in dairy animals from punjab,

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 893862 8 pageshttpdxdoiorg1011552013893862

Research ArticleMolecular Prevalence of Babesia bigemina andTrypanosoma evansi in Dairy Animals from PunjabIndia by Duplex PCR A Step Forward to the Detection andManagement of Concurrent Latent Infections

Amrita Sharma1 Lachhman Das Singla1 Ashuma Tuli1 Paramjit Kaur1

Balwinder Kaur Batth1 Mohammed Javed2 and Prayag Dutt Juyal1

1 Department of Veterinary Parasitology College of Veterinary Science Guru Angad Dev Veterinary andAnimal Sciences University Ludhiana Punjab 141004 India

2Department of Mathematics Statistics and Physics Punjab Agricultural University Ludhiana Punjab 141004 India

Correspondence should be addressed to Lachhman Das Singla ldsinglagmailcom

Received 30 April 2013 Revised 22 July 2013 Accepted 25 July 2013

Academic Editor Benoıt Stijlemans

Copyright copy 2013 Amrita Sharma 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

Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairyanimals from 9 districts of Punjab India for simultaneous detection of Babesia bigemina and Trypanosoma evansiThe results werecompared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterationsto know the clinical status and pathogenicity of infections The Bg3Bg4 and TR3TR4 primers were used in duplex PCR for Bbigemina and T evansi amplified products of 689 bp and 257 bp respectively The overall prevalence by duplex PCR was found tobe 3649 243 and 341 for T evansi B bigemina and dual infection respectively A more significant difference was observedfor dual infection status (119875 le 0005) as compared to T evansi (119875 le 005) and B bigemina (119875 le 001) among various districtsunder study A very low prevalence of T evansi (073) and B bigemina (048) was seen by GSTBSThe highly sensitive specificand cost-effective duplex PCR was able to detect latent T evansi and B bigemina infection in cattle and buffaloes Haematologicalevaluation revealed marked pathology in B bigemina infected group and in dual infected group in contrast to that infected with Tevansi alone

1 Introduction

Babesiosis and trypanosomosis are two economically impor-tant vector-borne diseases of tropical and subtropical partsof the world including India Bovine babesiosis caused byan apicomplexan haemoprotozoan parasite Babesia bigem-ina (family Babesiidae order Piroplasmida) is transmittedby brevirostrate tick Rhipicephalus (Boophilus) micropluscausing significant morbidity and mortality in cattle andbuffaloes Trypanosoma evansi the causative agent of theldquoSurrardquo ismechanically transmitted by tabanid flies [1] Dairyanimals especially bovines which are bearing productionstress along with other diseases are potential viable host tothese infections Bovines act as reservoir hosts of surra as the

course of disease remains subclinical India suffers losses ofabout 572 million US dollars annually due to babesiosis inlivestock [2] For African trypanosomosis estimated lossesare to the tune of US $13 billion [3] however no data isavailable on the economic losses due to T evansi

The diagnosis of T evansi and B bigemina is routinelydone by conventional parasitological techniques like Giemsastained thin blood smear (GSTBS) Surra is characterizedby fluctuating parasitaemia with periods of paroxysms andintermissions [4] Giemsa stained blood smear examinationis not a sensitive method to demonstrate parasites in thebloodmainly because of the periodically cryptic nature of theparasite The sensitivity of the technique may be enhancedby concentrating the blood (like buffy coat and minianion

2 BioMed Research International

Table 1 Oligonucleotide primers used for establishment of duplex PCR

Haemoprotozoan Primer Region amplified Product size

B bigemina [10] Bg3 51015840TAG TTG TAT TTC AGC CTC GCG 31015840 Small subunit ribosomal RNAsequence of B bigemina 689 bp

Bg4 51015840 AAC ATC CAA GCA GCT AHT TTA G 31015840

T evansi [11] TR3 51015840 GCG CGG ATT CTT TGC AGA CGA 31015840 Repetitive nucleotide sequences ofT evansi 257 bp

TR4 51015840 TGC AGA CAC TGG AAT GTT ACT 31015840

exchange chromatography) in place of using whole blood[5] Despite the improvement in parasitological techniques asignificantly high proportion of infections remain undetected[6] The recovered animals from B bigemina infection maysustain subclinical infection or become carrier for othersusceptible healthy animals in the herd and a source toinfect the tick vectors The serological tests including theindirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) are capable of detect-ing antibodies in carrier animals therefore they are oftenused for monitoring surveillance and export certification[7] Drawback of serological tests is that antibodies can bedetected even years after recovery of infection though noactive infection is prevalent so these methods cannot helpin revealing the exact picture of prevalence of infection atthat particular point For the diagnosis of subclinical andlatent infection nucleic acid based detection techniques likepolymerase chain reaction (PCR) assay permit the identifi-cation of parasite at levels far below those identified by thecommonly used conventional parasitological techniques forT evansi [8] and B bigemina [9] Further the detection ofboth of these haemoprotozoans simultaneously in a singlereaction by duplex PCR will be both time and cost effective

As both B bigemina and T evansi infections have almostsimilar types of signs and arthropod vectors of these twodiseases also exist together in suitable tropical climaticconditions it becomes necessary that an economical specificand sensitive technique be standardized and employed forsimultaneous detection of these two diseases in suspectedanimals Hence the purpose of the present study was tostandardize and employ a duplex PCR assay for simultaneousdetection of clinical subclinical and latent carrier forms of Tevansi andB bigemina infections for knowing the prevalenceof these infections in different districts of Punjab state andto further evaluate the haematological parameters to knowthe factors contributing to pathogenicity and to prevalenceof these infections in dairy animals for their proper treatmentand management

