canine filarial infections in a human brugia malayi

Research ArticleCanine Filarial Infections in a Human Brugia malayiEndemic Area of India

Reghu Ravindran1 Sincy Varghese1 Suresh N Nair2 Vimalkumar M Balan3

Bindu Lakshmanan1 Riyas M Ashruf3 Swaroop S Kumar1 Ajith Kumar K Gopalan1

Archana S Nair3 Aparna Malayil1 Leena Chandrasekhar4 Sanis Juliet2

Devada Kopparambil1 Rajendran Ramachandran5 Regu Kunjupillai5

and Showkath Ali M Kakada5

1 Department of Veterinary Parasitology College of Veterinary and Animal Sciences Pookode Wayanad Kerala 673576 India2Department of Veterinary Pharmacology and Toxicology College of Veterinary and Animal Sciences Pookode WayanadKerala 673 576 India

3 Department of Animal Husbandry Kerala 695 033 India4Department of Veterinary Anatomy College of Veterinary and Animal Sciences Pookode Wayanad Kerala 673 576 India5 National Centre for Diseases Control Kerala 673 003 India

Correspondence should be addressed to Reghu Ravindran drreghuraviyahoocom

Received 17 February 2014 Accepted 22 April 2014 Published 20 May 2014

Academic Editor Fabio Ribeiro Braga

Copyright copy 2014 Reghu Ravindran et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A very high prevalence of microfilaremia of 4268 per cent out of 164 canine blood samples examined was observed in Cherthala (ofAlappuzha district of Kerala state) a known human Brugia malayi endemic area of south IndiaThe species of canine microfilariaewere identified asDirofilaria repens Brugiamalayi andAcanthocheilonema reconditumD repenswas themost commonly detectedspecies followed by B pahangi D immitis was not detected in any of the samples examined Based on molecular techniquesmicrofilariae with histochemical staining pattern of ldquolocal staining at anal pore and diffuse staining at central bodyrdquowas identified asD repens in addition to those showing acid phosphatase activity only at the anal pore Even though B malayi like acid phosphataseactivity was observed in few dogs examined they were identified as genetically closer to B pahangi Hence the possibility of dogsacting as reservoirs of human B malayi in this area was ruled out

1 Introduction

Major filarial parasites of dogs are Acanthocheilonema recon-ditum Acanthocheilonema dracunculoides Brugia malayiBrugia pahangi Brugia ceylonensis Brugia patei Cercopithifi-laria grassii Dirofilaria immitis and Dirofilaria repens [1ndash4]Microfilariae ofBrugia spp are sheathedwhile those of othersare not sheathed

Canine filariosis was reported from various parts ofIndia mainly from states like Kerala Tamil Nadu KarnatakaOrissa West Bengal Bihar Uttar Pradesh and MaharashtraThe species ofmicrofilariae detected from these states includeD immitis from Kerala [5] C grassi from Tamil Nadu [6]

D immitis fromHimalayas [7]D immitis andA reconditumfrom West Bengal [8] D immitis and D repens from Orissa[9] D repens from Kerala [10] D repens and A reconditumfrom Karnataka [11] A reconditum D immitis and D repensfromMaharashtra and New Delhi and Microfilaria auquieriand a novel species of Acanthocheilonema from LadakhIndia [12] In general it is believed that D immitis is mostlyprevalent in north eastern India [13] while D repens isconfined to southern parts of the country [14 15]

Currently there is paucity of information on the preva-lence of filarial worms of dogs in the genus Brugia from IndiaThe disease caused in humans by subperiodic B malayiin Malaysia and Indonesia is considered zoonotic due to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 630160 9 pageshttpdxdoiorg1011552014630160

2 BioMed Research International

the existence of animal reservoir hosts like cats and dogs [16]Brugian filariosis is endemic in Alappuzha district of KeralaState [17ndash19] and thus the presence of B malayi in dogs isalways suspected Even though B malayi like microfilariae[20 21] were detected from cats and dogs from India theywere not confirmed asBmalayiThe present study focuses onthe detection and differentiation of microfilariae of domesticcanines inhabiting aBmalayi endemic area of southern India(Alappuzha district of Kerala state) based on histochemicalstaining and molecular techniques

