canine babesiosis in northwestern india: molecular detection and

Research ArticleCanine Babesiosis in Northwestern IndiaMolecular Detection and Assessment of Risk Factors

Amritpal Singh1 Harkirat Singh1 N K Singh1 N D Singh2 and S S Rath3

1 Department of Veterinary Parasitology College of Veterinary Science Guru Angad Dev Veterinary and Animal Sciences UniversityLudhiana Punjab 141 004 India

2Department of Veterinary Pathology College of Veterinary Science Guru Angad Dev Veterinary and Animal Sciences UniversityLudhiana Punjab 141 004 India

3 Animal Disease Research Centre College of Veterinary Science Guru Angad Dev Veterinary and Animal Sciences UniversityLudhiana Punjab 141 004 India

Correspondence should be addressed to S S Rath drssrath59rediffmailcom

Received 25 February 2014 Revised 5 May 2014 Accepted 27 May 2014 Published 12 June 2014

Academic Editor Stefano DrsquoAmelio

Copyright copy 2014 Amritpal Singh et alThis 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

In the current study a total of 214 blood samples from dogs in and around Ludhiana Punjab (India) suspected for canine babesiosiswere examined with conventional and molecular assays Examination of Giemsa-stained peripheral thin blood smears revealed anoverall prevalence of 747 (16214) for canine babesiosis encompassing 093 (2214) of largeBabesia and 654 (14214) ofBabesiagibsoni However molecular diagnosis revealed 1542 (33214) samples positive for B gibsoni infection as evident by the presenceof 671 bp amplicon The results of multivariate analysis showed that the prevalence of B gibsoni was associated with various riskfactors namely age (119875 lt 0001 OR 0398 CI 95 0080ndash1799) sex (119875 = 0022 OR 0849 CI 95 0403ndash1791) breed of host(119875 = 0371 OR 3345 CI 95 1045ndash10710) and season (119875 = 0230 OR 2143 CI 95 0788ndash5830) The prevalence of B gibsoniwas higher in summer as compared to winter season and in younger dogs while breed and sex of the host were not significantlyassociated with the occurrence of the disease

1 Introduction

Amongst the various prevalent canine vector-borne diseasescanine babesiosis is very common and clinically significantdisease caused by intraerythrocytic apicomplexan protozoabelonging to genus Babesia distributed worldwide includ-ing India Babesia species often referred to as piroplasmscomprise two main species B canis and B gibsoni basedon their size B canis is a large piroplasm (4-5 120583m) whichusually occurs as a single pear-shaped piroplasm or in pairs ofmerozoites divided by binary fission within the erythrocyte

Previous studies on the basis of differences in thegeographical distribution vector specificity and antigenicproperties [1 2] recognized that large canine piroplasms aresubdivided into three species namely B canis transmittedby Dermacentor reticulatus (in Europe) B vogeli transmittedby Rhipicephalus sanguineus (in tropical and subtropicalregions) and B rossi transmitted by Haemaphysalis elliptica(in South Africa) B gibsoni has been found to be associated

with infection of dogs in Asia North America northernand eastern Africa and Europe [3ndash5] It is a small parasitethat commonly appears as individual ring forms or pyriformbodies ranging between 10 and 25 120583m in size [3]

Clinically canine babesiosis has been found to resultin a wide range of presentations from subclinical dis-ease to serious illness characterised by fever pallor jaun-dice splenomegaly weakness and collapse associated withintra- and extravascular haemolysis hypoxic injury systemicinflammation thrombocytopenia and pigmenturia [6]

As far as the diagnosis of canine babesiosis is concerneddirect microscopic examination of the stained blood smear isthe most commonly used method as it is conclusive feasibleand cost effective diagnostic method but not necessarilydetects parasites in dogs with unapparent or chronic infec-tions since the level of parasitemia is very low [7] As regardsthe serological methods indirect fluorescent antibody test(IFAT) and enzyme linked immunosorbent assay (ELISA) forB gibsoni parasites are considered to be highly sensitive but

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 741785 5 pageshttpdxdoiorg1011552014741785

2 BioMed Research International

only moderately specific because of antigenic cross-reactionsto B canis [8] and normal dog erythrocytes [8 9] Thereforethe development of highly specific and sensitive system forthe diagnosis of canine babesiosis is still awaited In thisregard recent advances in molecular biology techniques likepolymerase chain reaction (PCR) have made it possible todetect and identify piroplasms with greater sensitivity andspecificity than traditional methods [10 11]

