Research Article Prevalence and Molecular Epidemiological Data on Dirofilaria immitis in Dogs from Northeastern States of India Sonjoy Kumar Borthakur, 1 Dilip Kumar Deka, 2 Saidul Islam, 2 Dilip Kumar Sarma, 3 and Prabhat Chandra Sarmah 2 1 College of Veterinary Sciences & A.H., Central Agricultural University, Selesih, Aizawl, Mizoram 796 014, India 2 College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam 781 022, India 3 National Research Centre on Pigs (ICAR), Rani, Guwahati, Assam 781 131, India Correspondence should be addressed to Sonjoy Kumar Borthakur; sanjoy barthakur@rediffmail.com Received 7 July 2014; Revised 19 October 2014; Accepted 12 November 2014 Academic Editor: Gabriella Cancrini Copyright © 2015 Sonjoy Kumar Borthakur et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e aim of the present study was to determine the prevalence of Dirofilaria immitis in stray, pet, and working dogs ( = 413, 266, and 103, resp.) from Guwahati (Assam) and Aizawl (Mizoram), areas located in two Northeastern States of India. Diagnostic methods applied were microscopy (wet film and Knott’s concentration technique), immunological test (Ag ELISA by SNAP 4Dx ELISA kit), and molecular tools (polymerase chain reaction and sequencing), which evidenced 11.38, 18.03, and 13.93% of positive animals, respectively. No significant differences were observed by area (18.23% versus 17.68%) nor by sex (18.1% versus 17.9%), whereas stray dogs proved more infected than other groups ( < 0.05). ELISA test evidenced an overall 22.69% of occult infections, mainly in working dogs (60%), and molecular techniques detected Dirofilaria (Nochtiella) repens in 4 stray dogs from Guwahati. Characterization of D. immitis isolates for ITS-2 region showed close identity with South Asian isolates. 1. Introduction Dirofilaria immitis, the heartworm of dog, is one of the most important filaroid nematodes responsible for causing canine dirofilariosis. Heartworm inhabits the right ventricle and pulmonary arteries of dogs and other animals. e heartworm parasite is transmitted by various mosquitoes belonging to the genera Culex, Aedes, and Anopheles. Adult female D. immitis lays microfilariae, which are taken up by suitable mosquito vectors and subsequently develop to the infective 3rd larval stage. Transmission takes place when a potential vector bites dogs or other hosts during a subsequent blood meal. It takes about 6-7 months to become an adult stage. Pathophysiological response to heartworm infection is mainly due to the presence of adult worm. e main clinical symptoms in dirofilariosis include persistent cough, difficult breathing, and poor exercise tolerance followed by ascites, anorexia, and weight loss. e symbiotic relationship with Wolbachia (a rickettsia) along with D. immitis amplifies disease severity [1]. e pathogenesis, pathology, and clinical manifestations of heartworm have been aptly reviewed [2]. Laboratory diagnosis of dirofilariosis in live animals is always in forefront in terms of demonstration and identifi- cation of microfilariae in tested blood sample. Radiography and cardiography aid in the diagnosis of D. immitis, but confirmatory and reliable diagnosis for heartworm disease is dependent on serology and molecular tests. Sometimes, in circulating blood of heartworm infected dogs microfilariae are absent and such condition is termed as “occult infection.” In this case, obviously microscopy and PCR give false neg- ative results. Several commercial ELISA based test kits are available to diagnose heartworm in dogs but these kits are not widely used in India. DNA based techniques provide an alternative approach which is very sensitive and accurate for identification of the filarial parasites [3]. Heartworm disease due to D. immitis has also been reported as an emerging zoonosis by several authors [4–8]. Human infection is mostly located in temperate, tropical, and Hindawi Publishing Corporation e Scientific World Journal Volume 2015, Article ID 265385, 7 pages http://dx.doi.org/10.1155/2015/265385
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Research ArticlePrevalence and Molecular Epidemiological Data onDirofilaria immitis in Dogs from Northeastern States of India
Sonjoy Kumar Borthakur1 Dilip Kumar Deka2 Saidul Islam2
Dilip Kumar Sarma3 and Prabhat Chandra Sarmah2
1College of Veterinary Sciences amp AH Central Agricultural University Selesih Aizawl Mizoram 796 014 India2College of Veterinary Science Assam Agricultural University Khanapara Guwahati Assam 781 022 India3National Research Centre on Pigs (ICAR) Rani Guwahati Assam 781 131 India
Correspondence should be addressed to Sonjoy Kumar Borthakur sanjoy barthakurrediffmailcom
Received 7 July 2014 Revised 19 October 2014 Accepted 12 November 2014
Academic Editor Gabriella Cancrini
Copyright copy 2015 Sonjoy Kumar Borthakur et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
The aim of the present study was to determine the prevalence of Dirofilaria immitis in stray pet and working dogs (119899 = 413266 and 103 resp) from Guwahati (Assam) and Aizawl (Mizoram) areas located in two Northeastern States of India Diagnosticmethods applied were microscopy (wet film and Knottrsquos concentration technique) immunological test (Ag ELISA by SNAP 4DxELISA kit) and molecular tools (polymerase chain reaction and sequencing) which evidenced 1138 1803 and 1393 of positiveanimals respectively No significant differences were observed by area (1823 versus 1768) nor by sex (181 versus 179)whereas stray dogs provedmore infected than other groups (119875 lt 005) ELISA test evidenced an overall 2269 of occult infectionsmainly in working dogs (60) and molecular techniques detected Dirofilaria (Nochtiella) repens in 4 stray dogs from GuwahatiCharacterization of D immitis isolates for ITS-2 region showed close identity with South Asian isolates
1 Introduction
Dirofilaria immitis the heartworm of dog is one of themost important filaroid nematodes responsible for causingcanine dirofilariosis Heartworm inhabits the right ventricleand pulmonary arteries of dogs and other animals Theheartworm parasite is transmitted by various mosquitoesbelonging to the genera Culex Aedes and Anopheles Adultfemale D immitis lays microfilariae which are taken up bysuitable mosquito vectors and subsequently develop to theinfective 3rd larval stage Transmission takes place when apotential vector bites dogs or other hosts during a subsequentblood meal It takes about 6-7 months to become an adultstage Pathophysiological response to heartworm infectionis mainly due to the presence of adult worm The mainclinical symptoms in dirofilariosis include persistent coughdifficult breathing and poor exercise tolerance followed byascites anorexia and weight loss The symbiotic relationshipwithWolbachia (a rickettsia) along with D immitis amplifies
disease severity [1] The pathogenesis pathology and clinicalmanifestations of heartworm have been aptly reviewed [2]
Laboratory diagnosis of dirofilariosis in live animals isalways in forefront in terms of demonstration and identifi-cation of microfilariae in tested blood sample Radiographyand cardiography aid in the diagnosis of D immitis butconfirmatory and reliable diagnosis for heartworm disease isdependent on serology and molecular tests Sometimes incirculating blood of heartworm infected dogs microfilariaeare absent and such condition is termed as ldquooccult infectionrdquoIn this case obviously microscopy and PCR give false neg-ative results Several commercial ELISA based test kits areavailable to diagnose heartworm in dogs but these kits arenot widely used in India DNA based techniques provide analternative approach which is very sensitive and accurate foridentification of the filarial parasites [3]
Heartworm disease due to D immitis has also beenreported as an emerging zoonosis by several authors [4ndash8]Human infection ismostly located in temperate tropical and
Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 265385 7 pageshttpdxdoiorg1011552015265385
2 The Scientific World Journal
subtropical areas of the world So far more than 1700 humancases of dirofilariosis (including gt370 pulmonary cases)have been documented worldwide suggesting that wherevercanine dirofilariosis is present humans are at risk of infection[9 10] Most D immitis human infections are asymptomaticshowing typical coin lesions on chest radiography and areoften mistakenly removed as neoplasm [11] In India the firstcase of human pulmonary dirofilariosis due to D immitiswas reported from Mumbai [12] After that several cases ofhuman dirofilariosis have been reported in India [13]
In animals there have been both epidemiological andclinical case studies of this worm worldwide [14ndash20] Preva-lence of this parasite in dogs from several parts from India hasbeen reported [21 22] Limited works revealed occurrenceof 3375 D immitis in Mizoram a Northeastern State ofIndia on the basis of examination of 240 dogs at slaughter[23] A recent study based on wet film and antigen testsrevealed 476 to 2954 prevalence ofD immitis in a hospitalpopulation of dogs fromAssam [24] But no systematic workon epidemiological aspect of D immitis has been carried outin the northeastern part of this country using combinationof conventional serological and molecular diagnostic tech-niques
The importance of this parasite and the paucity ofinformation about the prevalence of canine heartworm inthe Northeastern States of India inspired us to conductthe present plan of work We studied blood samples fromdogs that presented for veterinary attention and from dogscaptured by various nongovernmental organizations andmaintained in kennels The prevalence was studied by meansof conventional microscopy an immunological test andmolecular techniques and their efficacy was comparedFurther we explored some of the molecular characteristics ofD immitis in the studied area
2 Materials and Methods
21 Study Areas The study was undertaken systematicallyfor a period of one calendar year from August 2011 to July2012 in dogs from Guwahati and Aizawl Guwahati a city ofAssam having annual rainfall of 1500ndash2600mm is located at26∘111015840010158401015840 latitude N and 91∘441015840010158401015840 longitude E and Aizawl thecapital city of Mizoram State having annual rainfall of 2400ndash2962mm is located at 23∘4310158402710158401015840N and 92∘431015840210158401015840E The citiesare separated by surface distance of 550 km The location ofepidemiological study undertaken for the present study isshown in the map (Figure 1)
22 Selection of Dogs In the present investigation 3 cat-egories of dogs grouped as working dogs of military andparamilitary force pet dogs and stray dogs were selectedPet dogs of different breeds and paramilitary dogs mostlyof Labrador and German Shepherd breeds brought to theTeaching Veterinary Clinical Complexes (TVCC) of theCollege of Veterinary Science AssamAgricultural UniversityKhanapara Guwahati Assam and the College of VeterinarySciences amp AH Central Agricultural University SelesihAizawl Mizoram during the study period were taken for the
Northeast India
China (Tibet)
Bhutan
WestBengal
Itanagar
Arunachal Pradesh
Dispur Assam
Bangladesh
MeghalayaShillong
Myanmar (Burma)AgartalaTripura
AizawlMizoram
ManipurImphal
NagalandKohima
Figure 1 Map showing the study areas
study In case of military dogs most of the blood sampleswere directly sent to the Department of Parasitology forroutine check-up of heartworm infection The stray dogpopulation consisted of local nondescript street dogs of eithersex captured fromdifferent parts of the city for sterilization bylocal nongovernmental organizations like Peoples for Animal(PFA) and Just Be Friendly (JBF)
Three categories of dogs like working (103) pet (266) andstray (413) totaling 782 numbers were examined Dogs underthe study were of both sexes and a total of 488 dogs fromdifferent localities of Guwahati and 294 dogs from Aizawlformed the entire base of study during the programme
23 Blood Sampling Approximately 5mL of blood wasdrawn from the cephalic vein collected in disodium salt ofethylene diamine tetraacetic acid (Na
2EDTA) vacuum tubes
and stored at 4∘C until further use
24 Parasitological Investigation The prevalence study forD immitis was conducted on the basis ofconventionalimmunological and molecular techniques The conventionaltests were based onmicrofilarial availability in blood samplesassessed on the basis of wet blood film method and modifiedKnottrsquos concentration technique (KCT) [25] Subsequentlymicrofilarial identification was done [26] The immuno-logical evidence was based on the presence of heartwormantigens in tested blood samples and was performed with acommercially available ELISA test kit (SNAP 4Dx) followingmanufacturersquos test protocol Molecular evidence was basedon amplification of worm targeted DNA Blood samplesnegative for D immitis circulating antigen but positive atKCTwere processed for this technique Fresh adultDimmitisand positive blood samples collected from Aizawl slaughterhouse were used to standardize techniques and used aspositive control for the tests In addition blood samples weretaken from dogs clinically infected and found positive for Dimmitis being confirmed by KCT and SNAP tests Briefly themolecular technique for the present study was performed asfollows