2 Materials and Methods

21 Ethics Statement All study procedures and protocolswere approved by the institutional animal ethics committee ofCollege of Veterinary and Animal Sciences Pookode Kerala(which follows committee for the purpose of control andsupervision of experiments on animals (CPCSEA) guide-lines Ministry of Environment and Forest Government ofIndia) The committee did not deem it necessary for thisresearch group to obtain formal approval to conduct thisstudy National Centre for Diseases Control directly underDirectorate General of Health Services Ministry of Healthand Family Welfare Government of India is authorized tocollect microfilaria positive human blood samples Writteninformed consent was obtained from the human patients andowners of dogs before collection of samples

22 Preliminary Screening andCollection of Samples Sampleswere collected from dogs by collaborating veterinariansat ldquoprophylactic antirabies vaccination and blood parasitedetection campsrdquo in the south Indian panchayats of Keralastate namely Kuthiathode (KUT) Kadakkarappally (KAD)and Pattanakkad (PKT) Brugian filariosis is endemic in thisarea [17ndash19] Before sample collection data regarding the agesex and breed of these dogs were recorded

Blood samples were collected from the saphenous veinA drop of blood was examined by wet film techniqueat the camp site itself Thick smears were prepared fixedwith methanol stained using Giemsa and examined forthe presence of microfilariae with or without sheath Wholenonheparinized (1mL) andheparinized (1mL) blood sampleswere also collected from the saphenous vein of all these dogsA total of 164 blood samples of dogs (37 from Kadakkarap-pally 57 from Kuthiathode and 70 from Pattanakkad) werecollected

Blood sample of a known B malayi infected humanpatient from Alappuzha the endemic district of Kerala wasalso collected to serve as positive control for histochemicalstaining and PCR techniques

23 Histochemical Differentiation The nonheparinizedblood was allowed to clot and the serum fraction wascentrifuged in an Eppendorf tube for 5 minutes at 1000 rpmAfter discarding the supernatant the sediment containingthe microfilariae was resuspended in the remaining serumand a drop of this fluid was used for preparation of smearsThey were air dried fixed in absolute chilled acetone for

1 minute further air dried and stored in minus20∘C until usedThese smears were used for histochemical staining withintwo weeks after preparation

Histochemical staining was performed [22] to studythe differences in the acid phosphatase enzyme activity inmicrofilariae for identification of the species [22ndash26] Briefly20mL of solution I (Michaelis Veronal Acetate Buffer pH100) was mixed with 50mL of distilled water and 4mL ofsolution II (005 g Naphthol AS-TR phosphate sodium salt(Sigma) in 5mL of N N-dimethyl formamide) was added toit in a beaker In another beaker 32mL each of solutionsIII (10 g Pararosaniline hydrochloride 50mL concentratedhydrochloric acid and 200mL distilled water) and IV (4 percent sodiumnitrite) wasmixed and then added to themixturein the first beaker The pH of the mixture was adjustedto 50 with 01 N sodium hydroxide solution The finalsolution was prepared fresh every time before the stainingprocedure

The air dried smears were incubated in the substrate for1 hour at 37∘C rinsed in distilled water counterstained insolution V (mixture of 771mL of 02M sodium phosphate1229mL 01M nitric acid and 2 g methyl green) for 5minutes and rinsed in distilled water The slides were thendehydrated in 95 per cent and absolute ethyl alcohol respec-tively rinsed in xylene and mounted using DPXThe smearswere examined for the precipitated red azo dye indicating acidphosphatase activity

24 Polymerase Chain Reaction DNA isolation from theheparinised whole blood samples of dogs and human wasbased on phenol-chloroform isoamyl alcohol (PCI) method[27] The leukocyte DNA from a three-day-old pup servedas negative control For the Brugia specific PCR the DNAisolated from a known B malayi infected human patientrsquosblood served as positive control Canine blood samples withheavy (one microfilaria in every low power field) monoin-fection with D repens and A reconditum (confirmed byhistochemical staining) served as positive controls for thesespecies The reactions were carried out in a thermal cycler(Eppendorf Germany) the products were visualized (AlphaInnotech USA) after electrophoresis on a 15 per cent agarosegel and documented