Regarding Indian scenario though there are sporadicreports of canine babesiosis based on conventional diagnosticmethods [12ndash15] the true status of canine babesiosis is stillnot clear barring few reports [16 17] employing the PCRbased assays Furthermore molecular detection of caninebabesiosis has not yet been explored from Punjab north stateof India so the present work was carried out to know thestatus of canine babesiosis in this part of the country throughPCR based assays

2 Materials and Methods

21 Geographical Area The study was conducted from Lud-hiana district of Punjab state in the northwestern region ofIndia The climate of the region under study is excessivelyhot and dry during summers Winters are cool with somefrosts and the average annual rainfall is 5659mm Theseenvironmental conditions provide favourable and conduciveconditions for the survival and propagation of ticks andRhipicephalus sanguineus is the major tick infesting canines[18]

22 Samples A total of 214 blood samples were collectedaseptically from cephalic vein of the selected dogs in EDTAcoated vials from the dogs presented to Small Animal ClinicsTeaching Veterinary Clinical Complex GADVASU Ludhi-ana as well as local private veterinary clinics from a periodof one year (April 2012 to March 2013) Dogs were selectedon the basis of presence of naturally acquired tick infestationat the time of presentation andor showing clinical signsin accordance with the haemoprotozoan infection namelyfever haemoglobinuria anemia and so forth The collectedblood samples were utilized immediately for the preparationof thin blood smears and were then kept at minus20∘C until DNAextraction

Microscopic examination of blood sampleswas done afterstaining the prepared thin blood smears with Giemsa as perstandard protocol [19] and examined under oil immersionobjective of the microscope to detect the piroplasms and theresults obtained were compared to that of PCR assay

23 Genomic DNA Isolation For conducting the PCR assaygenomic DNAwas isolated from whole blood using QIAampDNA blood mini kit (QIAGEN GmbH Germany) followingthe manufacturerrsquos recommendations with minor modifi-cations and stored at minus20∘C till use Genomic DNA of Bgibsoni was isolated and utilized as a positive control frominfected blood sample showing parasitemia in blood smearexaminationGenomicDNAwas also isolated from thewholeblood of infection-free puppy and used as a negative controlalong with nuclease-free water

Table 1 Evaluation of diagnosticscreening PCR assays over bloodsmear examination

Parameter PCR (95 CI)Sensitivitylowast 100 (7847 100)Specificitylowast 905 (8564 9383)Diagnostic accuracylowast 9112 (8655 9424)lowastWilson score (httpwwwopenepicomv37DiagnosticTestDiagnostic-Testhtm)

24 PCR Protocol The PCR assay was optimized targeting aportion of the 18S rRNA gene to amplify B gibsoni as descri-bed by Inokuma et al [20]The sequences of the primers wereas follows

Gib599 Forward 51015840CTCGGCTACTTGCCTTGT-C31015840Gib1270 Reverse 51015840GCCGAAACTGAAATAACG-GC31015840

PCR assay in a final volume of 25 120583L was carried out in aPCR thermal cycler (Applied Biosystems USA) The mastermix consisted of 25 120583L of 10X PCR buffer (MBI Fermentas)05 120583L of 10mM dNTP mix (MBI Fermentas) 15 120583L of25mM MgCl

2(MBI Fermentas) 10U of recombinant Taq

DNA polymerase (MBI Fermentas) 1 120583L each (20 pmol) ofthe primers and 5 120583L of template DNA isolated from fieldsamplesThe volumewasmade up to 25120583Lwith nuclease-freewater The PCR cycling conditions were initial denaturationat 95∘C for 5min 40 cycles of denaturation at 95∘C for30 sec annealing at 56∘C for 30 sec and extension at 72∘Cfor 130min and the final extension was performed at 72∘Cfor 5min The PCR products obtained were checked foramplification by electrophoresis on a 15 agarose gel andvisualized using gel documentation system (Syngene UK) Inorder to check the specificity of the assays isolated genomicDNA of large Babesia Ehrlichia canis Hepatozoon canisand Trypanosoma evansi isolated from the microscopicallypositive cases were also employed in the PCR to see theamplification if any