2 Materials and Methods

21 Sample Collection Blood samples were randomly col-lected from 411 dairy animals (386 cattle and 25 buffaloes)during the period from May to October 2011 from 9 districtsof Punjab state Five mL of blood samples was collected inEDTAcoated vacutainers from the jugular vein of the animalsfor DNA isolation and hematological parameter evaluationSamples were brought from the field to laboratory in athermos containing ice cubes and processed for haematology

without delay The samples were stored at minus20∘C till isolationof DNA

22 Conventional Parasitological Method Thin blood smearswere prepared immediately after each blood collection Theblood smears were air-dried fixed in methanol stained withGiemsa and examined microscopically for the presence ofB bigemina and T evansi Morphometric measurement ofthese haemoprotozoans was done by software DPZ-BSW(OLYMPUS) for parasitological confirmation of the species

23 Molecular Diagnostic Method

231 DNA Template Preparation Genomic DNA wasextracted from the whole blood collected in EDTA-coatedvacutainers using HiMedia HiPurA Blood Genomic DNAMiniPrep Purification Spin Kit as per the given protocol(HiMedia Laboratories India) The blood leucocytes froma three-day-old neonatal calf were included as negativecontrol Trypanosoma evansi parasites of cattle strainpropagated in mice and purified by DEAE cellulose columnschromatographywere used asT evansi positive control whileB bigemina parasites isolated from infected erythrocytes ofclinically infected cattle were used as positive control for Bbigemina

232 Oligonucleotide Primers for Duplex-PCR Oligonu-cleotide primers used for the establishment of duplex-PCRfor T evansi and B bigemina targeted repetitive nucleotidesequences and small subunit ribosomal RNA sequencerespectively as mentioned in Table 1

233 Generation and Visualization of Duplex-PCR Amplifica-tion A total of 25120583L PCR reaction mixture was constitutedwith KAPA 2G fast hot start ready mix (1X containingKAPA2G fast hot start DNA polymerase KAPA 2G fast hotstart PCR buffer 02mM dNTP each and 15mM MgCl

2)

with 15 pmol of Bg3Bg4 primers and 125 pmol of TR3TR4primers and additional 1mM MgCl

2 The reaction was set

in automated thermocycler with the following programmeinitial denaturation at 95∘C (5min) 30 cycles of denaturationat 95∘C (30 sec) annealing at 57∘C (1min) and extension at72∘C (15min) with final extension at 72∘C for 10min Theamplified PCR products were separated by electrophoresis on1 agarose gel and visualized under UV transilluminator fordetection of 257 bp and 689 bp amplified product

BioMed Research International 3

234 Specificity of Primers Primers were examined for theirspecificity for each individual parasite species by amplifi-cation of DNA samples of individual parasite species aswell as from the mixture of DNA samples derived fromparasitologically positive samples of respective haemoproto-zoan with each set of primers DNA extracted from cattlestrain of T evansi was propagated in mice and purifiedby DEAE cellulose columns chromatography as mentionedabove DNA extracted from isolated Babesia bigemina para-sites frominfected erythrocytes from clinically infected cattleused as positive control for B bigemina Bovine leucocytesrsquoDNA from a three-day-old neonatal calf was used as negativecontrol DNA isolated from erythrocytes of cattle infectedwithAnaplasmamarginale andTheileria annulatawas furtherused to check the specificity of the primers

24 Haematological Analysis The haematology of the wholeblood was done with fully automated analyzed ADVIA 2120haematology system (Siemens Health Care Diagnostic IncDeerfield IL USA) as per the instructions of the manufac-turer

25 Statistical Analysis Chi-square test was employed tocompare prevalences of T evansi B bigemina and dualinfections diagnosed by conventional parasitological andmolecular techniques among various districts of PunjabOne-way analysis of variance (ANOVA) was applied onvarious hematological parameters to determine the variancein the animals of different groups that is group A (infectedwith T evansi alone) group B (infected with B bigeminaalone) and group C (infected with both T evansi and Bbigemina) as compared to the non-infected control animals(119875 lt 005) Animals free from external parasites (ticks) neg-ative for haemoprotozoans having hematological parameterswithin normal range free from any clinical sign and with nohistory of any treatment given in the past were kept undernoninfected control group All the values are expressed asmean plusmn standard deviation

3 Results

31 Specificity of PCR Primers PCR amplification employedon each individual DNA sample (T evansi and B bigemina)using their specific primers led to the detection of expectedfragments of size 257 and 689 bp respectively Each setof the primers was found to be specific for the respectiveparasite DNA and amplification of nontarget DNA samplesdid not lead to the production of PCR products whenother haemoparasite samples were used Primer specific forone parasite species did not produce PCR products fromany of the other parasite species (Figure 1) Parasite isolatesfrom different districts were found to show uniform PCRamplification as all the isolates were amplified with the sameset of primers used

32 Relative Efficacy of Conventional Parasitological Methodand Duplex PCR Assay and Corresponding Clinical PictureParasitaemia was observed only in 3 cases for T evansi and