The Scientific World Journal 3
Table 1 Prevalence of Dirofilaria immitis by category of dogs and area of study on the basis of Ag ELISA test
Dog categoryGuwahati Aizawl Overall
Numbers Positive Chi value Numbers Positive
Chi value Numbers Positive Chi value
Stray dogs 223 63 (2825)276139
190 44 (2315)110458
413 107 (2590)367706Pet dogs 174 17 (977) 92 7 (760) 266 24 (902)
Working dogs 91 9 (989) 12 1 (833) 103 10 (970)Total 488 89 (1823) 294 52 (1768) 782 141 (1803)
25 Isolation of Genomic DNA fromBlood andAdult ParasitesIsolation of genomic DNA from blood and adult parasite wascarried out using the DNeasy Blood and Tissue Kit (QiagenKit Catalogue number 69504) as per the protocols providedby the manufacturer The final templates were kept at minus20∘C
26 PCR Assays PCR technique targeted to amplify theITS-2 region of filarial wormsrsquo rDNA was applied accordingto previously described protocols [3 27] Briefly reactionmixture comprised 25120583L Taq polymerase buffer (10x) 01120583LdNTP (10mM) 05 120583L MgCl
2(50mM) 075 120583L of each
forward and reverse primer (60 pM) 1 UTaq polymerase and30 120583L template DNA (60 ng120583L concentration) by makingthe final volume up to 250 120583L with NFW The cyclingcondition used for amplifying the targeted product consistedof an initial denaturing step at 94∘C for 2min and 32 cyclesof denaturing (30 s at 94∘C) annealing (30 s at 60∘C for58S-ITS-2-28S-based primers and 30 s at 58∘C for ITS-2-based primers) and extension (30 s at 72∘C) a final extension(7min at 72∘C or 12min at 72∘C for cloning) and a soakat 4∘C in a Techne-5000 thermal cycler (Bibby Scientific)The confirmation of the amplified products was made by gelelectrophoresis of the PCRproduct in 15 agarose gel stainedwith ethidium bromide and visualized under gel doc (DNRBio-Imaging System MiniLumi)
The specificity of the PCR amplification for the corre-sponding D immitis target both on representative positiveblood samples and on adult worms was assessed by ampliconpurification followed by cloning and sequencing
27 Cloning of the Genomic Region Cloning of the PCRamplicon(s) for the genomic region as described above hasbeen performed using pDrive cloning vector (Qiagen PCRCloning Kit Catalog number 231124) DH5120572 E coli cell wasused for transformation of the plasmid using Transform AidBacterial Transformation Kit (Fermentas Catalog numberK2711) Subsequently clones were confirmed by clonesrsquo con-firmation PCR
28 Sequencing and Analysis of ITS-2 The recombinantclones were sent to the Department of Biochemistry Uni-versity of Delhi South Campus for automated sequencingThe sequences obtained were aligned and compared withother published sequences ofD immitis of dogs by ClustalWmethod using DNASTAR software and phylogenic analysiswas done Sequences were compared in silico with sequencesof D immitis (ITS-2) rDNA available in the gene bank for
each gene examined using the nucleotide-nucleotide ldquoBasicLocal Alignment Search Toolrdquo Sequences were submitted toNCBI to obtain accession numbers
3 Results
The study of the prevalence of canine heartworm carried outby means of different techniques obviously produced differ-ent results Overall 626 (49782) blood samples analysedproved microfilaraemic under wet film methods whereasinfection rates found by means of the other techniques werehigher As expected the highest number of positive animalswas detected by the ELISA test which evidenced an overallprevalence of 1803 (Table 1) without differences by areaand by sex On the contrary in both areas differences wereevidenced by group of dogs Table 2 summarizes data onthe efficacy of KCT ELISA test and PCR which classifiedas infected 1138 183 and 1393 of the examined dogsrespectively with stray dogs always more infected than othergroups Moreover PCR analysis with primers specific for Dimmitis detected in 88 animals the 302 bp expected band(Figure 2(a)) whereas that with panfilarial primers confirmedthe presence of D immitis DNA (band of 542 bp) in 88 dogsand recognized D repens DNA (band of 484 bp) in further 4animals (Figure 2(b))The present study also revealed overall2269 percent occult cases which were determined on thebasis of differences between heartworm positive cases in PCRtest and antigen detection test (SNAP 4Dx) The workingdogs had the highest occult infection (60) followed by pet(2916) and stray (1775) dogs
Phylogenetic analysis of two Guwahati isolates of Dirofi-laria immitis was comparedwith additional twelve sequencesfrom the NCBI GenBank by Clustal W of DNASTARSequences from isolates of India (EU087699) Taiwan(AF217800) China (EU182329 EU182330 and EU182331)Iran (JX889634 JN084166 and JN084168) Brazil (FJ263456FJ263464 and FJ263462) and Turkey (HM126607) wereincluded All the sequences fell under the same grouphowever the sequences from Southeast Asia were moreclosely related The phylogenetic tree constructed basedon this finding is depicted (Figure 3) Pairwise distanceanalysis of the ITS-2 sequences of Guwahati isolates showed847 to 998 identity and the divergence ranged from00 (Taiwan AF217800) to 136 (Iran JN084168) Onthe contrary sequences of Guwahati isolates (JX481279 andJX866681) were 986ndash989 identical to that of Taiwanspecies (AF217800) (Table 3) Accession numbers for each
4 The Scientific World Journal
Table 2 Comparative efficacy percentage of microscopy Ag ELISA and PCR in detecting Dirofilaria immitis infection in dogs
Detection methods
Dog category
Number ofbloodsamplestested
Microscopy (Knottrsquostechnique)
Ag ELISA(SNAP 4Dx test) PCR
Numbers of positive () Numbers ofpositive ()
Specific primer for Dimmitis ()
Panfilarial primers()
Stray 413 69 (1670) 107 (2590) 88 (2130) 92 (88 + 4lowast)(2227)
Pet 266 16 (601) 24 (902) 17 (639) 17 (639)Working 103 4 (388) 10 (970) 4 (388) 4 (388)Grand total 782 89 (1138) 141 (1803) 109 (1393) 113 (1445)lowastDirofilaria repens
A B C
302bp
(a)
A B C D E
484bp542bp
(b)
Figure 2 (a) Gel picture showing amplification of D immitis (specific primers) Lane A 100 bp ladder Lane B negative Lane C PCRproduct of ITS-2 (b) Gel picture showing amplification of D immitis and D repens (panfilarial primers) Lane A 100 bp ladder Lanes B andC amplification for D immitis Lanes D and E amplification for D repens
sequence for 2 isolates of D immitis (accession numbersJX481279 and JX866681) and forD repens (accession numberJX524743) were obtained from GenBank
4 Discussion
The present study provides the first comprehensive assess-ment of D immitis infection in dogs from the NortheasternStates of India The number of dogs proven positive forD immitis in one or more diagnostic tests was variableaccording to the test applied Recording of 626 bloodsamples microfilaraemic under wet film does not give theactual epidemiological situation of the studied areas owingto the low sensitivity of the method and to the failure inmicrofilarial species differentiation [28] Currently abundantliterature suggests detecting antigen test as the most sensitivediagnostic method for canine heartworm [29] The presentstudy showed prevalence of 902D immitis infection in petdogs and 2590 in stray dogs A similar type of varyingfrom 476 to 2954 D immitis infection was recorded inAssam in pet and street dogs respectively [24]The records of
highest prevalence in straystreet dogs are likely due to theirfree roaming habits making them vulnerable to being bittenby different mosquito vectors Moreover the present studywas carried out in a geographical location where subtropicalclimate and deciduous forest land prevail therefore in anenvironment where high rainfall and humidity create idealmosquito breeding places
Our present study also revealed a higher prevalence ofDimmitis in male dogs but we could not draw a conclusion onthe differences of prevalence amongst male and female dogsLike most record on heartworm prevalence [24 30ndash32] andunlike few cases from elsewhere [33 34] our study found anonsignificant higher prevalence of D immitis in male dogs
In the present investigation many dogs which werefound positive using SNAP 4Dx kit revealed occult infec-tions The same samples when subjected to PCR studiesrevealed lesser percent prevalenceOccult infections (amicro-filaraemic infections) could arise due to several causes likelow parasite burdens prepatent infection by young adultsinfection of dog by only male worm geriatric female wormand immune response from the host against microfilariae or
The Scientific World Journal 5
EU182329 ChinaEU182330 ChinaAF217800 Taiwan
EU087699 MizoramEU182331 China
JN084166 IranFJ263456 BrazilHM126607 TurkeyFJ263462 BrazilFJ263464 BrazilJN084168 Iran
GuwahatiGuwahati
02454
Nucleotide substitutions (
JX8666811JX4812791
JX889634 Iran
times100)
Figure 3 Phylogenetic tree constructed for Dirofilaria immitis from the ITS-2 region using Clustal W of DNASTAR
Table 3 ITS-2 sequence pair distance analysis of Dirofilaria immitis isolates compared with homologous isolates (slowaccurate IUB)
Percent identity1 2 3 4 5 6 7 8 9 10 11 12 13 14
Divergence
1 998 941 910 902 902 847 934 955 957 959 954 945 989 1 JX8666811 Guwahati2 02 941 910 902 902 847 934 955 954 957 952 943 986 2 JX4812791 Guwahati3 51 51 957 949 941 914 933 937 957 886 937 863 937 3 FJ2634568 Brazil4 87 87 46 976 937 890 902 906 922 855 906 831 906 4 FJ263462 Brazil5 96 96 55 25 953 883 902 898 914 848 898 824 898 5 FJ263464 Brazil6 96 96 63 68 50 879 898 895 914 848 895 832 898 6 HM126607 Turkey7 136 136 61 89 93 98 885 885 868 800 881 786 844 7 JN084168 Iran8 41 41 43 79 74 79 128 974 954 893 971 876 934 8 JN084166 Iran9 18 18 38 74 79 84 127 28 980 916 992 899 955 9 JX889634 Iran10 05 07 29 69 73 73 136 41 12 920 970 907 950 10 EU087699 Mizoram11 00 02 54 93 102 102 144 43 19 05 932 954 948 11 EU182329 China12 12 15 38 74 79 84 132 31 09 07 13 918 948 12 EU182331 China13 07 10 67 105 114 104 155 52 28 13 07 20 934 13 EU182330 China14 00 02 51 87 96 96 137 41 18 05 00 12 07 14 AF217800 Taiwan
1 2 3 4 5 6 7 8 9 10 11 12 13 14
under microfilaricidal therapy The high percentage of occultinfection is not uncommon and was previously reported byseveral authors from different parts of the world [35 36]Higher occult cases recorded in working (60) and pet(2916) dogs in comparison to stray dogs (1725) mightbe due to the fact that owners of pet and working dogs areverymuch concerned about the health status of their animalsHence there is regularity in their health check-up thatsurely often required administration of anthelmintic drugslike ivermectin an endectocide drug whose microfilaricidalactivity reduces the number of circulating microfilariae Onthe other hand stray dogs are seldom taken care of with suchtype of medications
The overall prevalence of D immitis detected by PCRwas 1393 (109782) lower than that evidenced by ELISAtest probably due to occult infections or to possible failurein DNA extraction Hence the main benefit of PCR in epi-demiological surveys on dirofilariosis is 100 confirmatoryspecies identification when mixed infections coexist [3 37]
Detection of 4 dogs infected byD repens supports the reportsof human infection in Northeastern States of India [38] andis an alarming finding since this species apparently moreadapted than D immitis to the human host often succeedsin its development to adult worm
Phylogenetic analysis of D immitis isolates of Guwahatishowed a close identity with certain South Asian isolates ofD immitis Pairwise homology analysis revealed 986ndash989identity with a few sequences available at NCBI GenBankPreviously 926 homology ofD immitis of Mizoram isolatewith D immitis of Taiwan isolate (AF217800) was docu-mented [39] In the present study between Guwahati andMizoram isolates the identity was 954 to 957 and thedivergence was 05 to 07
5 Conclusion
This study confirms the predominance of D immitis inthe northeastern region of India and reestablishes the area
6 The Scientific World Journal
as heartworm endemic KCT along with antigen ELISAdetection test confirmed their sensitivity whereas moleculartechniques confirmed their value in identification of caninefilarial wormsThepresence of bothDirofilaria species shouldalert physicians to the risk of human infections