25 Statistical Analysis All data analyses were performedby using statistical software program (SPSS for WindowsVersion 190 USA) Association between the prevalence ofB gibsoni by PCR and various risk factors namely sexage breed of the host and season was carried out by Chisquare (1205942-test) Variables with significant association at119875 lt 005 (two-sided) were subjected to the multivariatelogistic regression model The results were each expressed as119875 value and odds ratio (OR) with a 95 confidence interval(CI 95)

3 Results

31 Blood Smear Examination In the present study exami-nation of Giemsa-stained peripheral thin blood smears of 214canines revealed an overall prevalence of canine babesiosis as747with 093 (2214) positivity for the piroplasms of large

BioMed Research International 3

Table 2 Final logistic regression model for factors associated with prevalence of B gibsoni by PCR on animal levels

Variable Regression coefficient (120573) Standard error (SE) 119875 value Odds CI (95)lowast

Age minus1330 0199 0000 0398 0080ndash1799Sex 0346 0151 0022 0849 0403ndash1791Breed 0179 0200 0371 3345 1045ndash10710Season minus0223 0186 0230 2143 0788ndash5830lowastConfidence interval

1 2 3 4 5 6 M 7 8 9 10 11 12

671bp

Figure 1 Gel electrophoresis showing B gibsoni PCR assay LaneMGeneRuler 100 bp Ladder lanes 1ndash5 and 8ndash12 field collected sampleslane 6 negative control and lane 7 positive control

Babesia and 654 (14214) positivity for the piroplasms of Bgibsoni

32 PCR Protocol All the collected blood samples wereanalyzed by PCR assay to detect any amplification in theform of ethidium bromide-stained amplicons after stan-dardization Of the total samples subjected 1542 (33214)were positive for presence of B gibsoni infection as revealedby the amplification of a 671 bp product (Figure 1) Furtherthe PCR primers used in the present assay did not amplifyany product when the genomic DNA of large Babesia Ecanis H canis and T evansi were used as template revealingthe specificity of these primers The sensitivity specificityand diagnostic efficacy of PCR were determined by usingblood smear examination as the gold standard and results arepresented in Table 1

33 Correlation of Canine Babesiosis withVarious Risk FactorsThe correlation between the prevalence of B gibsoni andvarious risk factors was studied and the values of the corre-lation coefficients (120573) are presented in Table 2 Further theresults of multivariate analysis showed that the prevalence ofB gibsoniwas associatedwith various risk factors namely age(119875 lt 0001 OR 0398 CI 95 0080ndash1799) sex (119875 = 0022OR 0849 CI 95 0403ndash1791) breed of host (119875 = 0371OR 3345 CI 95 1045ndash10710) and season (119875 = 0230OR 2143 CI 95 0788ndash5830) The prevalence of B gibsoniwas higher in summer as compared to winter season andin younger dogs while breed and sex of the host were notsignificantly associated with the occurrence of the disease(Table 3)

4 Discussion

In the present study by using conventional parasitologi-cal techniques a statistically higher percent positivity wasrecorded for B gibsoni infection than large Babesia (119875 =00051) with the overall prevalence of canine babesiosis as747 (16214) Previously from the same region Eljadar[21] examined a total of 951 suspected dog samples fromSmall Animal Clinics GADVASU Ludhiana and three localprivate veterinary hospitals for haemoprotozoan infectionsand reported 126 samples to be positive for B canis and317 to be positive for B gibsoni The comparative higherprevalence of B gibsoni over B canis recorded by him is incongruence with that of the present study Similar findingswere recorded in earlier studies by Singh et al [15 22] fromthis region revealing the prevalence of B gibsoni and B canisin the range of 065ndash826 and 143ndash451 respectively

Microscopic detection of B gibsoni though smaller insize than large Babesia was easier because of its frequentappearance in the circulating host blood This might alsobe due to a low level parasitaemia in case of large Babesiainfection especially during very early or carrier stage whichis beyond the level of microscopic detection [6 10 23]The prevalence of canine babesiosis from various parts ofnorthern India has been reported to be ranging from 066 to89 [14 22 24 25]while fromSouthern India Senthil Kumaret al [13] recorded 39 and 849 prevalence of B canis andB gibsoni respectively Wide variation in climatic conditionsprevailing in different parts of India might be responsible forvarying percentage of these tick borne infections