M FEDCBA

689bp

257bp

Figure 1 Agarose gel (15) eletrophoresis showing amplified DNAfrom B bigemina (689 bp) and T evansi (257 bp) LaneMmolecularsize marker 100 bp plus Lane A showing no amplification for Thei-leria annulata genomic DNA Lane B showing amplification for Try-panosoma evansi genomic DNA Lane C showing no amplificationfor host leucocyte DNA Lane D showing amplification for Babesiabigemina genomic DNA Lane E showing no amplification forAnaplasma marginale genomic DNA Lane F showing amplificationfor Babesia bigemina and Trypanosoma evansi genomic DNA

in 2 cases for B bigemina piroplasms by GSTBS Commonclinical manifestations of fever pale mucous membranelacrimation depression and anorexia were observed inparasitologically positive animals for both types of infectionsFrom the clinically positive cases of trypanosomosis only onecattle was having corneal opacity and intermittent fever Twocows positive for babesiosis showed history of haemoglobin-uria and fever Overall it revealed the prevalence of T evansiand B bigemina to be only 073 and 048 respectively innine districts of Punjab under study (Table 2) The highestprevalence of T evansi was observed in district Jalandhar(75) and that of B bigemina was observed in districtLudhiana (206) No case of coinfection of the two parasiteswas revealed by this method

Out of the total sample size 355 of animals portrayedconsistent signs of anemia general weakness history ofreduced feed conversion and occasional episodes of feverTicks were observed on 786 of the affected cattle and 94of the affected buffaloes

Out of 411 samples examined 341 were found to haveconcurrent infection of both protozoans when targeted forduplex PCR (Figure 2) This PCR based diagnosis (Figure 2)revealed an overall prevalence of T evansi to be 3649 andthat of B bigemina to be only 243 A more significantdifference was observed for dual infection status (119875 le 0005)as compared to T evansi (119875 le 005) and B bigemina


M P N A B C D E

689bp

257bp

Figure 2 Agarose gel (15) eletrophoresis showing amplifiedDNAfrom B bigemina (689 bp) and T evansi (257 bp) LaneMmolecularsize marker 100 bp plus Lane P positive control Lane N negativecontrols (genomic DNA from host leucocytes) Lanes A and Bshowing simultaneous amplified B bigemina and T evansi genomicDNA from the blood of animals positive for infection Lane Cshowing amplified B bigemina genomic DNA from the blood ofanimal infected for B bigemina alone Lane D showing amplified Tevansi genomic DNA from the blood of animal infected for T evansialone Lane E showing no amplification indicating being negative fordual infection by PCR

(119875 le 001) among various districts under study withhighest prevalence of coprevalence of both protozoa indistrict Jalandhar (75) while that of T evansi and Bbigemina alone in district Sangrur (4782) and Mansa(741) respectively (Table 2) Based on the prevalence ofthe diseases the veterinarians of the particular regions wereasked to treat the animals specifically Population of the tickswas mainly constituted by R (Boophilus) microplus followedby Hyalomma anatolicum anatolicum Fourteen Tabanusspp flies could be collected only from Jalandhar Ludhianaand Patiala districts

PCR assay could effectively diagnose trypanosome infec-tion in animals under study revealing 4133 of subclinicalcases (with clinical signs but negative byGSTBS) and 566ofcarrier cases (GSTBS negative and with no clinical signs) thatharboured infection (Table 3) Correspondingly similar PCRassay report was observed in case of B bigemina infectionwhich diagnosed 125 clinical 1875 subclinical and 687carrier cases

33 Species-Wise Comparative Prevalence in Cattle and Buf-faloes Among cattle the prevalence of 3601 207 and 284was observed for infections of T evansi B bigemina andconcurrent infection of both parasites respectively by duplexPCR method of diagnosis Prevalence of these infections wasmore in buffaloes being 4400 800 and 1200 for T evansi

B bigemina and concurrent infection of both parasites(Table 2) The coprevalence of both haemoprotozoans wasfound to be higher in buffaloes (12) as compared to cattle(284) by duplex PCR

34 Correlation of the Infection Status with HaematologicalAlterations Leucogram (Table 4 Figure 3) showed signifi-cant (119875 lt 0001) leucocytosis in T evansi infected animals ofgroup A However in group B (B bigemina infected animals)neither leucocytosis nor leucopenia was seen as compared tohealthy animals of group D while animals of group A andgroup C showed significant leucocytosis as compared to thehealthy controls

The total erythrocyte count (TEC) was found reduced ininfected groups as compared to healthy animals (Table 4)Haemoglobin (Hb) was significantly lower (119875 lt 005)in all infected groups as compared to healthy controls InB bigemina infected and dual infected animals Hb wasdecreased more significantly Packed cell volume (PCV) wassignificantly lower in all the infected groups as comparedto healthy control animals the decrease being significant ingroup C animals as compared to group A animals Meancorpuscular volume (MCV) was significantly lower in Tevansi infected animals as compared to B bigemina infectedanimals Mean corpuscular haemoglobin (MCH) decreasedsignificantly in B bigemina and dual infected group ascompared to T evansi infected animals and healthy con-trol animals Mean corpuscular haemoglobin concentration(MCHC) was on higher side in infected groups as comparedto healthy control group with the increase being significantlyhigher in dual infected groups as compared to all othergroupsThrombocytosis was observed inB bigemina infectedgroup as compared to T evansi infected or dual infectedgroups (Table 4)