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge the Deans College of VeterinaryScience Assam Agricultural University Khanapara Guwa-hati and College of Veterinary Science amp AH Central Agri-cultural University Selesih Aizawl for providing facilitiesto carry out the research programme The first author alsoacknowledges the help received from Dr Rebecca J TraubSr Lecturer in Veterinary Public Health School of Vet-erinary Science University of Queensland QLD AustraliaFurther the authors are grateful toMrThomasMan GeneralManager of Other Asia and Mr Jerry Chang RegionalMarketing Manager of IDEXX Laboratories Inc for theirgenerosity in providing SNAP 4Dx as FOC kits to carry outthe epidemiological study
References
[1] R Morchon F Simon J Gonzalez-Miguel and I MelladoldquoRelationship DirofilariaHost cellular and molecular mecha-nisms of the heartworm disease vascular pathologyrdquo in Proceed-ings of the 2nd EuropeanDirofilariaDays RMorchon F SimonJ A Montoya and C Genchi Eds pp 116ndash123 SalamancaSpain September 2009
[2] W A Waren ldquoHeartworm diseaserdquo in Small Animal InternalMedicine R W Nelson and C G Couto Eds pp 169ndash184Mosby St Louis Mo USA 3rd edition 2003
[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L D Alpizar ldquoDiscrimination between six speciesof canine microfilariae by a single polymerase chain reactionrdquoVeterinary Parasitology vol 135 no 3-4 pp 303ndash314 2006
[4] N B Robinson C M Chavez and J H Conn ldquoPulmonarydirofilariasis in man A case report and review of the literaturerdquoJournal of Thoracic and Cardiovascular Surgery vol 74 no 3pp 403ndash408 1977
[5] J C Darrow and E E Lack ldquoSolitary lung nodule dueto Dirofilaria immitis (dog lsquoheartwormrsquo)rdquo Journal of SurgicalOncology vol 16 no 3 pp 219ndash224 1981
[6] F Simon AMuroM Cordero and JMartin ldquoA seroepidemio-logic survey of human dirofilariosis in Western Spainrdquo TropicalMedicine and Parasitology vol 42 no 2 pp 106ndash108 1991
[7] S Pampiglione F Rivasi G Angeli et al ldquoDirofilariasis due toDirofilaria repens in Italy an emergent zoonosis report of 60new casesrdquo Histopathology vol 38 no 4 pp 344ndash354 2001
[8] A Echeverri R F Long W Check and C M BurnettldquoPulmonary dirofilariasisrdquo Annals of Thoracic Surgery vol 67no 1 pp 201ndash202 1999
[9] J A Montoya-Alonso I Mellado E Carreton E D Cabrera-Pedrero R Morchon and F Simon ldquoCanine dirofilariosis
caused by Dirofilaria immitis is a risk factor for the humanpopulation on the island of Gran Canaria Canary IslandsSpainrdquo Parasitology Research vol 107 no 5 pp 1265ndash1269 2010
[10] F SimonM Siles-Lucas RMorchon et al ldquoHuman and animaldirofilariasis the emergence of a zoonotic mosaicrdquo ClinicalMicrobiology Reviews vol 25 no 3 pp 507ndash544 2012
[11] F Ciferri ldquoHumanpulmonary dirofilariasis in theUnited Statesa critical reviewrdquo American Journal of Tropical Medicine andHygiene vol 31 no 2 pp 302ndash308 1982
[12] B P Badhe and S Y Sane ldquoHuman pulmonary dirofilariasisin India a case reportrdquo The Journal of Tropical Medicine andHygiene vol 92 no 6 pp 425ndash426 1989
[13] T Dam and P Das ldquoThe importance of dirofilariasis in IndiardquoThe Internet Journal of Parasitic Diseases vol 1 no 1 2006
[14] R B Grieve L T Glickman A K Bater M Mika-GrieveC B Thomas and G J Patronek ldquoCanine Dirofilaria immitisinfection in a hyperenzootic area examination by parasitologicfindings at necropsy and by two serodiagnostic methodsrdquoAmerican Journal of Veterinary Research vol 47 no 2 pp 329ndash332 1986
[15] R Hatsushika T Okino and F Ohyama ldquoThe prevalence ofdog heartworm (Dirofilaria immitis) infection in stray dogs inOkayama Japanrdquo Kawasaki Medical Journal vol 18 pp 75ndash831992
[16] N Labarthe N Almosny J Guerrero andAMDuque-AraujoldquoDescription of the occurrence of canine Dirofilariasis in theState of Rio de Janeiro Brazilrdquo Memorias do Instituto OswaldoCruz vol 92 no 1 pp 47ndash51 1997
[17] L C Alves L V A de Silva M A da Gloria Faustino et alldquoSurvey of canine heartworm in the city of Recife PernambucoBrazilrdquo Memorias do Instituto Oswaldo Cruz vol 94 no 5 pp587ndash590 1999
[18] L Venco L Kramer and C Genchi ldquoHeartworm disease indogs unusual clinical casesrdquo Veterinary Parasitology vol 133no 2-3 pp 207ndash218 2005
[19] A Niwetpathomwat S Assarasakorn S Techangamsuwan SSuvarnavibhaja and M Kaewthamasorn ldquoCanine dirofilariasisand concurrent tick-borne transmitted diseases in BangkokThailandrdquo Journal of Comparative Clinical Pathology vol 15 no4 pp 249ndash253 2006
[20] M E Bolio-Gonzalez R I Rodriguez-Vivas C H Sauri-ArceoE Gutierrez-Blanco A Ortega-Pacheco and R F Colin-FloresldquoPrevalence of the Dirofilaria immitis infection in dogs fromMerida Yucatan Mexicordquo Veterinary Parasitology vol 148 no2 pp 166ndash169 2007
[21] A Chakrabarti and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983
[22] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMclnnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesand Vectors vol 3 article 30 2010
[23] S K Borthakur K Sarma T K Rajkhowa M R Das andS Rahman ldquoDirofilaria immitis infection in dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006
[24] K Bhattacharjee and P C Sarmah ldquoEpidemiological aspects ofDirofilaria immitis infection in dogs from Assam of NortheastIndiardquo Asian Pacific Journal of Tropical Disease vol 4 supple-ment 1 pp S255ndashS258 2014
[25] C M Hendrix Diagnostic Veterinary Parasitology Mosby StLouis Mo USA 2nd edition 1998
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal of
Microbiology
2 The Scientific World Journal
subtropical areas of the world So far more than 1700 humancases of dirofilariosis (including gt370 pulmonary cases)have been documented worldwide suggesting that wherevercanine dirofilariosis is present humans are at risk of infection[9 10] Most D immitis human infections are asymptomaticshowing typical coin lesions on chest radiography and areoften mistakenly removed as neoplasm [11] In India the firstcase of human pulmonary dirofilariosis due to D immitiswas reported from Mumbai [12] After that several cases ofhuman dirofilariosis have been reported in India [13]
In animals there have been both epidemiological andclinical case studies of this worm worldwide [14ndash20] Preva-lence of this parasite in dogs from several parts from India hasbeen reported [21 22] Limited works revealed occurrenceof 3375 D immitis in Mizoram a Northeastern State ofIndia on the basis of examination of 240 dogs at slaughter[23] A recent study based on wet film and antigen testsrevealed 476 to 2954 prevalence ofD immitis in a hospitalpopulation of dogs fromAssam [24] But no systematic workon epidemiological aspect of D immitis has been carried outin the northeastern part of this country using combinationof conventional serological and molecular diagnostic tech-niques
The importance of this parasite and the paucity ofinformation about the prevalence of canine heartworm inthe Northeastern States of India inspired us to conductthe present plan of work We studied blood samples fromdogs that presented for veterinary attention and from dogscaptured by various nongovernmental organizations andmaintained in kennels The prevalence was studied by meansof conventional microscopy an immunological test andmolecular techniques and their efficacy was comparedFurther we explored some of the molecular characteristics ofD immitis in the studied area
2 Materials and Methods
21 Study Areas The study was undertaken systematicallyfor a period of one calendar year from August 2011 to July2012 in dogs from Guwahati and Aizawl Guwahati a city ofAssam having annual rainfall of 1500ndash2600mm is located at26∘111015840010158401015840 latitude N and 91∘441015840010158401015840 longitude E and Aizawl thecapital city of Mizoram State having annual rainfall of 2400ndash2962mm is located at 23∘4310158402710158401015840N and 92∘431015840210158401015840E The citiesare separated by surface distance of 550 km The location ofepidemiological study undertaken for the present study isshown in the map (Figure 1)
22 Selection of Dogs In the present investigation 3 cat-egories of dogs grouped as working dogs of military andparamilitary force pet dogs and stray dogs were selectedPet dogs of different breeds and paramilitary dogs mostlyof Labrador and German Shepherd breeds brought to theTeaching Veterinary Clinical Complexes (TVCC) of theCollege of Veterinary Science AssamAgricultural UniversityKhanapara Guwahati Assam and the College of VeterinarySciences amp AH Central Agricultural University SelesihAizawl Mizoram during the study period were taken for the
Northeast India
China (Tibet)
Bhutan
WestBengal
Itanagar
Arunachal Pradesh
Dispur Assam
Bangladesh
MeghalayaShillong
Myanmar (Burma)AgartalaTripura
AizawlMizoram
ManipurImphal
NagalandKohima
Figure 1 Map showing the study areas
study In case of military dogs most of the blood sampleswere directly sent to the Department of Parasitology forroutine check-up of heartworm infection The stray dogpopulation consisted of local nondescript street dogs of eithersex captured fromdifferent parts of the city for sterilization bylocal nongovernmental organizations like Peoples for Animal(PFA) and Just Be Friendly (JBF)
Three categories of dogs like working (103) pet (266) andstray (413) totaling 782 numbers were examined Dogs underthe study were of both sexes and a total of 488 dogs fromdifferent localities of Guwahati and 294 dogs from Aizawlformed the entire base of study during the programme
23 Blood Sampling Approximately 5mL of blood wasdrawn from the cephalic vein collected in disodium salt ofethylene diamine tetraacetic acid (Na
2EDTA) vacuum tubes
and stored at 4∘C until further use
24 Parasitological Investigation The prevalence study forD immitis was conducted on the basis ofconventionalimmunological and molecular techniques The conventionaltests were based onmicrofilarial availability in blood samplesassessed on the basis of wet blood film method and modifiedKnottrsquos concentration technique (KCT) [25] Subsequentlymicrofilarial identification was done [26] The immuno-logical evidence was based on the presence of heartwormantigens in tested blood samples and was performed with acommercially available ELISA test kit (SNAP 4Dx) followingmanufacturersquos test protocol Molecular evidence was basedon amplification of worm targeted DNA Blood samplesnegative for D immitis circulating antigen but positive atKCTwere processed for this technique Fresh adultDimmitisand positive blood samples collected from Aizawl slaughterhouse were used to standardize techniques and used aspositive control for the tests In addition blood samples weretaken from dogs clinically infected and found positive for Dimmitis being confirmed by KCT and SNAP tests Briefly themolecular technique for the present study was performed asfollows
The Scientific World Journal 3
Table 1 Prevalence of Dirofilaria immitis by category of dogs and area of study on the basis of Ag ELISA test
Dog categoryGuwahati Aizawl Overall
Numbers Positive Chi value Numbers Positive
Chi value Numbers Positive Chi value
Stray dogs 223 63 (2825)276139
190 44 (2315)110458
413 107 (2590)367706Pet dogs 174 17 (977) 92 7 (760) 266 24 (902)
Working dogs 91 9 (989) 12 1 (833) 103 10 (970)Total 488 89 (1823) 294 52 (1768) 782 141 (1803)
25 Isolation of Genomic DNA fromBlood andAdult ParasitesIsolation of genomic DNA from blood and adult parasite wascarried out using the DNeasy Blood and Tissue Kit (QiagenKit Catalogue number 69504) as per the protocols providedby the manufacturer The final templates were kept at minus20∘C
26 PCR Assays PCR technique targeted to amplify theITS-2 region of filarial wormsrsquo rDNA was applied accordingto previously described protocols [3 27] Briefly reactionmixture comprised 25120583L Taq polymerase buffer (10x) 01120583LdNTP (10mM) 05 120583L MgCl
2(50mM) 075 120583L of each
forward and reverse primer (60 pM) 1 UTaq polymerase and30 120583L template DNA (60 ng120583L concentration) by makingthe final volume up to 250 120583L with NFW The cyclingcondition used for amplifying the targeted product consistedof an initial denaturing step at 94∘C for 2min and 32 cyclesof denaturing (30 s at 94∘C) annealing (30 s at 60∘C for58S-ITS-2-28S-based primers and 30 s at 58∘C for ITS-2-based primers) and extension (30 s at 72∘C) a final extension(7min at 72∘C or 12min at 72∘C for cloning) and a soakat 4∘C in a Techne-5000 thermal cycler (Bibby Scientific)The confirmation of the amplified products was made by gelelectrophoresis of the PCRproduct in 15 agarose gel stainedwith ethidium bromide and visualized under gel doc (DNRBio-Imaging System MiniLumi)
The specificity of the PCR amplification for the corre-sponding D immitis target both on representative positiveblood samples and on adult worms was assessed by ampliconpurification followed by cloning and sequencing