On the basis of present findings PCR based assay was ableto detect 1542 prevalence of B gibsoni Higher detectionof canine babesiosis by PCR based assays as compared tomicroscopy as observed in the present study has also beenreported by several authors worldwide indicating the highersensitivity levels of PCR [10 17 26ndash30] As far as the detectionof B gibsoni with PCR based assays is concerned manystudies have been carried out worldwide and the prevalencehas been recorded to be ranging from 33 to 55 [17 20 31ndash34]

As far as evaluation of various risk factors is concernedfor canine babesiosis several authors have observed theprevalence of the haemoprotozoan infections to be highestin young dogs [35 36] In terms of sex of the host from thedata obtained in the current study it can be concluded thatthe assays recorded no statistical significance difference inthe prevalence of the disease among males and female dogsThese results are incongruous with Amuta et al [28] andSingh et al [14]


Table 3 Assessment of various risk factors with regard to distribution of B gibsoni infection

Risk factor Parameter Number Blood smear () PCR ()

Age

0ndash6m 34 2 (588) 2 (588)6mndash1 y 40 6 (15) 12 (30)gt1 y 140 6 (428) 19 (1357)1205942 value 4653 8543lowast

SexMale 124 8 (645) 18 (1451)Female 90 6 (667) 15 (1667)1205942 value mdash 0066 0246

Breed

Labrador 71 7 (985) 16 (2253)German Shepherd 34 3 (882) 8 (2352)

Pug 24 1 (416) 2 (833)Others 35 mdash 3 (857)

Nondescript 50 3 (6) 4 (8)1205942 value mdash 5201 7829

Season

Summer 64 5 (781) 12 (1875)Rainy 78 6 (769) 14 (1794)Winter 72 3 (416) 7 (972)1205942 value mdash 0004 1827

Total 214 14 (654) 33 (1542)lowast

119875 lt 005 others include Pomeranian (8) Saint Bernard (9) Dalmatian (3) Boxer (3) Great Dane (3) Cocker Spaniel (2) Rottweiler (4) and NapoleonMastiff (2)

Regarding breed of the host the results revealed thatblood smear examination and PCR detected a statisticallynonsignificant difference in the prevalence of the B gibsoniamong the various breeds and nondescript dogs In seasonalprevalence of the disease the disease was most prevalent inwarm seasons as compared to winters The probable reasonbehind this trendmay be correlated to the seasonal activity ofthe brown dog tick Rhipicephalus sanguineus which is in itsabundance in hot and humid period of the year thus resultingin the higher incidence of haemoprotozoan infections inwarm months during warmer seasons [37]

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are grateful to the Director of Research GuruAngad Dev Veterinary and Animal Sciences University Lud-hiana Punjab India for providing the necessary facilities

References

[1] G Uilenberg F F J Franssen NM Perie andA AM SpanjerldquoThree groups of Babesia canis distinguished and a proposal fornomenclaturerdquo Veterinary Quarterly vol 11 no 1 pp 33ndash401989

[2] S Hauschild P Shayan and E Schein ldquoCharacterization andcomparison of merozoite antigens of different Babesia canisisolates by serological and immunological investigationsrdquo Par-asitology Research vol 81 no 8 pp 638ndash642 1995

[3] P A Conrad J Thomford I Yamane et al ldquoHemolytic anemiacaused by Babesia gibsoni infection in dogsrdquo Journal of theAmerican Veterinary Medical Association vol 199 no 5 pp601ndash605 1991

[4] RCasapulla L Baldi VAvallone R Sannino L Pazzanese andV Mizzoni ldquoCanine piroplasmosis due to Babesia gibsoniclinical and morphological aspectsrdquoVeterinary Record vol 142no 7 pp 168ndash169 1998

[5] A J Birkenheuer M G Levy K C M Savary R B Gagerand E B Breitschwerdt ldquoBabesia gibsoni infections in dogsfrom North Carolinardquo Journal of the American Animal HospitalAssociation vol 35 no 2 pp 125ndash128 1999

[6] P J Irwin ldquoCanine babesiosis from molecular taxonomy tocontrolrdquo Parasites and Vectors vol 2 supplement 1 article S42009

[7] S M Caccio B Antunovic A Moretti et al ldquoMolecular char-acterisation of Babesia canis canis and Babesia canis vogeli fromnaturally infected European dogsrdquo Veterinary Parasitology vol106 no 4 pp 285ndash292 2002