Overall microcytic normochromic type of anemia wasobserved in all the three infected groups due to significantreduction (119875 lt 001) in the mean corpuscular volume(MCV)

4 Discussion

The morphometric measurement of T evansi was 258 plusmn08 120583m and that of B bigemina was 29 plusmn 02 120583m in GSTBSThese measurements were in accordance with the standarddimensions described for T evansi and B bigemina PCRbased diagnosis showed the prevalence of T evansi and Bbigemina to be 3649 and 243 respectively as comparedto much lower prevalences (T evansi 073 and B bigemina048) observed by GSTBS It justifies the sensitivity ofduplex PCR as it was able to expose the subclinical and carriercases of both T evansi and B bigemina which would haveled to further attention for treatment and control in lieubeing obscured This very well corroborates with the factthat due to antigenic variations cryptic infections are seen inT evansi leading to immunosuppression and nondetectableparasitaemia during the infection processes [4 12 13] Ahigher subclinical incidence of T evansi (346) than Tannulata (16) and B bigemina (06) in cattle has been


Table 2 Prevalence of B bigemina and T evansi alone and in combination by Giemsa stained thin blood smear (GSTBS) examination andduplex polymerase chain reaction (PCR)

Districts covered Samples tested Positive for T evansi alone Positive for B bigemina Positive for both T evansi and B bigeminaBy GSTBS By PCR By GSTBS By PCR By GSTBS By PCR

Ferozepur 25 0 0 0 0 0 0Mansa 54 0 20 (3703) 0 4 (741) 0 1 (185)Moga 18 0 6 (3333) 0 0 0 0Sangrur 23 0 11 (4782) 0 1 (434) 0 1 (434)Bathinda 66 0 23 (3484) 0 0 0 0Fatehgarh Sahib 54 0 22 (4074) 0 1 (185) 0 0Patiala 34 0 15 (4411) 0 0 0 2 (588)Jalandhar 40 3 (75) 12 (30) 0 0 0 3 (75)Ludhiana 97 0 41 (4226) 2 (206) 4 (412) 0 7 (722)Total 411 3 (073) 150 (3649) 2 (048) 10 (243) 0 14 (341)1205942 24lowast 672lowast 16lowastlowast 206lowastlowastlowast NS 2714lowast

Cow 386 3 (077) 139 (3601) 1 (025) 8 (207) 0 11 (284)Buffalo 25 0 11 (44) 1 (4) 2 (8) 0 3 (12)1205942 NS 10922lowast NS NS NS 457lowastlowastlowast119875 le 0005 lowastlowast119875 le 005 and lowastlowastlowast119875 le 001

Table 3 Correlation of infection of T evansi B bigemina and dual infection with sensitivity of PCR

T evansi positive (150) B bigemina positive (10) Mixed infection positive (14)+CS minusCS minusNCS +CS minusCS minusNCS +CS minusCS minusNCS

PCR positive 3 (2) 62 (4133) 85 (566) 2 (200) 3 (30) 5 (50) 0 9 (6485) 5 (3571)+CS sample positive by slide and animal showed clinical signs (clinical cases)minusCS sample negative by slide and animal showed clinical signs (subclinical cases)minusNCS sample negative by slide and animal showed no clinical signs (latent carriers)

Table 4 Haematological values of animals infected with B bigemina T evansi and dual infection

ParameterTLC(times 103cells120583l)

TEC(times106

cells120583l)

Hb(gdL)

PCV()

MCV(fL)

MCHpg

MCHCgdl

Plt(times103

cells120583l)Positive for Tevansi alone(group A)

Total samplestested = 92

1539a plusmn559

550a plusmn163

851a plusmn214

2161a plusmn928

3812a plusmn492

1581a plusmn217

4102a plusmn320

34151a plusmn21497

Positive for Bbigemina alone(group B)


7376b plusmn361

3846b plusmn144

660b plusmn195

1610ab plusmn469

4416b plusmn324

1816b plusmn171

4058a plusmn312

63200b plusmn34859

Positive for Tevansi and Bbigemina (groupC)


2187c plusmn5439

359bc plusmn143

696b plusmn162

1591b plusmn318

4075ab plusmn603

1801bc plusmn573

4428b plusmn596

24087a plusmn23359

Non infectedcontrol group(group D)

Total numberof samples =

30

1106b plusmn125

668d plusmn129

1009c plusmn139

2876c plusmn412

4399bc plusmn604

1542a plusmn255

3512c plusmn318

33683a plusmn11262

Normal range(NR) [22] 4ndash12 5ndash10 8ndash15 24ndash46 40ndash60 144ndash186 26ndash34 100ndash800

Values of TLC TEC Hb PCV MCV MCH MCHC and Plt with different superscripts andashd differ significantly at 119875 le 005 through the columnsTLC total leucocyte count TEC total erythrocyte count HB haemoglobin PCV packed cell volume MCV mean corpuscular volume MCH meancorpuscular haemoglobin MCHC mean corpuscular haemoglobin concentration and Plt platelets