27 Cloning of the Genomic Region Cloning of the PCRamplicon(s) for the genomic region as described above hasbeen performed using pDrive cloning vector (Qiagen PCRCloning Kit Catalog number 231124) DH5120572 E coli cell wasused for transformation of the plasmid using Transform AidBacterial Transformation Kit (Fermentas Catalog numberK2711) Subsequently clones were confirmed by clonesrsquo con-firmation PCR
28 Sequencing and Analysis of ITS-2 The recombinantclones were sent to the Department of Biochemistry Uni-versity of Delhi South Campus for automated sequencingThe sequences obtained were aligned and compared withother published sequences ofD immitis of dogs by ClustalWmethod using DNASTAR software and phylogenic analysiswas done Sequences were compared in silico with sequencesof D immitis (ITS-2) rDNA available in the gene bank for
each gene examined using the nucleotide-nucleotide ldquoBasicLocal Alignment Search Toolrdquo Sequences were submitted toNCBI to obtain accession numbers
3 Results
The study of the prevalence of canine heartworm carried outby means of different techniques obviously produced differ-ent results Overall 626 (49782) blood samples analysedproved microfilaraemic under wet film methods whereasinfection rates found by means of the other techniques werehigher As expected the highest number of positive animalswas detected by the ELISA test which evidenced an overallprevalence of 1803 (Table 1) without differences by areaand by sex On the contrary in both areas differences wereevidenced by group of dogs Table 2 summarizes data onthe efficacy of KCT ELISA test and PCR which classifiedas infected 1138 183 and 1393 of the examined dogsrespectively with stray dogs always more infected than othergroups Moreover PCR analysis with primers specific for Dimmitis detected in 88 animals the 302 bp expected band(Figure 2(a)) whereas that with panfilarial primers confirmedthe presence of D immitis DNA (band of 542 bp) in 88 dogsand recognized D repens DNA (band of 484 bp) in further 4animals (Figure 2(b))The present study also revealed overall2269 percent occult cases which were determined on thebasis of differences between heartworm positive cases in PCRtest and antigen detection test (SNAP 4Dx) The workingdogs had the highest occult infection (60) followed by pet(2916) and stray (1775) dogs
Phylogenetic analysis of two Guwahati isolates of Dirofi-laria immitis was comparedwith additional twelve sequencesfrom the NCBI GenBank by Clustal W of DNASTARSequences from isolates of India (EU087699) Taiwan(AF217800) China (EU182329 EU182330 and EU182331)Iran (JX889634 JN084166 and JN084168) Brazil (FJ263456FJ263464 and FJ263462) and Turkey (HM126607) wereincluded All the sequences fell under the same grouphowever the sequences from Southeast Asia were moreclosely related The phylogenetic tree constructed basedon this finding is depicted (Figure 3) Pairwise distanceanalysis of the ITS-2 sequences of Guwahati isolates showed847 to 998 identity and the divergence ranged from00 (Taiwan AF217800) to 136 (Iran JN084168) Onthe contrary sequences of Guwahati isolates (JX481279 andJX866681) were 986ndash989 identical to that of Taiwanspecies (AF217800) (Table 3) Accession numbers for each
4 The Scientific World Journal
Table 2 Comparative efficacy percentage of microscopy Ag ELISA and PCR in detecting Dirofilaria immitis infection in dogs
Detection methods
Dog category
Number ofbloodsamplestested
Microscopy (Knottrsquostechnique)
Ag ELISA(SNAP 4Dx test) PCR
Numbers of positive () Numbers ofpositive ()
Specific primer for Dimmitis ()
Panfilarial primers()
Stray 413 69 (1670) 107 (2590) 88 (2130) 92 (88 + 4lowast)(2227)
Pet 266 16 (601) 24 (902) 17 (639) 17 (639)Working 103 4 (388) 10 (970) 4 (388) 4 (388)Grand total 782 89 (1138) 141 (1803) 109 (1393) 113 (1445)lowastDirofilaria repens
A B C
302bp
(a)
A B C D E
484bp542bp
(b)
Figure 2 (a) Gel picture showing amplification of D immitis (specific primers) Lane A 100 bp ladder Lane B negative Lane C PCRproduct of ITS-2 (b) Gel picture showing amplification of D immitis and D repens (panfilarial primers) Lane A 100 bp ladder Lanes B andC amplification for D immitis Lanes D and E amplification for D repens
sequence for 2 isolates of D immitis (accession numbersJX481279 and JX866681) and forD repens (accession numberJX524743) were obtained from GenBank
4 Discussion
The present study provides the first comprehensive assess-ment of D immitis infection in dogs from the NortheasternStates of India The number of dogs proven positive forD immitis in one or more diagnostic tests was variableaccording to the test applied Recording of 626 bloodsamples microfilaraemic under wet film does not give theactual epidemiological situation of the studied areas owingto the low sensitivity of the method and to the failure inmicrofilarial species differentiation [28] Currently abundantliterature suggests detecting antigen test as the most sensitivediagnostic method for canine heartworm [29] The presentstudy showed prevalence of 902D immitis infection in petdogs and 2590 in stray dogs A similar type of varyingfrom 476 to 2954 D immitis infection was recorded inAssam in pet and street dogs respectively [24]The records of
highest prevalence in straystreet dogs are likely due to theirfree roaming habits making them vulnerable to being bittenby different mosquito vectors Moreover the present studywas carried out in a geographical location where subtropicalclimate and deciduous forest land prevail therefore in anenvironment where high rainfall and humidity create idealmosquito breeding places
Our present study also revealed a higher prevalence ofDimmitis in male dogs but we could not draw a conclusion onthe differences of prevalence amongst male and female dogsLike most record on heartworm prevalence [24 30ndash32] andunlike few cases from elsewhere [33 34] our study found anonsignificant higher prevalence of D immitis in male dogs
In the present investigation many dogs which werefound positive using SNAP 4Dx kit revealed occult infec-tions The same samples when subjected to PCR studiesrevealed lesser percent prevalenceOccult infections (amicro-filaraemic infections) could arise due to several causes likelow parasite burdens prepatent infection by young adultsinfection of dog by only male worm geriatric female wormand immune response from the host against microfilariae or
The Scientific World Journal 5
EU182329 ChinaEU182330 ChinaAF217800 Taiwan
EU087699 MizoramEU182331 China
JN084166 IranFJ263456 BrazilHM126607 TurkeyFJ263462 BrazilFJ263464 BrazilJN084168 Iran
GuwahatiGuwahati
02454
Nucleotide substitutions (
JX8666811JX4812791
JX889634 Iran
times100)
Figure 3 Phylogenetic tree constructed for Dirofilaria immitis from the ITS-2 region using Clustal W of DNASTAR
Table 3 ITS-2 sequence pair distance analysis of Dirofilaria immitis isolates compared with homologous isolates (slowaccurate IUB)
Percent identity1 2 3 4 5 6 7 8 9 10 11 12 13 14
Divergence
1 998 941 910 902 902 847 934 955 957 959 954 945 989 1 JX8666811 Guwahati2 02 941 910 902 902 847 934 955 954 957 952 943 986 2 JX4812791 Guwahati3 51 51 957 949 941 914 933 937 957 886 937 863 937 3 FJ2634568 Brazil4 87 87 46 976 937 890 902 906 922 855 906 831 906 4 FJ263462 Brazil5 96 96 55 25 953 883 902 898 914 848 898 824 898 5 FJ263464 Brazil6 96 96 63 68 50 879 898 895 914 848 895 832 898 6 HM126607 Turkey7 136 136 61 89 93 98 885 885 868 800 881 786 844 7 JN084168 Iran8 41 41 43 79 74 79 128 974 954 893 971 876 934 8 JN084166 Iran9 18 18 38 74 79 84 127 28 980 916 992 899 955 9 JX889634 Iran10 05 07 29 69 73 73 136 41 12 920 970 907 950 10 EU087699 Mizoram11 00 02 54 93 102 102 144 43 19 05 932 954 948 11 EU182329 China12 12 15 38 74 79 84 132 31 09 07 13 918 948 12 EU182331 China13 07 10 67 105 114 104 155 52 28 13 07 20 934 13 EU182330 China14 00 02 51 87 96 96 137 41 18 05 00 12 07 14 AF217800 Taiwan
1 2 3 4 5 6 7 8 9 10 11 12 13 14
under microfilaricidal therapy The high percentage of occultinfection is not uncommon and was previously reported byseveral authors from different parts of the world [35 36]Higher occult cases recorded in working (60) and pet(2916) dogs in comparison to stray dogs (1725) mightbe due to the fact that owners of pet and working dogs areverymuch concerned about the health status of their animalsHence there is regularity in their health check-up thatsurely often required administration of anthelmintic drugslike ivermectin an endectocide drug whose microfilaricidalactivity reduces the number of circulating microfilariae Onthe other hand stray dogs are seldom taken care of with suchtype of medications
The overall prevalence of D immitis detected by PCRwas 1393 (109782) lower than that evidenced by ELISAtest probably due to occult infections or to possible failurein DNA extraction Hence the main benefit of PCR in epi-demiological surveys on dirofilariosis is 100 confirmatoryspecies identification when mixed infections coexist [3 37]
Detection of 4 dogs infected byD repens supports the reportsof human infection in Northeastern States of India [38] andis an alarming finding since this species apparently moreadapted than D immitis to the human host often succeedsin its development to adult worm
Phylogenetic analysis of D immitis isolates of Guwahatishowed a close identity with certain South Asian isolates ofD immitis Pairwise homology analysis revealed 986ndash989identity with a few sequences available at NCBI GenBankPreviously 926 homology ofD immitis of Mizoram isolatewith D immitis of Taiwan isolate (AF217800) was docu-mented [39] In the present study between Guwahati andMizoram isolates the identity was 954 to 957 and thedivergence was 05 to 07
5 Conclusion
This study confirms the predominance of D immitis inthe northeastern region of India and reestablishes the area
6 The Scientific World Journal
as heartworm endemic KCT along with antigen ELISAdetection test confirmed their sensitivity whereas moleculartechniques confirmed their value in identification of caninefilarial wormsThepresence of bothDirofilaria species shouldalert physicians to the risk of human infections
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge the Deans College of VeterinaryScience Assam Agricultural University Khanapara Guwa-hati and College of Veterinary Science amp AH Central Agri-cultural University Selesih Aizawl for providing facilitiesto carry out the research programme The first author alsoacknowledges the help received from Dr Rebecca J TraubSr Lecturer in Veterinary Public Health School of Vet-erinary Science University of Queensland QLD AustraliaFurther the authors are grateful toMrThomasMan GeneralManager of Other Asia and Mr Jerry Chang RegionalMarketing Manager of IDEXX Laboratories Inc for theirgenerosity in providing SNAP 4Dx as FOC kits to carry outthe epidemiological study
References
[1] R Morchon F Simon J Gonzalez-Miguel and I MelladoldquoRelationship DirofilariaHost cellular and molecular mecha-nisms of the heartworm disease vascular pathologyrdquo in Proceed-ings of the 2nd EuropeanDirofilariaDays RMorchon F SimonJ A Montoya and C Genchi Eds pp 116ndash123 SalamancaSpain September 2009
[2] W A Waren ldquoHeartworm diseaserdquo in Small Animal InternalMedicine R W Nelson and C G Couto Eds pp 169ndash184Mosby St Louis Mo USA 3rd edition 2003
[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L D Alpizar ldquoDiscrimination between six speciesof canine microfilariae by a single polymerase chain reactionrdquoVeterinary Parasitology vol 135 no 3-4 pp 303ndash314 2006
[4] N B Robinson C M Chavez and J H Conn ldquoPulmonarydirofilariasis in man A case report and review of the literaturerdquoJournal of Thoracic and Cardiovascular Surgery vol 74 no 3pp 403ndash408 1977
[5] J C Darrow and E E Lack ldquoSolitary lung nodule dueto Dirofilaria immitis (dog lsquoheartwormrsquo)rdquo Journal of SurgicalOncology vol 16 no 3 pp 219ndash224 1981
[6] F Simon AMuroM Cordero and JMartin ldquoA seroepidemio-logic survey of human dirofilariosis in Western Spainrdquo TropicalMedicine and Parasitology vol 42 no 2 pp 106ndash108 1991
[7] S Pampiglione F Rivasi G Angeli et al ldquoDirofilariasis due toDirofilaria repens in Italy an emergent zoonosis report of 60new casesrdquo Histopathology vol 38 no 4 pp 344ndash354 2001
[8] A Echeverri R F Long W Check and C M BurnettldquoPulmonary dirofilariasisrdquo Annals of Thoracic Surgery vol 67no 1 pp 201ndash202 1999
[9] J A Montoya-Alonso I Mellado E Carreton E D Cabrera-Pedrero R Morchon and F Simon ldquoCanine dirofilariosis
caused by Dirofilaria immitis is a risk factor for the humanpopulation on the island of Gran Canaria Canary IslandsSpainrdquo Parasitology Research vol 107 no 5 pp 1265ndash1269 2010