[8] I Yamane P A Conrad and I A Gardner ldquoBabesia gibsoniinfection in dogsrdquo Journal of Protozoology Research vol 3 pp111ndash125 1993

[9] K Adachi M Tateishi Y Horii H Nagatomo T Shimizu andS Makimura ldquoReactivity of serum anti-erythrocyte membraneantibody in Babesia gibsoni-infected dogsrdquo The Journal ofVeterinary Medical Science vol 56 no 5 pp 997ndash999 1994

[10] A J Birkenheuer M G Levy M Stebbins M Poore and EBreitschwerdt ldquoSerosurvey of anti-Babesia antibodies in straydogs and American pit bull terriers and American Staffordshireterriers from North Carolinardquo Journal of the American AnimalHospital Association vol 39 no 6 pp 551ndash557 2003

[11] R Jefferies U M Ryan C J Muhlnickel and P J IrwinldquoTwo species of canine Babesia in Australia detection and


characterization by PCRrdquo Journal of Parasitology vol 89 no 2pp 409ndash412 2003

[12] N Sundar C Balachandran and A Senthivelan ldquoIncidence ofBabesia gibsoni infection in dogs in Tamil Nadurdquo Journal ofVeterinary Parasitology vol 18 pp 79ndash80 2004

[13] K Senthil Kumar S Vairamuthu and D Kathiresanl ldquoPreva-lence of haemoprotozoans in canines in Chennai Cityrdquo Tamil-nadu Journal of Veterinary and Animal Science vol 5 no 3 pp104ndash108 2009

[14] N K Singh J M Haque H Singh and S S Rath ldquoPrevalenceof canine parasitic infectionsrdquo IndianVeterinary Journal vol 88no 6 pp 76ndash77 2011

[15] H Singh M Haque Jyoti N K Singh and S S Rath ldquoOccur-rence of parasitic infections in dogs in and around LudhianaPunjab (India)rdquo Applied Biological Research vol 14 no 1 pp108ndash110 2012

[16] P A M Abd Rani P J Irwin G T Coleman M Gatne andR J Traub ldquoA survey of canine tick-borne diseases in IndiardquoParasites and Vectors vol 4 article 141 2011

[17] R Laha K Bhattacharjee P C Sarmah et al ldquoBabesia infectionin naturally exposed pet dogs from a north-eastern state(Assam) of India detection by microscopy and polymerasechain reactionrdquo Journal of Parasitic Diseases 2013

[18] H S Gill and B S Gill ldquoQualitative district-wise distributionof adult ixodid ticks in the Punjab staterdquo in Ixodid Ticks ofDomestic Animals in the Punjab State pp 2ndash14 PAU LudhianaIndia 1977

[19] E H Coles Veterinary Clinical Pathology WB Saunders Lon-don UK 4th edition 1986

[20] H Inokuma Y Yoshizaki K Matsumoto et al ldquoMolecular sur-vey of Babesia infection in dogs in Okinawa Japanrdquo VeterinaryParasitology vol 121 no 3-4 pp 341ndash346 2004

[21] M S M Eljadar Clinico-diagnostic studies on vector transmittedhaemoprotozoan diseases in dogs [MS thesis] GADVASULudhiana India 2010

[22] H Singh M Haque N K Singh and S S Rath ldquoPrevalenceof canine parasitic infections in and around Ludhiana PunjabrdquoJournal of Veterinary Parasitology vol 25 no 2 pp 179ndash1802011

[23] G Bourdoiseau ldquoCanine babesiosis in Francerdquo VeterinaryParasitology vol 138 no 1-2 pp 118ndash125 2006

[24] J P Varshney and S Dey ldquoA clinical study on haemoprotozoaninfections in referral caninesrdquo Journal of Remount and Veteri-nary Corps vol 37 pp 83ndash89 1998

[25] S Chaudhuri Studies on clinico-therapeutic aspects of babesiosisin dogs [MS thesis] Indian Veterinary Research InstituteIzatnagar India 2006

[26] S Gotsch M Leschnik G Duscher J P Burgstaller W Wille-Piazzai and A Joachim ldquoTicks and haemoparasites of dogsfrom Praia Cape Verderdquo Veterinary Parasitology vol 166 no1-2 pp 171ndash174 2009

[27] L H ODwyer V V A Lopes A S Rubini K D S Paduan andP E M Ribolla ldquoBabesia spp infection in dogs from rural areasof Sao Paulo State Brazilrdquo Revista Brasileira de ParasitologiaVeterinaria vol 18 no 2 pp 23ndash26 2009