0

10

20

30

40

50

60

TLC TEC Hb PCV MCV MCH MCHC Plt100

Group AGroup B

Group CGroup D

a

b

c

ba

b dbc

ab b c

a

b

c

ab

ab ab

bc

a b a

bc

a ab

c

aa

ab

Figure 3 Haematological parameters of animals infected with T evansi (group a) B bigemina (group b) both T evansi and B bigemina(group c) and non infected control (group d) Values of TLC TEC Hb PCV MCV MCH MCHC and Plt with different letters andashd differsignificantly at 119875 le 005

reported in Karnataka (India) [14] by conventional single-plex PCR Exploring of the carrier and subclinical cases bymore sensitive techniques namely PCR will be helpful injudging the potential threat of the diseases in a particulararea or farm This will further help in the quick and accuratetreatment strategies and management of the diseases [15] aswas advised in the present study

Epidemiological findings [16] have revealed R microplusas the predominant tick population (8528) in Punjab(India) which is responsible for transmission of B bigeminain cattle and buffaloes By using GSTBS as well as PCR ahigher number of cases of haemoprotozoa infection wereobserved in buffaloes than in cattle This may be due to greatvariation in the sample size of cattle and buffaloes [17] Pre-viously parasitological prevalences by GSTBS examinationwere reported to be 792 for T evansi [18] and 594 forB bigemina in dairy animals [19] in Punjab state History ofpresence of tabanid fly in the vicinity was frequently recordedfrom the area of Moga Ferozepur and Sangrur districtsFarms at district Jalandhar were in the vicinity of paddy fieldswhich are conducive for breeding of tabanid fliesThismay beattributed to high prevalence ofT evansi in Jalandhar district

No observation of mixed infection of T evansi andB bigemina by GSTBS examination may be due to lowsensitivity of this conventional parasitological technique[15] Buffaloes are considered to be more resistant towardsthe haemoprotozoan diseases [20] Hence buffaloes act asreservoir hosts to other animals and may become clinicallyill under stress conditions Further it was observed thatcrossbred cattle are more sensitive to these haemoprotozoaninfections as compared to buffaloes as the clinical cases wereencountered only in crossbred cattle Infection rate of Tevansi and B bovis has been reported to be 137 and 74 inbuffaloes as depicted by duplex PCR [21] A very little or nomixed infection was observed in dairy cows it was 37 and0 respectively for T evansi and B bovis

Duplex PCR due to its high sensitivity and specificity isable to detect low level of infections and can be suitable for

epidemiological study of T evansi and B bigemina infectionin cattle and buffaloes The incidence of coinfection of bothprotozoa is lesser which may be due to different vectorsresponsible for their transmission The reason for highermolecular prevalence of T evansi as compared to B bigeminacan be corroborated with the fact that the tabanid flies haveinterrupted feeding habit and can travel longer distances ascompared to the ticks

The observed leucocytosis in animals of groups C andA is in agreement with some previous reports [23ndash25] Itmay be due to the multiplicity of the antigens resultinginto more stimulation of immune system The insignificantchanges in total leucocyte count in B bigemina infectedgroup may be due to the breakdown of red blood cellsby the protozoan stimulating phagocytic cells such as lym-phocytes and monocytes to clean up the toxic remnants ofruptured red blood cell [26] The significant decrease in HbTEC and PCV in B bigemina infected group as comparedto other groups may be due to intravascular haemolysiswhich is supported by the previous reports [27] Overallhematological revelations showed that anemia was moresevere in B bigemina infected and dual infected animals ascompared to the animals infectedwithT evansi alone Resultsindicated that even subclinical and latent carrier infectionsdiagnosed by molecular means are responsible for inducingpathogenicity

5 Conclusion

The present study reveals the concurrent latent infectionof babesiosis and trypanosomosis in cattle and buffaloes inPunjab The diagnosis of the dual latent infections by duplexPCR will be helpful in control and prevention of these deadlydiseases as this technique is both time and cost effective Thetechnique is useful in the detection of molecular prevalenceof the haemoprotozoan diseasesThe haematological analysissupported the pathogenicity of the dual latent infection


Abbreviations

PCR Polymerase chain reactionGSTBS Giemsa stained thin blood smear

Conflict of Interests

The authors declare that there is no professional or financialconflict of interests related to this paper

Acknowledgment

Thanks are due to Director of Research Guru Angad DevVeterinary and Animal Sciences University Ludhiana forproviding financial support under Rashtriya Krishi VikasYojna project ldquoEmployment of immunomolecular diagnostictools for haemoprotozoan diseases of livestock in Punjabstaterdquo to carry out the research work

References

[1] A L Sumba S Mihok and F A Oyieke ldquoMechanical trans-mission of Trypanosoma evansi and T congolense by Stomoxysniger and S taeniatus in a laboratory mouse modelrdquo Medicaland Veterinary Entomology vol 12 no 4 pp 417ndash422 1998

[2] R McLeod and P Kristjanson Final Report of Joint ESYSInternational Livestock Research InstituteAustralian Centre forInternational Agricultural Research Tick Cost Project-EconomicImpact of Ticks and Tick-Borne Diseases to Livestock in AfricaAsia and Australia International Livestock Research InstituteNairobi Kenya 1999

[3] OIE ldquoTrypanosoma evansi infections (surra)rdquo in TerrestrialManual Chapter 2117 Office International Des Epizootiesvol 1 pp 1ndash14 World Health Organization for Animal HealthParis France 2010 httpwwwoieinteninternational-stand-ard-settingterrestrial-manualaccess-online

[4] B S Gill Trypanosomes and Trypanosomiases of Indian Live-stock Indian Council for Agricultural Research New DelhiIndia 1991