[10] F SimonM Siles-Lucas RMorchon et al ldquoHuman and animaldirofilariasis the emergence of a zoonotic mosaicrdquo ClinicalMicrobiology Reviews vol 25 no 3 pp 507ndash544 2012
[11] F Ciferri ldquoHumanpulmonary dirofilariasis in theUnited Statesa critical reviewrdquo American Journal of Tropical Medicine andHygiene vol 31 no 2 pp 302ndash308 1982
[12] B P Badhe and S Y Sane ldquoHuman pulmonary dirofilariasisin India a case reportrdquo The Journal of Tropical Medicine andHygiene vol 92 no 6 pp 425ndash426 1989
[13] T Dam and P Das ldquoThe importance of dirofilariasis in IndiardquoThe Internet Journal of Parasitic Diseases vol 1 no 1 2006
[14] R B Grieve L T Glickman A K Bater M Mika-GrieveC B Thomas and G J Patronek ldquoCanine Dirofilaria immitisinfection in a hyperenzootic area examination by parasitologicfindings at necropsy and by two serodiagnostic methodsrdquoAmerican Journal of Veterinary Research vol 47 no 2 pp 329ndash332 1986
[15] R Hatsushika T Okino and F Ohyama ldquoThe prevalence ofdog heartworm (Dirofilaria immitis) infection in stray dogs inOkayama Japanrdquo Kawasaki Medical Journal vol 18 pp 75ndash831992
[16] N Labarthe N Almosny J Guerrero andAMDuque-AraujoldquoDescription of the occurrence of canine Dirofilariasis in theState of Rio de Janeiro Brazilrdquo Memorias do Instituto OswaldoCruz vol 92 no 1 pp 47ndash51 1997
[17] L C Alves L V A de Silva M A da Gloria Faustino et alldquoSurvey of canine heartworm in the city of Recife PernambucoBrazilrdquo Memorias do Instituto Oswaldo Cruz vol 94 no 5 pp587ndash590 1999
[18] L Venco L Kramer and C Genchi ldquoHeartworm disease indogs unusual clinical casesrdquo Veterinary Parasitology vol 133no 2-3 pp 207ndash218 2005
[19] A Niwetpathomwat S Assarasakorn S Techangamsuwan SSuvarnavibhaja and M Kaewthamasorn ldquoCanine dirofilariasisand concurrent tick-borne transmitted diseases in BangkokThailandrdquo Journal of Comparative Clinical Pathology vol 15 no4 pp 249ndash253 2006
[20] M E Bolio-Gonzalez R I Rodriguez-Vivas C H Sauri-ArceoE Gutierrez-Blanco A Ortega-Pacheco and R F Colin-FloresldquoPrevalence of the Dirofilaria immitis infection in dogs fromMerida Yucatan Mexicordquo Veterinary Parasitology vol 148 no2 pp 166ndash169 2007
[21] A Chakrabarti and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983
[22] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMclnnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesand Vectors vol 3 article 30 2010
[23] S K Borthakur K Sarma T K Rajkhowa M R Das andS Rahman ldquoDirofilaria immitis infection in dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006
[24] K Bhattacharjee and P C Sarmah ldquoEpidemiological aspects ofDirofilaria immitis infection in dogs from Assam of NortheastIndiardquo Asian Pacific Journal of Tropical Disease vol 4 supple-ment 1 pp S255ndashS258 2014
[25] C M Hendrix Diagnostic Veterinary Parasitology Mosby StLouis Mo USA 2nd edition 1998
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
The Scientific World Journal 3
Table 1 Prevalence of Dirofilaria immitis by category of dogs and area of study on the basis of Ag ELISA test
Dog categoryGuwahati Aizawl Overall
Numbers Positive Chi value Numbers Positive
Chi value Numbers Positive Chi value
Stray dogs 223 63 (2825)276139
190 44 (2315)110458
413 107 (2590)367706Pet dogs 174 17 (977) 92 7 (760) 266 24 (902)
Working dogs 91 9 (989) 12 1 (833) 103 10 (970)Total 488 89 (1823) 294 52 (1768) 782 141 (1803)
25 Isolation of Genomic DNA fromBlood andAdult ParasitesIsolation of genomic DNA from blood and adult parasite wascarried out using the DNeasy Blood and Tissue Kit (QiagenKit Catalogue number 69504) as per the protocols providedby the manufacturer The final templates were kept at minus20∘C
26 PCR Assays PCR technique targeted to amplify theITS-2 region of filarial wormsrsquo rDNA was applied accordingto previously described protocols [3 27] Briefly reactionmixture comprised 25120583L Taq polymerase buffer (10x) 01120583LdNTP (10mM) 05 120583L MgCl
2(50mM) 075 120583L of each
forward and reverse primer (60 pM) 1 UTaq polymerase and30 120583L template DNA (60 ng120583L concentration) by makingthe final volume up to 250 120583L with NFW The cyclingcondition used for amplifying the targeted product consistedof an initial denaturing step at 94∘C for 2min and 32 cyclesof denaturing (30 s at 94∘C) annealing (30 s at 60∘C for58S-ITS-2-28S-based primers and 30 s at 58∘C for ITS-2-based primers) and extension (30 s at 72∘C) a final extension(7min at 72∘C or 12min at 72∘C for cloning) and a soakat 4∘C in a Techne-5000 thermal cycler (Bibby Scientific)The confirmation of the amplified products was made by gelelectrophoresis of the PCRproduct in 15 agarose gel stainedwith ethidium bromide and visualized under gel doc (DNRBio-Imaging System MiniLumi)
The specificity of the PCR amplification for the corre-sponding D immitis target both on representative positiveblood samples and on adult worms was assessed by ampliconpurification followed by cloning and sequencing
27 Cloning of the Genomic Region Cloning of the PCRamplicon(s) for the genomic region as described above hasbeen performed using pDrive cloning vector (Qiagen PCRCloning Kit Catalog number 231124) DH5120572 E coli cell wasused for transformation of the plasmid using Transform AidBacterial Transformation Kit (Fermentas Catalog numberK2711) Subsequently clones were confirmed by clonesrsquo con-firmation PCR
28 Sequencing and Analysis of ITS-2 The recombinantclones were sent to the Department of Biochemistry Uni-versity of Delhi South Campus for automated sequencingThe sequences obtained were aligned and compared withother published sequences ofD immitis of dogs by ClustalWmethod using DNASTAR software and phylogenic analysiswas done Sequences were compared in silico with sequencesof D immitis (ITS-2) rDNA available in the gene bank for
each gene examined using the nucleotide-nucleotide ldquoBasicLocal Alignment Search Toolrdquo Sequences were submitted toNCBI to obtain accession numbers
3 Results
The study of the prevalence of canine heartworm carried outby means of different techniques obviously produced differ-ent results Overall 626 (49782) blood samples analysedproved microfilaraemic under wet film methods whereasinfection rates found by means of the other techniques werehigher As expected the highest number of positive animalswas detected by the ELISA test which evidenced an overallprevalence of 1803 (Table 1) without differences by areaand by sex On the contrary in both areas differences wereevidenced by group of dogs Table 2 summarizes data onthe efficacy of KCT ELISA test and PCR which classifiedas infected 1138 183 and 1393 of the examined dogsrespectively with stray dogs always more infected than othergroups Moreover PCR analysis with primers specific for Dimmitis detected in 88 animals the 302 bp expected band(Figure 2(a)) whereas that with panfilarial primers confirmedthe presence of D immitis DNA (band of 542 bp) in 88 dogsand recognized D repens DNA (band of 484 bp) in further 4animals (Figure 2(b))The present study also revealed overall2269 percent occult cases which were determined on thebasis of differences between heartworm positive cases in PCRtest and antigen detection test (SNAP 4Dx) The workingdogs had the highest occult infection (60) followed by pet(2916) and stray (1775) dogs
Phylogenetic analysis of two Guwahati isolates of Dirofi-laria immitis was comparedwith additional twelve sequencesfrom the NCBI GenBank by Clustal W of DNASTARSequences from isolates of India (EU087699) Taiwan(AF217800) China (EU182329 EU182330 and EU182331)Iran (JX889634 JN084166 and JN084168) Brazil (FJ263456FJ263464 and FJ263462) and Turkey (HM126607) wereincluded All the sequences fell under the same grouphowever the sequences from Southeast Asia were moreclosely related The phylogenetic tree constructed basedon this finding is depicted (Figure 3) Pairwise distanceanalysis of the ITS-2 sequences of Guwahati isolates showed847 to 998 identity and the divergence ranged from00 (Taiwan AF217800) to 136 (Iran JN084168) Onthe contrary sequences of Guwahati isolates (JX481279 andJX866681) were 986ndash989 identical to that of Taiwanspecies (AF217800) (Table 3) Accession numbers for each
4 The Scientific World Journal
Table 2 Comparative efficacy percentage of microscopy Ag ELISA and PCR in detecting Dirofilaria immitis infection in dogs
Detection methods
Dog category
Number ofbloodsamplestested
Microscopy (Knottrsquostechnique)
Ag ELISA(SNAP 4Dx test) PCR
Numbers of positive () Numbers ofpositive ()
Specific primer for Dimmitis ()
Panfilarial primers()
Stray 413 69 (1670) 107 (2590) 88 (2130) 92 (88 + 4lowast)(2227)
Pet 266 16 (601) 24 (902) 17 (639) 17 (639)Working 103 4 (388) 10 (970) 4 (388) 4 (388)Grand total 782 89 (1138) 141 (1803) 109 (1393) 113 (1445)lowastDirofilaria repens
A B C
302bp
(a)
A B C D E
484bp542bp
(b)
Figure 2 (a) Gel picture showing amplification of D immitis (specific primers) Lane A 100 bp ladder Lane B negative Lane C PCRproduct of ITS-2 (b) Gel picture showing amplification of D immitis and D repens (panfilarial primers) Lane A 100 bp ladder Lanes B andC amplification for D immitis Lanes D and E amplification for D repens
sequence for 2 isolates of D immitis (accession numbersJX481279 and JX866681) and forD repens (accession numberJX524743) were obtained from GenBank
4 Discussion
The present study provides the first comprehensive assess-ment of D immitis infection in dogs from the NortheasternStates of India The number of dogs proven positive forD immitis in one or more diagnostic tests was variableaccording to the test applied Recording of 626 bloodsamples microfilaraemic under wet film does not give theactual epidemiological situation of the studied areas owingto the low sensitivity of the method and to the failure inmicrofilarial species differentiation [28] Currently abundantliterature suggests detecting antigen test as the most sensitivediagnostic method for canine heartworm [29] The presentstudy showed prevalence of 902D immitis infection in petdogs and 2590 in stray dogs A similar type of varyingfrom 476 to 2954 D immitis infection was recorded inAssam in pet and street dogs respectively [24]The records of
highest prevalence in straystreet dogs are likely due to theirfree roaming habits making them vulnerable to being bittenby different mosquito vectors Moreover the present studywas carried out in a geographical location where subtropicalclimate and deciduous forest land prevail therefore in anenvironment where high rainfall and humidity create idealmosquito breeding places
Our present study also revealed a higher prevalence ofDimmitis in male dogs but we could not draw a conclusion onthe differences of prevalence amongst male and female dogsLike most record on heartworm prevalence [24 30ndash32] andunlike few cases from elsewhere [33 34] our study found anonsignificant higher prevalence of D immitis in male dogs
In the present investigation many dogs which werefound positive using SNAP 4Dx kit revealed occult infec-tions The same samples when subjected to PCR studiesrevealed lesser percent prevalenceOccult infections (amicro-filaraemic infections) could arise due to several causes likelow parasite burdens prepatent infection by young adultsinfection of dog by only male worm geriatric female wormand immune response from the host against microfilariae or
The Scientific World Journal 5
EU182329 ChinaEU182330 ChinaAF217800 Taiwan
EU087699 MizoramEU182331 China
JN084166 IranFJ263456 BrazilHM126607 TurkeyFJ263462 BrazilFJ263464 BrazilJN084168 Iran
GuwahatiGuwahati
02454
Nucleotide substitutions (
JX8666811JX4812791
JX889634 Iran
times100)
Figure 3 Phylogenetic tree constructed for Dirofilaria immitis from the ITS-2 region using Clustal W of DNASTAR
Table 3 ITS-2 sequence pair distance analysis of Dirofilaria immitis isolates compared with homologous isolates (slowaccurate IUB)
Percent identity1 2 3 4 5 6 7 8 9 10 11 12 13 14
Divergence
1 998 941 910 902 902 847 934 955 957 959 954 945 989 1 JX8666811 Guwahati2 02 941 910 902 902 847 934 955 954 957 952 943 986 2 JX4812791 Guwahati3 51 51 957 949 941 914 933 937 957 886 937 863 937 3 FJ2634568 Brazil4 87 87 46 976 937 890 902 906 922 855 906 831 906 4 FJ263462 Brazil5 96 96 55 25 953 883 902 898 914 848 898 824 898 5 FJ263464 Brazil6 96 96 63 68 50 879 898 895 914 848 895 832 898 6 HM126607 Turkey7 136 136 61 89 93 98 885 885 868 800 881 786 844 7 JN084168 Iran8 41 41 43 79 74 79 128 974 954 893 971 876 934 8 JN084166 Iran9 18 18 38 74 79 84 127 28 980 916 992 899 955 9 JX889634 Iran10 05 07 29 69 73 73 136 41 12 920 970 907 950 10 EU087699 Mizoram11 00 02 54 93 102 102 144 43 19 05 932 954 948 11 EU182329 China12 12 15 38 74 79 84 132 31 09 07 13 918 948 12 EU182331 China13 07 10 67 105 114 104 155 52 28 13 07 20 934 13 EU182330 China14 00 02 51 87 96 96 137 41 18 05 00 12 07 14 AF217800 Taiwan