241 D repens Specific PCR PCR assay for the amplificationof 246 bp direct tandem repeats of D repens using specificprimers [28] 51015840-CCGGTAGACCATGGCATTAT-31015840 (For-ward) and 51015840-CGGTCTTGGACGTTTGGTTA-31015840 (Reverse)custom synthesized from IDT USA was standardized ThePCR amplification was performed in 25 120583L reaction volumecontaining 25 120583L 10x PCR buffer 1 120583L (025mM) dNTP and30 pmol of each primer 15 U Taq polymerase and 5 120583L oftemplate DNA Reaction conditions were as follows after theinitial denaturation at 94∘C for 5 minutes 40 cycles eachwith 94∘C for 30 seconds 55∘C for 30 seconds and 72∘Cfor 25 seconds were run A final extension at 72∘C for 5minutes was also given One product of D repens specificPCR was directly sent to Chromos Biotech Bangalore forsequencing

BioMed Research International 3

Table 1 Prevalence of canine microfilariae in a human B malayi endemic area of Kerala India based on wet film examination Giemsastaining histochemical staining and polymerase chain reaction

Place Samplesexamined W f G

Histochemical reactionacid phosphatase reaction Polymerase chain reaction

Drepens

Arecondi-tum

Bpahangi

Newreaction

Bmalayilike

Drepens

A recondi-tum Brugia sp (Hha1)

Kadakkarappally 37 11 16 11 0 3 11 3 10 0 6

Kuthiathode 57 23 29 18 2 3 28 0 28 2 7

Pattanakkad 70 17 25 16 0 12 19 3 26 0 10

Total 164 51 70 45 2 18 58 6 64 2 23W f wet film examination G Giemsa staining

242 A reconditum Specific PCR PCR assay for the ampli-fication of 348 bp ITS2 fragment of A reconditum usingprimers [29] 51015840-CAGGTGATGGTTTGATGTGC-31015840 (For-ward) and 51015840-CACTCGCACTGCTTCACTTC-31015840 (Reverse)custom synthesized from IDT USA was standardized ThePCR amplification was performed in 25 120583L reaction volumecontaining 25 120583L 10x PCR buffer 1 120583L (025mM) dNTP and03mM of each primer 25U Taq polymerase and 5 120583L oftemplate DNA The PCR cycling consisted of a denaturationstep at 94∘C for 3 minutes and 30 cycles each with denaturingat 94∘C for 3 minutes annealing at 63∘C for 1 minuteextension at 72∘C for 30 seconds and final extension at72∘C for 7 minutes One product of A reconditum specificPCR was directly sent to Chromos Biotech Bangalore forsequencing

243 Brugia Specific PCR PCR amplification of a 322 bpHha1 fragment [30] using specific primers 51015840-GCGCAT-AAATTCATCAGC-31015840 (Forward) and 51015840-GCGCAAAAC-TTAATTACAAAAGC-31015840 (Reverse) constituted the geneticdiagnostic criterion for Brugia infection in the study dogsThe PCR amplification was performed in 25 120583L reactionvolume containing 25 120583L 10x PCR buffer 1 120583L (025mM)dNTP and 10 pmol of each primer 25U Taq polymeraseand 5 120583L of template DNA The PCR procedure consistedof an initial denaturation step at 94∘C for 5 minutes and 35cycles each of denaturation at 94∘C for 1 minute annealing at56∘C for 1 minute extension at 72∘C for 1 minute and finalextension at 72∘C for 10 minutes PCR products were clonedand sequenced

244 Amplification of 58S-ITS2-28S In order to confirm theidentity of microfilariae with new acid phosphatase stainingpattern PCR assay for the amplification of 58S-ITS2-28Sfragment of rDNA gene ofsuspected samplesusing specificprimers [3] 51015840-AGTGCGAATTGCAGACGCATTGAG-31015840(Forward) 51015840-AGCGGGTAATCACGACTGAGTTGA-31015840(Reverse) was standardizedThe PCR amplification was per-formed in 25 120583L reaction volume containing 25 120583L 10x PCRbuffer 1 120583L (025mM) dNTP and 100 pmol of each primers15 U Taq polymerase and 5 120583L of template DNA Reactionconditions were as follows after the initial denaturation at94∘C for 2 minutes 32 cycles each with 94∘C for 30 seconds60∘C for 30 seconds and 72∘C for 30 secondswere run Afinal

extension at 72∘C for 7 minutes was also given PCR productswere cloned and sequenced