[5] S A Reid A Husein and D B Copeman ldquoEvaluation andimprovement of parasitological tests for Trypanosoma evansiinfectionrdquo Veterinary Parasitology vol 102 no 4 pp 291ndash2972001

[6] A G Luckins ldquoProtozoal diseases of camelsrdquo in Proceedings ofthe 1st International Conference W R Allen A J Higgins I GMayhew D H Snow and J F Wade Eds pp 23ndash27 R and WPublication New Market Limited Suffolk UK 1992

[7] F R Araujo C R Madruga C R B Leal et al ldquoComparisonbetween enzyme-linked immunosorbent assay indirect fluo-rescent antibody and rapid conglutination tests in detectingantibodies against Babesia bovisrdquo Veterinary Parasitology vol74 no 2-4 pp 101ndash108 1998

[8] MDesquesnes andAMRDavila ldquoApplications of PCR-basedtools for detection and identification of animal trypanosomesa review and perspectivesrdquo Veterinary Parasitology vol 109 no3-4 pp 213ndash231 2002

[9] J V Figueroa L P Chieves G S Johnson and G M BueningldquoMultiplex polymerase chain reaction based assay for thedetection of Babesia bigemina Babesia bovis and Anaplasmamarginale DNA in bovine bloodrdquo Veterinary Parasitology vol50 no 1-2 pp 69ndash81 1993

[10] J Ellis C Hefford P R Baverstock B P Dalrymple and AM Johnson ldquoRibosomal DNA sequence comparison of Babesiaand TheileriardquoMolecular and Biochemical Parasitology vol 54no 1 pp 87ndash95 1992

[11] N Wuyts N Chokesajjawatee and S Panyim ldquoA simplifiedand highly sensitive detection of Trypanosoma evansi by DNAamplificationrdquoThe Southeast Asian Journal of Tropical Medicineand Public Health vol 25 no 2 pp 266ndash271 1994

[12] L D Singla S K Sharma and P D Juyal ldquoPharmacokineticsof diminazene in combination with procaine and antipyrine inTrypanosoma evansi infected buffalo calvesrdquo Indian Journal ofAnimal Sciences vol 78 no 6 pp 571ndash575 2008

[13] L D Singla P D Juyal and N S Sharma ldquoImmune responsesto haemorrhagic septicaemia (HS) vaccination in Trypanosomaevansi infected buffalo-calvesrdquo Tropical Animal Health andProduction vol 42 no 4 pp 589ndash595 2010

[14] A SNair R Ravindran B Lakshmanan et al ldquoHaemoprotozoaof cattle in Northern Kerala Indiardquo Tropical Biomedicine vol28 no 1 pp 68ndash75 2011

[15] P D Juyal L D Singla and P Kaur ldquoManagement of surradue to Trypanosoma evansi in India an overviewrdquo in InfectiousDiseases of Domestic Animals and Zoonosis in India V Tandonand B N Dhawan Eds vol 75 pp 109ndash120 Proceedings of theNational Academy of Sciences New Delhi India 2005

[16] M Haque J Jyoti N K Singh S S Rath and S GhoshldquoEpidemiology and seasonal dynamics of Ixodid ticks of dairyanimals of Punjab state Indiardquo Indian Journal of AnimalSciences vol 81 no 7 pp 661ndash664 2011

[17] C L Yadav R P Gupta and N S Ruprah ldquoThe prevalenceof haemoprotozoan infections in cattle and buffaloesrdquo IndianVeterinary Medical Journal vol 9 pp 205ndash209 1985

[18] G S Aulakh L D Singla P Kaur and A Alka ldquoBovinebabesiosis due to Babesia bigemina haematobiochemical andtherapeutic studiesrdquo Indian Journal of Animal Sciences vol 75no 6 pp 617ndash622 2005

[19] G S Aulakh L D Singla and J Singh ldquoBovine trypanosomosisdue to Trypanosoma evansi clinical haematobiochemical andtherapeutic studiesrdquo in New Horizons in Animal Sciences RC Sobti and V L Sharma Eds Vishal Publishing JalandharIndia 2005

[20] DK Singh ldquoRecent developmentsrdquo inTheResearch andControlofTheileria Annulata Proceedings of aWorkshop Held at Ilrad TT Dolan Ed pp 17ndash19 Nairobi Kenya September 1990

[21] Y ShiWHuangW Zhang et al ldquoRapid and sensitive detectionof Trypanosoma evansi and Babesia bovis in bovine by doublePCRrdquo Guangxi Agricultural Sciences vol 41 pp 163ndash166 2010

[22] CM Kahn S Line and S E Aiello Eds Reference Guide Table6 in Merck Manual Merck amp Co Whitehousestation NJ USA9th edition 2005

[23] P S Walia I S Kalra P D Juyal and S P Ahuja ldquoRoleof activity of Trypanosoma evansi in inducing anemia andimmunomodulation in buffalo calvesrdquo Journal of VeterinaryParasitology vol 10 pp 1ndash9 1996

[24] M D Kulkarni M B Nisal M K J Rao and P E KulkarnildquoHaemodynamic studies of trypanosomiasis in buffalo (bubalusbublais)rdquo Indian Veterinary Journal vol 74 no 11 pp 981ndash9821997