1 2 3 4 5 6 7 8 9 10 11 12 13 14
under microfilaricidal therapy The high percentage of occultinfection is not uncommon and was previously reported byseveral authors from different parts of the world [35 36]Higher occult cases recorded in working (60) and pet(2916) dogs in comparison to stray dogs (1725) mightbe due to the fact that owners of pet and working dogs areverymuch concerned about the health status of their animalsHence there is regularity in their health check-up thatsurely often required administration of anthelmintic drugslike ivermectin an endectocide drug whose microfilaricidalactivity reduces the number of circulating microfilariae Onthe other hand stray dogs are seldom taken care of with suchtype of medications
The overall prevalence of D immitis detected by PCRwas 1393 (109782) lower than that evidenced by ELISAtest probably due to occult infections or to possible failurein DNA extraction Hence the main benefit of PCR in epi-demiological surveys on dirofilariosis is 100 confirmatoryspecies identification when mixed infections coexist [3 37]
Detection of 4 dogs infected byD repens supports the reportsof human infection in Northeastern States of India [38] andis an alarming finding since this species apparently moreadapted than D immitis to the human host often succeedsin its development to adult worm
Phylogenetic analysis of D immitis isolates of Guwahatishowed a close identity with certain South Asian isolates ofD immitis Pairwise homology analysis revealed 986ndash989identity with a few sequences available at NCBI GenBankPreviously 926 homology ofD immitis of Mizoram isolatewith D immitis of Taiwan isolate (AF217800) was docu-mented [39] In the present study between Guwahati andMizoram isolates the identity was 954 to 957 and thedivergence was 05 to 07
5 Conclusion
This study confirms the predominance of D immitis inthe northeastern region of India and reestablishes the area
6 The Scientific World Journal
as heartworm endemic KCT along with antigen ELISAdetection test confirmed their sensitivity whereas moleculartechniques confirmed their value in identification of caninefilarial wormsThepresence of bothDirofilaria species shouldalert physicians to the risk of human infections
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge the Deans College of VeterinaryScience Assam Agricultural University Khanapara Guwa-hati and College of Veterinary Science amp AH Central Agri-cultural University Selesih Aizawl for providing facilitiesto carry out the research programme The first author alsoacknowledges the help received from Dr Rebecca J TraubSr Lecturer in Veterinary Public Health School of Vet-erinary Science University of Queensland QLD AustraliaFurther the authors are grateful toMrThomasMan GeneralManager of Other Asia and Mr Jerry Chang RegionalMarketing Manager of IDEXX Laboratories Inc for theirgenerosity in providing SNAP 4Dx as FOC kits to carry outthe epidemiological study
References
[1] R Morchon F Simon J Gonzalez-Miguel and I MelladoldquoRelationship DirofilariaHost cellular and molecular mecha-nisms of the heartworm disease vascular pathologyrdquo in Proceed-ings of the 2nd EuropeanDirofilariaDays RMorchon F SimonJ A Montoya and C Genchi Eds pp 116ndash123 SalamancaSpain September 2009
[2] W A Waren ldquoHeartworm diseaserdquo in Small Animal InternalMedicine R W Nelson and C G Couto Eds pp 169ndash184Mosby St Louis Mo USA 3rd edition 2003
[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L D Alpizar ldquoDiscrimination between six speciesof canine microfilariae by a single polymerase chain reactionrdquoVeterinary Parasitology vol 135 no 3-4 pp 303ndash314 2006
[4] N B Robinson C M Chavez and J H Conn ldquoPulmonarydirofilariasis in man A case report and review of the literaturerdquoJournal of Thoracic and Cardiovascular Surgery vol 74 no 3pp 403ndash408 1977
[5] J C Darrow and E E Lack ldquoSolitary lung nodule dueto Dirofilaria immitis (dog lsquoheartwormrsquo)rdquo Journal of SurgicalOncology vol 16 no 3 pp 219ndash224 1981
[6] F Simon AMuroM Cordero and JMartin ldquoA seroepidemio-logic survey of human dirofilariosis in Western Spainrdquo TropicalMedicine and Parasitology vol 42 no 2 pp 106ndash108 1991
[7] S Pampiglione F Rivasi G Angeli et al ldquoDirofilariasis due toDirofilaria repens in Italy an emergent zoonosis report of 60new casesrdquo Histopathology vol 38 no 4 pp 344ndash354 2001
[8] A Echeverri R F Long W Check and C M BurnettldquoPulmonary dirofilariasisrdquo Annals of Thoracic Surgery vol 67no 1 pp 201ndash202 1999
[9] J A Montoya-Alonso I Mellado E Carreton E D Cabrera-Pedrero R Morchon and F Simon ldquoCanine dirofilariosis
caused by Dirofilaria immitis is a risk factor for the humanpopulation on the island of Gran Canaria Canary IslandsSpainrdquo Parasitology Research vol 107 no 5 pp 1265ndash1269 2010
[10] F SimonM Siles-Lucas RMorchon et al ldquoHuman and animaldirofilariasis the emergence of a zoonotic mosaicrdquo ClinicalMicrobiology Reviews vol 25 no 3 pp 507ndash544 2012
[11] F Ciferri ldquoHumanpulmonary dirofilariasis in theUnited Statesa critical reviewrdquo American Journal of Tropical Medicine andHygiene vol 31 no 2 pp 302ndash308 1982
[12] B P Badhe and S Y Sane ldquoHuman pulmonary dirofilariasisin India a case reportrdquo The Journal of Tropical Medicine andHygiene vol 92 no 6 pp 425ndash426 1989
[13] T Dam and P Das ldquoThe importance of dirofilariasis in IndiardquoThe Internet Journal of Parasitic Diseases vol 1 no 1 2006
[14] R B Grieve L T Glickman A K Bater M Mika-GrieveC B Thomas and G J Patronek ldquoCanine Dirofilaria immitisinfection in a hyperenzootic area examination by parasitologicfindings at necropsy and by two serodiagnostic methodsrdquoAmerican Journal of Veterinary Research vol 47 no 2 pp 329ndash332 1986
[15] R Hatsushika T Okino and F Ohyama ldquoThe prevalence ofdog heartworm (Dirofilaria immitis) infection in stray dogs inOkayama Japanrdquo Kawasaki Medical Journal vol 18 pp 75ndash831992
[16] N Labarthe N Almosny J Guerrero andAMDuque-AraujoldquoDescription of the occurrence of canine Dirofilariasis in theState of Rio de Janeiro Brazilrdquo Memorias do Instituto OswaldoCruz vol 92 no 1 pp 47ndash51 1997
[17] L C Alves L V A de Silva M A da Gloria Faustino et alldquoSurvey of canine heartworm in the city of Recife PernambucoBrazilrdquo Memorias do Instituto Oswaldo Cruz vol 94 no 5 pp587ndash590 1999
[18] L Venco L Kramer and C Genchi ldquoHeartworm disease indogs unusual clinical casesrdquo Veterinary Parasitology vol 133no 2-3 pp 207ndash218 2005
[19] A Niwetpathomwat S Assarasakorn S Techangamsuwan SSuvarnavibhaja and M Kaewthamasorn ldquoCanine dirofilariasisand concurrent tick-borne transmitted diseases in BangkokThailandrdquo Journal of Comparative Clinical Pathology vol 15 no4 pp 249ndash253 2006
[20] M E Bolio-Gonzalez R I Rodriguez-Vivas C H Sauri-ArceoE Gutierrez-Blanco A Ortega-Pacheco and R F Colin-FloresldquoPrevalence of the Dirofilaria immitis infection in dogs fromMerida Yucatan Mexicordquo Veterinary Parasitology vol 148 no2 pp 166ndash169 2007
[21] A Chakrabarti and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983
[22] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMclnnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesand Vectors vol 3 article 30 2010
[23] S K Borthakur K Sarma T K Rajkhowa M R Das andS Rahman ldquoDirofilaria immitis infection in dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006
[24] K Bhattacharjee and P C Sarmah ldquoEpidemiological aspects ofDirofilaria immitis infection in dogs from Assam of NortheastIndiardquo Asian Pacific Journal of Tropical Disease vol 4 supple-ment 1 pp S255ndashS258 2014
[25] C M Hendrix Diagnostic Veterinary Parasitology Mosby StLouis Mo USA 2nd edition 1998
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 The Scientific World Journal
Table 2 Comparative efficacy percentage of microscopy Ag ELISA and PCR in detecting Dirofilaria immitis infection in dogs
Detection methods
Dog category
Number ofbloodsamplestested
Microscopy (Knottrsquostechnique)
Ag ELISA(SNAP 4Dx test) PCR
Numbers of positive () Numbers ofpositive ()
Specific primer for Dimmitis ()
Panfilarial primers()
Stray 413 69 (1670) 107 (2590) 88 (2130) 92 (88 + 4lowast)(2227)
Pet 266 16 (601) 24 (902) 17 (639) 17 (639)Working 103 4 (388) 10 (970) 4 (388) 4 (388)Grand total 782 89 (1138) 141 (1803) 109 (1393) 113 (1445)lowastDirofilaria repens
A B C
302bp
(a)
A B C D E
484bp542bp
(b)
Figure 2 (a) Gel picture showing amplification of D immitis (specific primers) Lane A 100 bp ladder Lane B negative Lane C PCRproduct of ITS-2 (b) Gel picture showing amplification of D immitis and D repens (panfilarial primers) Lane A 100 bp ladder Lanes B andC amplification for D immitis Lanes D and E amplification for D repens
sequence for 2 isolates of D immitis (accession numbersJX481279 and JX866681) and forD repens (accession numberJX524743) were obtained from GenBank
4 Discussion
The present study provides the first comprehensive assess-ment of D immitis infection in dogs from the NortheasternStates of India The number of dogs proven positive forD immitis in one or more diagnostic tests was variableaccording to the test applied Recording of 626 bloodsamples microfilaraemic under wet film does not give theactual epidemiological situation of the studied areas owingto the low sensitivity of the method and to the failure inmicrofilarial species differentiation [28] Currently abundantliterature suggests detecting antigen test as the most sensitivediagnostic method for canine heartworm [29] The presentstudy showed prevalence of 902D immitis infection in petdogs and 2590 in stray dogs A similar type of varyingfrom 476 to 2954 D immitis infection was recorded inAssam in pet and street dogs respectively [24]The records of
highest prevalence in straystreet dogs are likely due to theirfree roaming habits making them vulnerable to being bittenby different mosquito vectors Moreover the present studywas carried out in a geographical location where subtropicalclimate and deciduous forest land prevail therefore in anenvironment where high rainfall and humidity create idealmosquito breeding places
Our present study also revealed a higher prevalence ofDimmitis in male dogs but we could not draw a conclusion onthe differences of prevalence amongst male and female dogsLike most record on heartworm prevalence [24 30ndash32] andunlike few cases from elsewhere [33 34] our study found anonsignificant higher prevalence of D immitis in male dogs
In the present investigation many dogs which werefound positive using SNAP 4Dx kit revealed occult infec-tions The same samples when subjected to PCR studiesrevealed lesser percent prevalenceOccult infections (amicro-filaraemic infections) could arise due to several causes likelow parasite burdens prepatent infection by young adultsinfection of dog by only male worm geriatric female wormand immune response from the host against microfilariae or
The Scientific World Journal 5
EU182329 ChinaEU182330 ChinaAF217800 Taiwan
EU087699 MizoramEU182331 China
JN084166 IranFJ263456 BrazilHM126607 TurkeyFJ263462 BrazilFJ263464 BrazilJN084168 Iran
GuwahatiGuwahati
02454
Nucleotide substitutions (
JX8666811JX4812791
JX889634 Iran
times100)
Figure 3 Phylogenetic tree constructed for Dirofilaria immitis from the ITS-2 region using Clustal W of DNASTAR
Table 3 ITS-2 sequence pair distance analysis of Dirofilaria immitis isolates compared with homologous isolates (slowaccurate IUB)
Percent identity1 2 3 4 5 6 7 8 9 10 11 12 13 14
Divergence
1 998 941 910 902 902 847 934 955 957 959 954 945 989 1 JX8666811 Guwahati2 02 941 910 902 902 847 934 955 954 957 952 943 986 2 JX4812791 Guwahati3 51 51 957 949 941 914 933 937 957 886 937 863 937 3 FJ2634568 Brazil4 87 87 46 976 937 890 902 906 922 855 906 831 906 4 FJ263462 Brazil5 96 96 55 25 953 883 902 898 914 848 898 824 898 5 FJ263464 Brazil6 96 96 63 68 50 879 898 895 914 848 895 832 898 6 HM126607 Turkey7 136 136 61 89 93 98 885 885 868 800 881 786 844 7 JN084168 Iran8 41 41 43 79 74 79 128 974 954 893 971 876 934 8 JN084166 Iran9 18 18 38 74 79 84 127 28 980 916 992 899 955 9 JX889634 Iran10 05 07 29 69 73 73 136 41 12 920 970 907 950 10 EU087699 Mizoram11 00 02 54 93 102 102 144 43 19 05 932 954 948 11 EU182329 China12 12 15 38 74 79 84 132 31 09 07 13 918 948 12 EU182331 China13 07 10 67 105 114 104 155 52 28 13 07 20 934 13 EU182330 China14 00 02 51 87 96 96 137 41 18 05 00 12 07 14 AF217800 Taiwan
1 2 3 4 5 6 7 8 9 10 11 12 13 14