25 Cloning and Sequencing of PCR Products PCR productswere eluted from the gel using QIA quick gel extraction kit(QIAgen Germany) based on manufacturerrsquos protocol Theeluted product (8 120583L) was used for cloning into a pTZ57RTvector (InsTAclone PCR cloning kit Fermentas USA) whichwas used to transform the JM107 strain of E coli (FermentasUSA) The transformed cells were plated on an LB agarwith ampicillin (50 120583gmL) as the selective antibiotic Afterovernight incubation at 37∘C positive white colonies wereselected and subcultured in LB broth overnightThe presenceof the insert was confirmed by restriction digestion of theisolated plasmid from the subculture using EcoR1 and Sal Ienzymes (GeneJET plasmid mini prep kit Fermentas USA)The stab culture of the colony containing the insert was sentfor automated sequencing to Chromos Biotech Bangalore Inorder to verify mixed infection or recombination 2 clonesfrom each PCR products were sequenced

26 Sequence Analysis The sequence analysis was done usingClustalV method of MegAlign programme (DNASTARUSA)

3 Results

31 Preliminary Screening A total of 164 blood samples ofdogs were collected from Kadakkarappally (37) Kuthiathode(57) and Pattanakkad (70) Out of 164 dogs 114 were fromnondescript animals while 50 were from exotic breeds (SpitzGerman shepherd Dachshund and Doberman)

Wet film examination revealed microfilariae in 11 (2972per cent) samples from Kadakkarappally 23 (4035 per cent)from Kuthiathode and 17 (2429 per cent) from Pattanakkad(Table 1) The overall prevalence of microfilaremia based onwet film examination was 3117 per cent

Giemsa staining technique revealed microfilariae in 16(4324 per cent) samples from Kadakkarappally 29 (5087per cent) from Kuthiathode and 25 (3571 per cent) fromPattanakkad Overall the prevalence of microfilaremia basedon Giemsa staining was 4268 per cent Four dogs revealedmicrofilariae with sheath


An

Figure 1 D repens microfilaria showing acid phosphatase activityat the anal pore (An)

An

E

Figure 2 A reconditum microfilaria showing acid phosphataseactivity throughout the entire body especially between the excretory(E) and anal pores (An)

Fifty two per cent of exotic breeds and 38 per cent ofnondescript breeds of dogs harboured microfilariae Preva-lence of microfilaraemia was more common in dogs abovetwo years of age Also male dogs exhibited higher prevalence(Table 2)

32 Histochemical Differentiation The D repens microfilar-iae showed single locus of intense acid phosphatase stainingin the region of the anal pore (Figure 1) This type of reactionwas observed in 274 per cent of dogs

The entire body especially between the excretory and analpores was stained bright red inmicrofilariae ofA reconditum(Figure 2) Only two out of 164 smears examined revealed thistype of reaction

For B pahangi microfilariae the heavy and diffuse acidphosphatase activity was observed along the entire bodylength although the excretory and anal pores were stillrecognizable (Figure 3) A total of 18 smears (11 per cent)showed this type of reaction

Am

AnE

Figure 3 B pahangi microfilaria showing heavy and diffuse acidphosphatase activity along the entire body lengthThe excretory (E)and anal pores (An) are recognizable

An

Figure 4 Microfilaria showing acid phosphatase activity with redspot at anal pore (An) region and diffuse red staining at the centralbody

The acid phosphatase staining pattern with local stainingat the anal pore and diffuse staining in the central body(Figure 4) was observed in 58 cases (354 per cent) Most ofthe samples contained a mixture of D repens and this type ofmicrofilariae

Blood smears prepared from human patients with con-firmed B malayi infection contained microfilariae with lociof acid phosphatase activity in the areas of the amphids theexcretory pore the anal pore and the phasmids (Figure 5)Out of 164 dogs tested 6 harboured microfilariae withhistochemical reaction similar to B malayi (Figure 6)

33 Polymerase Chain Reaction

331 D repens PCR detected the 246 bp D repens-specificproduct in 64 out of 164 samples PCR product from a samplethat containedmicrofilariae showing a single locus of stainingin the region of the anal pore was sequenced for confirmation(accession number JN830762)


