































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of



























4 Results






Organsexamined


()




5 Discussion



(a) (b)


(a) (b)















M 1 2 3 4 5 6 7 8 9 10 11 M

M 12 13 14 15 16 17 18 19 20 21 22 M





6 Conclusion


7 Recommendation






Acknowledgments



References











































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








4 Results






Organsexamined


()




5 Discussion



(a) (b)


(a) (b)















M 1 2 3 4 5 6 7 8 9 10 11 M

M 12 13 14 15 16 17 18 19 20 21 22 M





6 Conclusion


7 Recommendation






Acknowledgments



References











































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a) (b)


(a) (b)















M 1 2 3 4 5 6 7 8 9 10 11 M

M 12 13 14 15 16 17 18 19 20 21 22 M





6 Conclusion


7 Recommendation






Acknowledgments



References











































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













M 1 2 3 4 5 6 7 8 9 10 11 M

M 12 13 14 15 16 17 18 19 20 21 22 M





6 Conclusion


7 Recommendation






Acknowledgments



References











































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







References











































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






Detection ofMycobacteriumbovis in Organs of Slaughtered Cattle by DNA-Based Polymerase Chain Reaction and Ziehl-Neelsen Techniques in Bauchi State, Nigeria

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