under microfilaricidal therapy The high percentage of occultinfection is not uncommon and was previously reported byseveral authors from different parts of the world [35 36]Higher occult cases recorded in working (60) and pet(2916) dogs in comparison to stray dogs (1725) mightbe due to the fact that owners of pet and working dogs areverymuch concerned about the health status of their animalsHence there is regularity in their health check-up thatsurely often required administration of anthelmintic drugslike ivermectin an endectocide drug whose microfilaricidalactivity reduces the number of circulating microfilariae Onthe other hand stray dogs are seldom taken care of with suchtype of medications
The overall prevalence of D immitis detected by PCRwas 1393 (109782) lower than that evidenced by ELISAtest probably due to occult infections or to possible failurein DNA extraction Hence the main benefit of PCR in epi-demiological surveys on dirofilariosis is 100 confirmatoryspecies identification when mixed infections coexist [3 37]
Detection of 4 dogs infected byD repens supports the reportsof human infection in Northeastern States of India [38] andis an alarming finding since this species apparently moreadapted than D immitis to the human host often succeedsin its development to adult worm
Phylogenetic analysis of D immitis isolates of Guwahatishowed a close identity with certain South Asian isolates ofD immitis Pairwise homology analysis revealed 986ndash989identity with a few sequences available at NCBI GenBankPreviously 926 homology ofD immitis of Mizoram isolatewith D immitis of Taiwan isolate (AF217800) was docu-mented [39] In the present study between Guwahati andMizoram isolates the identity was 954 to 957 and thedivergence was 05 to 07
5 Conclusion
This study confirms the predominance of D immitis inthe northeastern region of India and reestablishes the area
6 The Scientific World Journal
as heartworm endemic KCT along with antigen ELISAdetection test confirmed their sensitivity whereas moleculartechniques confirmed their value in identification of caninefilarial wormsThepresence of bothDirofilaria species shouldalert physicians to the risk of human infections
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge the Deans College of VeterinaryScience Assam Agricultural University Khanapara Guwa-hati and College of Veterinary Science amp AH Central Agri-cultural University Selesih Aizawl for providing facilitiesto carry out the research programme The first author alsoacknowledges the help received from Dr Rebecca J TraubSr Lecturer in Veterinary Public Health School of Vet-erinary Science University of Queensland QLD AustraliaFurther the authors are grateful toMrThomasMan GeneralManager of Other Asia and Mr Jerry Chang RegionalMarketing Manager of IDEXX Laboratories Inc for theirgenerosity in providing SNAP 4Dx as FOC kits to carry outthe epidemiological study
References
[1] R Morchon F Simon J Gonzalez-Miguel and I MelladoldquoRelationship DirofilariaHost cellular and molecular mecha-nisms of the heartworm disease vascular pathologyrdquo in Proceed-ings of the 2nd EuropeanDirofilariaDays RMorchon F SimonJ A Montoya and C Genchi Eds pp 116ndash123 SalamancaSpain September 2009
[2] W A Waren ldquoHeartworm diseaserdquo in Small Animal InternalMedicine R W Nelson and C G Couto Eds pp 169ndash184Mosby St Louis Mo USA 3rd edition 2003
[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L D Alpizar ldquoDiscrimination between six speciesof canine microfilariae by a single polymerase chain reactionrdquoVeterinary Parasitology vol 135 no 3-4 pp 303ndash314 2006
[4] N B Robinson C M Chavez and J H Conn ldquoPulmonarydirofilariasis in man A case report and review of the literaturerdquoJournal of Thoracic and Cardiovascular Surgery vol 74 no 3pp 403ndash408 1977
[5] J C Darrow and E E Lack ldquoSolitary lung nodule dueto Dirofilaria immitis (dog lsquoheartwormrsquo)rdquo Journal of SurgicalOncology vol 16 no 3 pp 219ndash224 1981
[6] F Simon AMuroM Cordero and JMartin ldquoA seroepidemio-logic survey of human dirofilariosis in Western Spainrdquo TropicalMedicine and Parasitology vol 42 no 2 pp 106ndash108 1991
[7] S Pampiglione F Rivasi G Angeli et al ldquoDirofilariasis due toDirofilaria repens in Italy an emergent zoonosis report of 60new casesrdquo Histopathology vol 38 no 4 pp 344ndash354 2001
[8] A Echeverri R F Long W Check and C M BurnettldquoPulmonary dirofilariasisrdquo Annals of Thoracic Surgery vol 67no 1 pp 201ndash202 1999
[9] J A Montoya-Alonso I Mellado E Carreton E D Cabrera-Pedrero R Morchon and F Simon ldquoCanine dirofilariosis
caused by Dirofilaria immitis is a risk factor for the humanpopulation on the island of Gran Canaria Canary IslandsSpainrdquo Parasitology Research vol 107 no 5 pp 1265ndash1269 2010
[10] F SimonM Siles-Lucas RMorchon et al ldquoHuman and animaldirofilariasis the emergence of a zoonotic mosaicrdquo ClinicalMicrobiology Reviews vol 25 no 3 pp 507ndash544 2012
[11] F Ciferri ldquoHumanpulmonary dirofilariasis in theUnited Statesa critical reviewrdquo American Journal of Tropical Medicine andHygiene vol 31 no 2 pp 302ndash308 1982
[12] B P Badhe and S Y Sane ldquoHuman pulmonary dirofilariasisin India a case reportrdquo The Journal of Tropical Medicine andHygiene vol 92 no 6 pp 425ndash426 1989
[13] T Dam and P Das ldquoThe importance of dirofilariasis in IndiardquoThe Internet Journal of Parasitic Diseases vol 1 no 1 2006
[14] R B Grieve L T Glickman A K Bater M Mika-GrieveC B Thomas and G J Patronek ldquoCanine Dirofilaria immitisinfection in a hyperenzootic area examination by parasitologicfindings at necropsy and by two serodiagnostic methodsrdquoAmerican Journal of Veterinary Research vol 47 no 2 pp 329ndash332 1986
[15] R Hatsushika T Okino and F Ohyama ldquoThe prevalence ofdog heartworm (Dirofilaria immitis) infection in stray dogs inOkayama Japanrdquo Kawasaki Medical Journal vol 18 pp 75ndash831992
[16] N Labarthe N Almosny J Guerrero andAMDuque-AraujoldquoDescription of the occurrence of canine Dirofilariasis in theState of Rio de Janeiro Brazilrdquo Memorias do Instituto OswaldoCruz vol 92 no 1 pp 47ndash51 1997
[17] L C Alves L V A de Silva M A da Gloria Faustino et alldquoSurvey of canine heartworm in the city of Recife PernambucoBrazilrdquo Memorias do Instituto Oswaldo Cruz vol 94 no 5 pp587ndash590 1999
[18] L Venco L Kramer and C Genchi ldquoHeartworm disease indogs unusual clinical casesrdquo Veterinary Parasitology vol 133no 2-3 pp 207ndash218 2005
[19] A Niwetpathomwat S Assarasakorn S Techangamsuwan SSuvarnavibhaja and M Kaewthamasorn ldquoCanine dirofilariasisand concurrent tick-borne transmitted diseases in BangkokThailandrdquo Journal of Comparative Clinical Pathology vol 15 no4 pp 249ndash253 2006
[20] M E Bolio-Gonzalez R I Rodriguez-Vivas C H Sauri-ArceoE Gutierrez-Blanco A Ortega-Pacheco and R F Colin-FloresldquoPrevalence of the Dirofilaria immitis infection in dogs fromMerida Yucatan Mexicordquo Veterinary Parasitology vol 148 no2 pp 166ndash169 2007
[21] A Chakrabarti and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983
[22] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMclnnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesand Vectors vol 3 article 30 2010
[23] S K Borthakur K Sarma T K Rajkhowa M R Das andS Rahman ldquoDirofilaria immitis infection in dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006
[24] K Bhattacharjee and P C Sarmah ldquoEpidemiological aspects ofDirofilaria immitis infection in dogs from Assam of NortheastIndiardquo Asian Pacific Journal of Tropical Disease vol 4 supple-ment 1 pp S255ndashS258 2014
[25] C M Hendrix Diagnostic Veterinary Parasitology Mosby StLouis Mo USA 2nd edition 1998
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
The Scientific World Journal 5
EU182329 ChinaEU182330 ChinaAF217800 Taiwan
EU087699 MizoramEU182331 China
JN084166 IranFJ263456 BrazilHM126607 TurkeyFJ263462 BrazilFJ263464 BrazilJN084168 Iran
GuwahatiGuwahati
02454
Nucleotide substitutions (
JX8666811JX4812791
JX889634 Iran
times100)
Figure 3 Phylogenetic tree constructed for Dirofilaria immitis from the ITS-2 region using Clustal W of DNASTAR
Table 3 ITS-2 sequence pair distance analysis of Dirofilaria immitis isolates compared with homologous isolates (slowaccurate IUB)
Percent identity1 2 3 4 5 6 7 8 9 10 11 12 13 14
Divergence
1 998 941 910 902 902 847 934 955 957 959 954 945 989 1 JX8666811 Guwahati2 02 941 910 902 902 847 934 955 954 957 952 943 986 2 JX4812791 Guwahati3 51 51 957 949 941 914 933 937 957 886 937 863 937 3 FJ2634568 Brazil4 87 87 46 976 937 890 902 906 922 855 906 831 906 4 FJ263462 Brazil5 96 96 55 25 953 883 902 898 914 848 898 824 898 5 FJ263464 Brazil6 96 96 63 68 50 879 898 895 914 848 895 832 898 6 HM126607 Turkey7 136 136 61 89 93 98 885 885 868 800 881 786 844 7 JN084168 Iran8 41 41 43 79 74 79 128 974 954 893 971 876 934 8 JN084166 Iran9 18 18 38 74 79 84 127 28 980 916 992 899 955 9 JX889634 Iran10 05 07 29 69 73 73 136 41 12 920 970 907 950 10 EU087699 Mizoram11 00 02 54 93 102 102 144 43 19 05 932 954 948 11 EU182329 China12 12 15 38 74 79 84 132 31 09 07 13 918 948 12 EU182331 China13 07 10 67 105 114 104 155 52 28 13 07 20 934 13 EU182330 China14 00 02 51 87 96 96 137 41 18 05 00 12 07 14 AF217800 Taiwan
1 2 3 4 5 6 7 8 9 10 11 12 13 14
under microfilaricidal therapy The high percentage of occultinfection is not uncommon and was previously reported byseveral authors from different parts of the world [35 36]Higher occult cases recorded in working (60) and pet(2916) dogs in comparison to stray dogs (1725) mightbe due to the fact that owners of pet and working dogs areverymuch concerned about the health status of their animalsHence there is regularity in their health check-up thatsurely often required administration of anthelmintic drugslike ivermectin an endectocide drug whose microfilaricidalactivity reduces the number of circulating microfilariae Onthe other hand stray dogs are seldom taken care of with suchtype of medications
The overall prevalence of D immitis detected by PCRwas 1393 (109782) lower than that evidenced by ELISAtest probably due to occult infections or to possible failurein DNA extraction Hence the main benefit of PCR in epi-demiological surveys on dirofilariosis is 100 confirmatoryspecies identification when mixed infections coexist [3 37]
Detection of 4 dogs infected byD repens supports the reportsof human infection in Northeastern States of India [38] andis an alarming finding since this species apparently moreadapted than D immitis to the human host often succeedsin its development to adult worm
Phylogenetic analysis of D immitis isolates of Guwahatishowed a close identity with certain South Asian isolates ofD immitis Pairwise homology analysis revealed 986ndash989identity with a few sequences available at NCBI GenBankPreviously 926 homology ofD immitis of Mizoram isolatewith D immitis of Taiwan isolate (AF217800) was docu-mented [39] In the present study between Guwahati andMizoram isolates the identity was 954 to 957 and thedivergence was 05 to 07
5 Conclusion
This study confirms the predominance of D immitis inthe northeastern region of India and reestablishes the area
6 The Scientific World Journal
as heartworm endemic KCT along with antigen ELISAdetection test confirmed their sensitivity whereas moleculartechniques confirmed their value in identification of caninefilarial wormsThepresence of bothDirofilaria species shouldalert physicians to the risk of human infections
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge the Deans College of VeterinaryScience Assam Agricultural University Khanapara Guwa-hati and College of Veterinary Science amp AH Central Agri-cultural University Selesih Aizawl for providing facilitiesto carry out the research programme The first author alsoacknowledges the help received from Dr Rebecca J TraubSr Lecturer in Veterinary Public Health School of Vet-erinary Science University of Queensland QLD AustraliaFurther the authors are grateful toMrThomasMan GeneralManager of Other Asia and Mr Jerry Chang RegionalMarketing Manager of IDEXX Laboratories Inc for theirgenerosity in providing SNAP 4Dx as FOC kits to carry outthe epidemiological study
References
[1] R Morchon F Simon J Gonzalez-Miguel and I MelladoldquoRelationship DirofilariaHost cellular and molecular mecha-nisms of the heartworm disease vascular pathologyrdquo in Proceed-ings of the 2nd EuropeanDirofilariaDays RMorchon F SimonJ A Montoya and C Genchi Eds pp 116ndash123 SalamancaSpain September 2009
[2] W A Waren ldquoHeartworm diseaserdquo in Small Animal InternalMedicine R W Nelson and C G Couto Eds pp 169ndash184Mosby St Louis Mo USA 3rd edition 2003
[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L D Alpizar ldquoDiscrimination between six speciesof canine microfilariae by a single polymerase chain reactionrdquoVeterinary Parasitology vol 135 no 3-4 pp 303ndash314 2006