[28] E U Amuta B O Atu R S Houmsou and J G AyasharldquoRhipicephalus sanguineus infestation and Babesia canis infec-tion among domestic dogs in Makurdi Benue State-NigeriardquoInternational Journal of Academic Research vol 2 no 3 pp 170ndash172 2010

[29] M Ionita I LMitrea K Pfister DHamel CM Buzatu andCSilaghi ldquoCanine babesiosis in Romania due toBabesia canis andBabesia vogeli a molecular approachrdquo Parasitology Researchvol 110 no 5 pp 1659ndash1664 2012

[30] A Matsuu S Ono H Ikadai et al ldquoDevelopment of a SYBRgreen real-time polymerase chain reaction assay for quantitativedetection of Babesia gibsoni (Asian genotype) DNArdquo Journal ofVeterinary Diagnostic Investigation vol 17 no 6 pp 569ndash5732005

[31] A S Mokhtar S F Lim and S T Tay ldquoMolecular detectionof Anaplasma platys and Babesia gibsoni in dogs in MalaysiardquoTropical Biomedicine vol 30 no 2 pp 345ndash348 2013

[32] M H Talukder A Matsuu A Iguchi B C Roy N Nishiiand Y Hikasa ldquoPCR-based survey of vector-borne pathogens indogs in Dhaka Bangladeshrdquo Journal of Bangladesh AgriculturalUniversity vol 10 pp 249ndash253 2012

[33] S Fukumoto X Xuan S Shigeno et al ldquoDevelopment ofa polymerase chain reaction method for diagnosing Babesiagibsoni infection in dogsrdquo Journal of Veterinary Medical Sciencevol 63 no 9 pp 977ndash981 2001

[34] D K Macintire M K Boudreaux G D West C Bourne JC Wright and P A Conrad ldquoBabesia gibsoni infection amongdogs in the southeastern United Statesrdquo Journal of the AmericanVeterinary Medical Association vol 220 no 3 pp 325ndash3292002

[35] S U Abdullahi A R Mohammed A Trimnell R Sannusiand R Alafiatayo ldquoClinical and haematological findings in 70naturally occurring cases of canine babesiosis due to Babesiacanisrdquo Journal of Small Animal Practice vol 31 pp 145ndash147 1990

[36] D Samradhni D K Maske R Shobha and P N ShindeldquoBionomics and haemodynamics in blood protozoal infectionsin dogs from Nagpurrdquo Indian Journal of Animal Health vol 44no 1 pp 57ndash66 2005

[37] E J L SoulsbyHelminths Arthropods and Protozoa of Domesti-cated Animals Bailliere-Tindall London UK 7th edition 1982

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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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


only moderately specific because of antigenic cross-reactionsto B canis [8] and normal dog erythrocytes [8 9] Thereforethe development of highly specific and sensitive system forthe diagnosis of canine babesiosis is still awaited In thisregard recent advances in molecular biology techniques likepolymerase chain reaction (PCR) have made it possible todetect and identify piroplasms with greater sensitivity andspecificity than traditional methods [10 11]

Regarding Indian scenario though there are sporadicreports of canine babesiosis based on conventional diagnosticmethods [12ndash15] the true status of canine babesiosis is stillnot clear barring few reports [16 17] employing the PCRbased assays Furthermore molecular detection of caninebabesiosis has not yet been explored from Punjab north stateof India so the present work was carried out to know thestatus of canine babesiosis in this part of the country throughPCR based assays

2 Materials and Methods

21 Geographical Area The study was conducted from Lud-hiana district of Punjab state in the northwestern region ofIndia The climate of the region under study is excessivelyhot and dry during summers Winters are cool with somefrosts and the average annual rainfall is 5659mm Theseenvironmental conditions provide favourable and conduciveconditions for the survival and propagation of ticks andRhipicephalus sanguineus is the major tick infesting canines[18]

22 Samples A total of 214 blood samples were collectedaseptically from cephalic vein of the selected dogs in EDTAcoated vials from the dogs presented to Small Animal ClinicsTeaching Veterinary Clinical Complex GADVASU Ludhi-ana as well as local private veterinary clinics from a periodof one year (April 2012 to March 2013) Dogs were selectedon the basis of presence of naturally acquired tick infestationat the time of presentation andor showing clinical signsin accordance with the haemoprotozoan infection namelyfever haemoglobinuria anemia and so forth The collectedblood samples were utilized immediately for the preparationof thin blood smears and were then kept at minus20∘C until DNAextraction