[25] P Kaur and P D Juyal ldquoHaematobiochemical changes inexperimental surra in cow calves treated with diminazeneaceturate alongwith antipyrine andprocainerdquo IndianVeterinaryJournal vol 80 no 10 pp 975ndash978 2003


[26] A H Hussein N A E S Mohammed and H K MohammedldquoTheileriosis and babesiosis in cattle haemogram and somebiochemical parametersrdquo in Proceedings of XIII InternationalCongress of International Society of AnimalHygiene pp 143ndash150Tartu Estonia June2007

[27] A K Sharma R C Katoch K B Nagal R S Kishtwaria andS K Sharma ldquoBovine Babesiosis in Palam Valley of HimachalPradeshrdquo Indian Veterinary Journal vol 77 no 8 pp 731ndash7322000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Zoology


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Microbiology


Table 1 Oligonucleotide primers used for establishment of duplex PCR

Haemoprotozoan Primer Region amplified Product size

B bigemina [10] Bg3 51015840TAG TTG TAT TTC AGC CTC GCG 31015840 Small subunit ribosomal RNAsequence of B bigemina 689 bp

Bg4 51015840 AAC ATC CAA GCA GCT AHT TTA G 31015840

T evansi [11] TR3 51015840 GCG CGG ATT CTT TGC AGA CGA 31015840 Repetitive nucleotide sequences ofT evansi 257 bp

TR4 51015840 TGC AGA CAC TGG AAT GTT ACT 31015840

exchange chromatography) in place of using whole blood[5] Despite the improvement in parasitological techniques asignificantly high proportion of infections remain undetected[6] The recovered animals from B bigemina infection maysustain subclinical infection or become carrier for othersusceptible healthy animals in the herd and a source toinfect the tick vectors The serological tests including theindirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) are capable of detect-ing antibodies in carrier animals therefore they are oftenused for monitoring surveillance and export certification[7] Drawback of serological tests is that antibodies can bedetected even years after recovery of infection though noactive infection is prevalent so these methods cannot helpin revealing the exact picture of prevalence of infection atthat particular point For the diagnosis of subclinical andlatent infection nucleic acid based detection techniques likepolymerase chain reaction (PCR) assay permit the identifi-cation of parasite at levels far below those identified by thecommonly used conventional parasitological techniques forT evansi [8] and B bigemina [9] Further the detection ofboth of these haemoprotozoans simultaneously in a singlereaction by duplex PCR will be both time and cost effective

As both B bigemina and T evansi infections have almostsimilar types of signs and arthropod vectors of these twodiseases also exist together in suitable tropical climaticconditions it becomes necessary that an economical specificand sensitive technique be standardized and employed forsimultaneous detection of these two diseases in suspectedanimals Hence the purpose of the present study was tostandardize and employ a duplex PCR assay for simultaneousdetection of clinical subclinical and latent carrier forms of Tevansi andB bigemina infections for knowing the prevalenceof these infections in different districts of Punjab state andto further evaluate the haematological parameters to knowthe factors contributing to pathogenicity and to prevalenceof these infections in dairy animals for their proper treatmentand management

2 Materials and Methods

21 Sample Collection Blood samples were randomly col-lected from 411 dairy animals (386 cattle and 25 buffaloes)during the period from May to October 2011 from 9 districtsof Punjab state Five mL of blood samples was collected inEDTAcoated vacutainers from the jugular vein of the animalsfor DNA isolation and hematological parameter evaluationSamples were brought from the field to laboratory in athermos containing ice cubes and processed for haematology

without delay The samples were stored at minus20∘C till isolationof DNA

22 Conventional Parasitological Method Thin blood smearswere prepared immediately after each blood collection Theblood smears were air-dried fixed in methanol stained withGiemsa and examined microscopically for the presence ofB bigemina and T evansi Morphometric measurement ofthese haemoprotozoans was done by software DPZ-BSW(OLYMPUS) for parasitological confirmation of the species

23 Molecular Diagnostic Method

231 DNA Template Preparation Genomic DNA wasextracted from the whole blood collected in EDTA-coatedvacutainers using HiMedia HiPurA Blood Genomic DNAMiniPrep Purification Spin Kit as per the given protocol(HiMedia Laboratories India) The blood leucocytes froma three-day-old neonatal calf were included as negativecontrol Trypanosoma evansi parasites of cattle strainpropagated in mice and purified by DEAE cellulose columnschromatographywere used asT evansi positive control whileB bigemina parasites isolated from infected erythrocytes ofclinically infected cattle were used as positive control for Bbigemina

232 Oligonucleotide Primers for Duplex-PCR Oligonu-cleotide primers used for the establishment of duplex-PCRfor T evansi and B bigemina targeted repetitive nucleotidesequences and small subunit ribosomal RNA sequencerespectively as mentioned in Table 1

233 Generation and Visualization of Duplex-PCR Amplifica-tion A total of 25120583L PCR reaction mixture was constitutedwith KAPA 2G fast hot start ready mix (1X containingKAPA2G fast hot start DNA polymerase KAPA 2G fast hotstart PCR buffer 02mM dNTP each and 15mM MgCl

2)

with 15 pmol of Bg3Bg4 primers and 125 pmol of TR3TR4primers and additional 1mM MgCl

2 The reaction was set

in automated thermocycler with the following programmeinitial denaturation at 95∘C (5min) 30 cycles of denaturationat 95∘C (30 sec) annealing at 57∘C (1min) and extension at72∘C (15min) with final extension at 72∘C for 10min Theamplified PCR products were separated by electrophoresis on1 agarose gel and visualized under UV transilluminator fordetection of 257 bp and 689 bp amplified product