[4] N B Robinson C M Chavez and J H Conn ldquoPulmonarydirofilariasis in man A case report and review of the literaturerdquoJournal of Thoracic and Cardiovascular Surgery vol 74 no 3pp 403ndash408 1977
[5] J C Darrow and E E Lack ldquoSolitary lung nodule dueto Dirofilaria immitis (dog lsquoheartwormrsquo)rdquo Journal of SurgicalOncology vol 16 no 3 pp 219ndash224 1981
[6] F Simon AMuroM Cordero and JMartin ldquoA seroepidemio-logic survey of human dirofilariosis in Western Spainrdquo TropicalMedicine and Parasitology vol 42 no 2 pp 106ndash108 1991
[7] S Pampiglione F Rivasi G Angeli et al ldquoDirofilariasis due toDirofilaria repens in Italy an emergent zoonosis report of 60new casesrdquo Histopathology vol 38 no 4 pp 344ndash354 2001
[8] A Echeverri R F Long W Check and C M BurnettldquoPulmonary dirofilariasisrdquo Annals of Thoracic Surgery vol 67no 1 pp 201ndash202 1999
[9] J A Montoya-Alonso I Mellado E Carreton E D Cabrera-Pedrero R Morchon and F Simon ldquoCanine dirofilariosis
caused by Dirofilaria immitis is a risk factor for the humanpopulation on the island of Gran Canaria Canary IslandsSpainrdquo Parasitology Research vol 107 no 5 pp 1265ndash1269 2010
[10] F SimonM Siles-Lucas RMorchon et al ldquoHuman and animaldirofilariasis the emergence of a zoonotic mosaicrdquo ClinicalMicrobiology Reviews vol 25 no 3 pp 507ndash544 2012
[11] F Ciferri ldquoHumanpulmonary dirofilariasis in theUnited Statesa critical reviewrdquo American Journal of Tropical Medicine andHygiene vol 31 no 2 pp 302ndash308 1982
[12] B P Badhe and S Y Sane ldquoHuman pulmonary dirofilariasisin India a case reportrdquo The Journal of Tropical Medicine andHygiene vol 92 no 6 pp 425ndash426 1989
[13] T Dam and P Das ldquoThe importance of dirofilariasis in IndiardquoThe Internet Journal of Parasitic Diseases vol 1 no 1 2006
[14] R B Grieve L T Glickman A K Bater M Mika-GrieveC B Thomas and G J Patronek ldquoCanine Dirofilaria immitisinfection in a hyperenzootic area examination by parasitologicfindings at necropsy and by two serodiagnostic methodsrdquoAmerican Journal of Veterinary Research vol 47 no 2 pp 329ndash332 1986
[15] R Hatsushika T Okino and F Ohyama ldquoThe prevalence ofdog heartworm (Dirofilaria immitis) infection in stray dogs inOkayama Japanrdquo Kawasaki Medical Journal vol 18 pp 75ndash831992
[16] N Labarthe N Almosny J Guerrero andAMDuque-AraujoldquoDescription of the occurrence of canine Dirofilariasis in theState of Rio de Janeiro Brazilrdquo Memorias do Instituto OswaldoCruz vol 92 no 1 pp 47ndash51 1997
[17] L C Alves L V A de Silva M A da Gloria Faustino et alldquoSurvey of canine heartworm in the city of Recife PernambucoBrazilrdquo Memorias do Instituto Oswaldo Cruz vol 94 no 5 pp587ndash590 1999
[18] L Venco L Kramer and C Genchi ldquoHeartworm disease indogs unusual clinical casesrdquo Veterinary Parasitology vol 133no 2-3 pp 207ndash218 2005
[19] A Niwetpathomwat S Assarasakorn S Techangamsuwan SSuvarnavibhaja and M Kaewthamasorn ldquoCanine dirofilariasisand concurrent tick-borne transmitted diseases in BangkokThailandrdquo Journal of Comparative Clinical Pathology vol 15 no4 pp 249ndash253 2006
[20] M E Bolio-Gonzalez R I Rodriguez-Vivas C H Sauri-ArceoE Gutierrez-Blanco A Ortega-Pacheco and R F Colin-FloresldquoPrevalence of the Dirofilaria immitis infection in dogs fromMerida Yucatan Mexicordquo Veterinary Parasitology vol 148 no2 pp 166ndash169 2007
[21] A Chakrabarti and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983
[22] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMclnnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesand Vectors vol 3 article 30 2010
[23] S K Borthakur K Sarma T K Rajkhowa M R Das andS Rahman ldquoDirofilaria immitis infection in dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006
[24] K Bhattacharjee and P C Sarmah ldquoEpidemiological aspects ofDirofilaria immitis infection in dogs from Assam of NortheastIndiardquo Asian Pacific Journal of Tropical Disease vol 4 supple-ment 1 pp S255ndashS258 2014
[25] C M Hendrix Diagnostic Veterinary Parasitology Mosby StLouis Mo USA 2nd edition 1998
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 The Scientific World Journal
as heartworm endemic KCT along with antigen ELISAdetection test confirmed their sensitivity whereas moleculartechniques confirmed their value in identification of caninefilarial wormsThepresence of bothDirofilaria species shouldalert physicians to the risk of human infections
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors acknowledge the Deans College of VeterinaryScience Assam Agricultural University Khanapara Guwa-hati and College of Veterinary Science amp AH Central Agri-cultural University Selesih Aizawl for providing facilitiesto carry out the research programme The first author alsoacknowledges the help received from Dr Rebecca J TraubSr Lecturer in Veterinary Public Health School of Vet-erinary Science University of Queensland QLD AustraliaFurther the authors are grateful toMrThomasMan GeneralManager of Other Asia and Mr Jerry Chang RegionalMarketing Manager of IDEXX Laboratories Inc for theirgenerosity in providing SNAP 4Dx as FOC kits to carry outthe epidemiological study
References
[1] R Morchon F Simon J Gonzalez-Miguel and I MelladoldquoRelationship DirofilariaHost cellular and molecular mecha-nisms of the heartworm disease vascular pathologyrdquo in Proceed-ings of the 2nd EuropeanDirofilariaDays RMorchon F SimonJ A Montoya and C Genchi Eds pp 116ndash123 SalamancaSpain September 2009
[2] W A Waren ldquoHeartworm diseaserdquo in Small Animal InternalMedicine R W Nelson and C G Couto Eds pp 169ndash184Mosby St Louis Mo USA 3rd edition 2003
[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L D Alpizar ldquoDiscrimination between six speciesof canine microfilariae by a single polymerase chain reactionrdquoVeterinary Parasitology vol 135 no 3-4 pp 303ndash314 2006
[4] N B Robinson C M Chavez and J H Conn ldquoPulmonarydirofilariasis in man A case report and review of the literaturerdquoJournal of Thoracic and Cardiovascular Surgery vol 74 no 3pp 403ndash408 1977
[5] J C Darrow and E E Lack ldquoSolitary lung nodule dueto Dirofilaria immitis (dog lsquoheartwormrsquo)rdquo Journal of SurgicalOncology vol 16 no 3 pp 219ndash224 1981
[6] F Simon AMuroM Cordero and JMartin ldquoA seroepidemio-logic survey of human dirofilariosis in Western Spainrdquo TropicalMedicine and Parasitology vol 42 no 2 pp 106ndash108 1991
[7] S Pampiglione F Rivasi G Angeli et al ldquoDirofilariasis due toDirofilaria repens in Italy an emergent zoonosis report of 60new casesrdquo Histopathology vol 38 no 4 pp 344ndash354 2001
[8] A Echeverri R F Long W Check and C M BurnettldquoPulmonary dirofilariasisrdquo Annals of Thoracic Surgery vol 67no 1 pp 201ndash202 1999
[9] J A Montoya-Alonso I Mellado E Carreton E D Cabrera-Pedrero R Morchon and F Simon ldquoCanine dirofilariosis
caused by Dirofilaria immitis is a risk factor for the humanpopulation on the island of Gran Canaria Canary IslandsSpainrdquo Parasitology Research vol 107 no 5 pp 1265ndash1269 2010
[10] F SimonM Siles-Lucas RMorchon et al ldquoHuman and animaldirofilariasis the emergence of a zoonotic mosaicrdquo ClinicalMicrobiology Reviews vol 25 no 3 pp 507ndash544 2012
[11] F Ciferri ldquoHumanpulmonary dirofilariasis in theUnited Statesa critical reviewrdquo American Journal of Tropical Medicine andHygiene vol 31 no 2 pp 302ndash308 1982
[12] B P Badhe and S Y Sane ldquoHuman pulmonary dirofilariasisin India a case reportrdquo The Journal of Tropical Medicine andHygiene vol 92 no 6 pp 425ndash426 1989
[13] T Dam and P Das ldquoThe importance of dirofilariasis in IndiardquoThe Internet Journal of Parasitic Diseases vol 1 no 1 2006
[14] R B Grieve L T Glickman A K Bater M Mika-GrieveC B Thomas and G J Patronek ldquoCanine Dirofilaria immitisinfection in a hyperenzootic area examination by parasitologicfindings at necropsy and by two serodiagnostic methodsrdquoAmerican Journal of Veterinary Research vol 47 no 2 pp 329ndash332 1986
[15] R Hatsushika T Okino and F Ohyama ldquoThe prevalence ofdog heartworm (Dirofilaria immitis) infection in stray dogs inOkayama Japanrdquo Kawasaki Medical Journal vol 18 pp 75ndash831992
[16] N Labarthe N Almosny J Guerrero andAMDuque-AraujoldquoDescription of the occurrence of canine Dirofilariasis in theState of Rio de Janeiro Brazilrdquo Memorias do Instituto OswaldoCruz vol 92 no 1 pp 47ndash51 1997
[17] L C Alves L V A de Silva M A da Gloria Faustino et alldquoSurvey of canine heartworm in the city of Recife PernambucoBrazilrdquo Memorias do Instituto Oswaldo Cruz vol 94 no 5 pp587ndash590 1999
[18] L Venco L Kramer and C Genchi ldquoHeartworm disease indogs unusual clinical casesrdquo Veterinary Parasitology vol 133no 2-3 pp 207ndash218 2005
[19] A Niwetpathomwat S Assarasakorn S Techangamsuwan SSuvarnavibhaja and M Kaewthamasorn ldquoCanine dirofilariasisand concurrent tick-borne transmitted diseases in BangkokThailandrdquo Journal of Comparative Clinical Pathology vol 15 no4 pp 249ndash253 2006
[20] M E Bolio-Gonzalez R I Rodriguez-Vivas C H Sauri-ArceoE Gutierrez-Blanco A Ortega-Pacheco and R F Colin-FloresldquoPrevalence of the Dirofilaria immitis infection in dogs fromMerida Yucatan Mexicordquo Veterinary Parasitology vol 148 no2 pp 166ndash169 2007
[21] A Chakrabarti and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983
[22] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMclnnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesand Vectors vol 3 article 30 2010
[23] S K Borthakur K Sarma T K Rajkhowa M R Das andS Rahman ldquoDirofilaria immitis infection in dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006
[24] K Bhattacharjee and P C Sarmah ldquoEpidemiological aspects ofDirofilaria immitis infection in dogs from Assam of NortheastIndiardquo Asian Pacific Journal of Tropical Disease vol 4 supple-ment 1 pp S255ndashS258 2014
[25] C M Hendrix Diagnostic Veterinary Parasitology Mosby StLouis Mo USA 2nd edition 1998
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
The Scientific World Journal 7
[26] E J L Soulsby Helminths Arthropods and Protozoa of Domes-ticated Animals ELBS Bailliere and Tindall London UK 7thedition 1982
[27] P-H Mar I-C Yang G-N Chang and A C-Y Fei ldquoSpe-cific polymerase chain reaction for differential diagnosis ofDirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region 2 (ITS2)rdquoVeterinary Parasitology vol 106 no 3 pp 243ndash252 2002
[28] C Genchi L Rinaldi and G Cringoli Mappe Parassitologiche8 Dirofilaria immitis and D repens in Dog and Cat and HumanInfections Rolando Editore Naples Italy 2007
[29] C Datz ldquoUpdate on canine and feline heartworm testsrdquoCompendium on Continuing Education for the Practicing Veteri-narian vol 25 no 1 pp 30ndash40 2003
[30] C K Fan K E Su Y H Lin C W Liao W Y Du andH Y Chiou ldquoSeroepidemiologic survey of Dirofilaria immitisinfection among domestic dogs in Taipei City and mountainaboriginal districts in Taiwan (1998-1999)rdquoVeterinary Parasitol-ogy vol 102 no 1-2 pp 113ndash120 2001
[31] K H Song S E Lee M Hayasaki K Shiramizu D H KimandKW Cho ldquoSeroprevalence of canine dirofilariosis in SouthKoreardquoVeterinary Parasitology vol 114 no 3 pp 231ndash236 2003
[32] H Hou G Shen W Wu et al ldquoPrevalence of Dirofilariaimmitis infection in dogs from Dandong Chinardquo VeterinaryParasitology vol 183 no 1-2 pp 189ndash193 2011
[33] J A Montoya M Morales O Ferrer J M Molina and J ACorbera ldquoThe prevalence ofDirofilaria immitis in gran canariaCanary Islands Spain (1994ndash1996)rdquo Veterinary Parasitologyvol 75 no 2-3 pp 221ndash226 1998
[34] A Yildirim A Ica O Atalay O Duzlu and A Inci ldquoPrevalenceand epidemiological aspects ofDirofilaria immitis in dogs fromKayseri Province Turkeyrdquo Research in Veterinary Science vol82 no 3 pp 358ndash363 2007
[35] C-H Lai C-H Ting K-C Tung and J-S Wang ldquoVariationin the prevalence of dirofilariasis in stray dogs from CentralTaiwanrdquo Journal of the Chinese Society of Veterinary Science vol27 pp 69ndash73 2001
[36] K Yildiz S Y Duru B B Yagci N Ocal and A N GazyagcildquoThe prevalence of Dirofilaria immitis in dogs in KirikkalerdquoTurkiye Parazitoloji Dergisi vol 32 no 3 pp 225ndash228 2008
[37] G Favia A Lanfrancotti A Della Torre G Cancrini and MColuzzi ldquoPolymerase chain reaction-identification of Dirofi-laria repens and Dirofilaria immitisrdquo Parasitology vol 113 no6 pp 567ndash571 1996
[38] R Nath R Gogoi N Bordoloi and T Gogoi ldquoOcular dirofilar-iasisrdquo Indian Journal of Pathology and Microbiology vol 53 no1 pp 157ndash159 2010
[39] J BamMolecular cloning and characterization of ITS1 and ITS2regions of ribosomal gene of Dirofilaria immitis [MS thesis]Deemed University Indian Veterinary Research Institute 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
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