Microscopic examination of blood sampleswas done afterstaining the prepared thin blood smears with Giemsa as perstandard protocol [19] and examined under oil immersionobjective of the microscope to detect the piroplasms and theresults obtained were compared to that of PCR assay

23 Genomic DNA Isolation For conducting the PCR assaygenomic DNAwas isolated from whole blood using QIAampDNA blood mini kit (QIAGEN GmbH Germany) followingthe manufacturerrsquos recommendations with minor modifi-cations and stored at minus20∘C till use Genomic DNA of Bgibsoni was isolated and utilized as a positive control frominfected blood sample showing parasitemia in blood smearexaminationGenomicDNAwas also isolated from thewholeblood of infection-free puppy and used as a negative controlalong with nuclease-free water

Table 1 Evaluation of diagnosticscreening PCR assays over bloodsmear examination

Parameter PCR (95 CI)Sensitivitylowast 100 (7847 100)Specificitylowast 905 (8564 9383)Diagnostic accuracylowast 9112 (8655 9424)lowastWilson score (httpwwwopenepicomv37DiagnosticTestDiagnostic-Testhtm)

24 PCR Protocol The PCR assay was optimized targeting aportion of the 18S rRNA gene to amplify B gibsoni as descri-bed by Inokuma et al [20]The sequences of the primers wereas follows

Gib599 Forward 51015840CTCGGCTACTTGCCTTGT-C31015840Gib1270 Reverse 51015840GCCGAAACTGAAATAACG-GC31015840

PCR assay in a final volume of 25 120583L was carried out in aPCR thermal cycler (Applied Biosystems USA) The mastermix consisted of 25 120583L of 10X PCR buffer (MBI Fermentas)05 120583L of 10mM dNTP mix (MBI Fermentas) 15 120583L of25mM MgCl

2(MBI Fermentas) 10U of recombinant Taq

DNA polymerase (MBI Fermentas) 1 120583L each (20 pmol) ofthe primers and 5 120583L of template DNA isolated from fieldsamplesThe volumewasmade up to 25120583Lwith nuclease-freewater The PCR cycling conditions were initial denaturationat 95∘C for 5min 40 cycles of denaturation at 95∘C for30 sec annealing at 56∘C for 30 sec and extension at 72∘Cfor 130min and the final extension was performed at 72∘Cfor 5min The PCR products obtained were checked foramplification by electrophoresis on a 15 agarose gel andvisualized using gel documentation system (Syngene UK) Inorder to check the specificity of the assays isolated genomicDNA of large Babesia Ehrlichia canis Hepatozoon canisand Trypanosoma evansi isolated from the microscopicallypositive cases were also employed in the PCR to see theamplification if any

25 Statistical Analysis All data analyses were performedby using statistical software program (SPSS for WindowsVersion 190 USA) Association between the prevalence ofB gibsoni by PCR and various risk factors namely sexage breed of the host and season was carried out by Chisquare (1205942-test) Variables with significant association at119875 lt 005 (two-sided) were subjected to the multivariatelogistic regression model The results were each expressed as119875 value and odds ratio (OR) with a 95 confidence interval(CI 95)

3 Results

31 Blood Smear Examination In the present study exami-nation of Giemsa-stained peripheral thin blood smears of 214canines revealed an overall prevalence of canine babesiosis as747with 093 (2214) positivity for the piroplasms of large





1 2 3 4 5 6 M 7 8 9 10 11 12

671bp





4 Discussion








Age



Breed


Pug 24 1 (416) 2 (833)Others 35 mdash 3 (857)


Season


Total 214 14 (654) 33 (1542)lowast





Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1 2 3 4 5 6 M 7 8 9 10 11 12

671bp





4 Discussion








Age



Breed


Pug 24 1 (416) 2 (833)Others 35 mdash 3 (857)


Season


Total 214 14 (654) 33 (1542)lowast





Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Age



Breed


Pug 24 1 (416) 2 (833)Others 35 mdash 3 (857)


Season


Total 214 14 (654) 33 (1542)lowast





Acknowledgment


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

canine babesiosis in northwestern india: molecular detection and

Documents