3 Results



M FEDCBA

689bp

257bp






M P N A B C D E

689bp

257bp









4 Discussion












TEC(times106

cells120583l)

Hb(gdL)

PCV()

MCV(fL)

MCHpg

MCHCgdl

Plt(times103



1539a plusmn559

550a plusmn163

851a plusmn214

2161a plusmn928

3812a plusmn492

1581a plusmn217

4102a plusmn320

34151a plusmn21497



7376b plusmn361

3846b plusmn144

660b plusmn195

1610ab plusmn469

4416b plusmn324

1816b plusmn171

4058a plusmn312

63200b plusmn34859



2187c plusmn5439

359bc plusmn143

696b plusmn162

1591b plusmn318

4075ab plusmn603

1801bc plusmn573

4428b plusmn596

24087a plusmn23359



30

1106b plusmn125

668d plusmn129

1009c plusmn139

2876c plusmn412

4399bc plusmn604

1542a plusmn255

3512c plusmn318

33683a plusmn11262




0

10

20

30

40

50

60


Group AGroup B

Group CGroup D

a

b

c

ba

b dbc

ab b c

a

b

c

ab

ab ab

bc

a b a

bc

a ab

c

aa

ab








5 Conclusion



Abbreviations




Acknowledgment


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results



M FEDCBA

689bp

257bp






M P N A B C D E

689bp

257bp









4 Discussion












TEC(times106

cells120583l)

Hb(gdL)

PCV()

MCV(fL)

MCHpg

MCHCgdl

Plt(times103



1539a plusmn559

550a plusmn163

851a plusmn214

2161a plusmn928

3812a plusmn492

1581a plusmn217

4102a plusmn320

34151a plusmn21497



7376b plusmn361

3846b plusmn144

660b plusmn195

1610ab plusmn469

4416b plusmn324

1816b plusmn171

4058a plusmn312

63200b plusmn34859



2187c plusmn5439

359bc plusmn143

696b plusmn162

1591b plusmn318

4075ab plusmn603

1801bc plusmn573

4428b plusmn596

24087a plusmn23359



30

1106b plusmn125

668d plusmn129

1009c plusmn139

2876c plusmn412

4399bc plusmn604

1542a plusmn255

3512c plusmn318

33683a plusmn11262




0

10

20

30

40

50

60


Group AGroup B

Group CGroup D

a

b

c

ba

b dbc

ab b c

a

b

c

ab

ab ab

bc

a b a

bc

a ab

c

aa

ab








5 Conclusion



Abbreviations




Acknowledgment


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M P N A B C D E

689bp

257bp









4 Discussion












TEC(times106

cells120583l)

Hb(gdL)

PCV()

MCV(fL)

MCHpg

MCHCgdl

Plt(times103



1539a plusmn559

550a plusmn163

851a plusmn214

2161a plusmn928

3812a plusmn492

1581a plusmn217

4102a plusmn320

34151a plusmn21497



7376b plusmn361

3846b plusmn144

660b plusmn195

1610ab plusmn469

4416b plusmn324

1816b plusmn171

4058a plusmn312

63200b plusmn34859



2187c plusmn5439

359bc plusmn143

696b plusmn162

1591b plusmn318

4075ab plusmn603

1801bc plusmn573

4428b plusmn596

24087a plusmn23359



30

1106b plusmn125

668d plusmn129

1009c plusmn139

2876c plusmn412

4399bc plusmn604

1542a plusmn255

3512c plusmn318

33683a plusmn11262




0

10

20

30

40

50

60


Group AGroup B

Group CGroup D

a

b

c

ba

b dbc

ab b c

a

b

c

ab

ab ab

bc

a b a

bc

a ab

c

aa

ab








5 Conclusion



Abbreviations




Acknowledgment


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











TEC(times106

cells120583l)

Hb(gdL)

PCV()

MCV(fL)

MCHpg

MCHCgdl

Plt(times103



1539a plusmn559

550a plusmn163

851a plusmn214

2161a plusmn928

3812a plusmn492

1581a plusmn217

4102a plusmn320

34151a plusmn21497



7376b plusmn361

3846b plusmn144

660b plusmn195

1610ab plusmn469

4416b plusmn324

1816b plusmn171

4058a plusmn312

63200b plusmn34859



2187c plusmn5439

359bc plusmn143

696b plusmn162

1591b plusmn318

4075ab plusmn603

1801bc plusmn573

4428b plusmn596

24087a plusmn23359



30

1106b plusmn125

668d plusmn129

1009c plusmn139

2876c plusmn412

4399bc plusmn604

1542a plusmn255

3512c plusmn318

33683a plusmn11262




0

10

20

30

40

50

60


Group AGroup B

Group CGroup D

a

b

c

ba

b dbc

ab b c

a

b

c

ab

ab ab

bc

a b a

bc

a ab

c

aa

ab








5 Conclusion



Abbreviations




Acknowledgment


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40

50

60


Group AGroup B

Group CGroup D

a

b

c

ba

b dbc

ab b c

a

b

c

ab

ab ab

bc

a b a

bc

a ab

c

aa

ab








5 Conclusion



Abbreviations




Acknowledgment


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abbreviations




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

in dairy animals from punjab,

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