Table2Microfilaraemiain

different

breedsage

grou

psand

sexeso

fdogsinaB

malayiend

emicarea

ofKe

ralaInd

iabased

onGiemsa

staining

techniqu

e

Bloo

dsm

ears

Area

Breed

Age

grou

pSex

Total

ND

Exotic

Spitz

GSD

Dach

Dob

lt2years

gt2years

Male

Female

Exam

ined

Kadakkarappally

296

20

014

2333

437

Kuthiathod

e36

105

42

1344

3918

57Patta

nakk

ad49

172

11

3139

5614

70To

tal

114

339

53

58106

128

36164

Positive

Kadakkarappally

113

20

04

1216

016

(4324)

Kuthiathod

e16

62

32

425

245

29(5087)

Patta

nakk

ad17

61

01

322

205

25(3571)

Total

44(386

)15

(4546

)

5(5556

)

3(60

)3(100

)

11(1897

)

59(5566

)

60(4688

)

10(277

8)

70(4268

)

NDN

ondescrip

tGSDG

erman

shepherdD

achDachshu

ndD

obD

oberman


PAm

AnE

Figure 5 Human B malayimicrofilaria showing acid phosphataseactivity in the areas of amphid (Am) excretory pore (E) anal pore(An) and phasmid (P)

332 A reconditum PCR with primers specific for Areconditum amplified the expected 348 bp product fromtwo samples containing microfilariae with histochemicalreactions typical ofA reconditumThe partial sequence of thePCR product was submitted to GenBank (accession numberJQ039745)

333 Brugia Species PCRwith primers specific forBrugia spamplified the expected 322 bp product from 23 (14 per cent)samples

OneHha1 PCR product each from samples showing onlyB pahangi (PKT 94) (accession number JN601135) humanB malayi (accession number JN413104) and B malayi likeparasite of dog (two clones fromKAD 19) (accession numbersJN601136 and JN601137) were cloned and sequenced Thephylogenetic tree (Figure 7) and distance matrix (Figure 8)plotted based on ClustalV method of MegAlign programme(DNASTAR USA) demonstrated that the microfilariae withhistochemical reaction similar to B malayi were geneticallycloser to B pahangi of dogs

334 Amplification of 58S-ITS2-28S For confirmation of thespecies of microfilariae with the acid phosphatase stainingreaction pattern ldquolocal staining at the anal pore and diffusestaining in the central bodyrdquo sequence analysis of PCR prod-uct of 58S-ITS2-28S fragment of rDNA gene was resortedto The 584 bp fragment was sent for sequencing Whensubmitted to NCBI-BLAST the 492498 bp stretches of the584 bp product (accession numbers JQ039743 and JQ039744)shared 97 per cent identity with the previously reported Drepens sequence AY 693808

4 Discussion

Filarial infections in dogs can be diagnosed throughmorpho-logical observations of the circulatingmicrofilariae detectionof circulating antigens histochemical staining of circulatingmicrofilariae or throughmolecular approaches Proper iden-tification of circulating microfilariae based on morphologyrequires the involvement of a well-trained parasitologistFilarial infection with multiple species and morphologicalalterations of microfilariae due to incorrect preventive treat-ment of dogs are not easily differentiated morphologicallyeven by trained persons [31]

Histochemical staining to detect acid phosphatase activ-ity can overcome most of these problems however this

P

AnAm

E

Figure 6 Canine B malayi like microfilaria showing acid phos-phatase activity in the areas of amphid (Am) excretory pore (E)anal pore (An) and phasmid (P)

technique requires fresh samples to yield optimal resultsBesides being time consuming and labour intensive bothstaining methods require expertise to identify and confirmthe species [32] Molecular methods based on species specificPCRs are simple and easy to perform Use of properlydesigned primers and control template DNAs can make PCRassays species specific PCR assays are very sensitive due tothe exponential amplification of target DNA Additionallyit may be possible with a PCR to detect circulating DNAliberated from host destroyed microfilariae or from adultworms [30]

Microscopical examination of Giemsa stained bloodsmears were used previously for detection of the presence ofmicrofilaremia in dogs of Thrissur Kerala The prevalencevaried from 7 to 265 per cent [10 12 33] In the presentstudy using the same technique the overall prevalence ofmicrofilaremia in dogs was 4268 per cent and the highestprevalence (5087 per cent) was observed in KuthiathodeOnly two samples revealed sheathed microfilariae Othertechniques (histochemical staining and PCR) employed inthe study detected more number of dogs positive for microfi-lariae especially Brugia species

Even though the most pathogenic canine filariaD immi-tis was reported previously from the Kerala state [5] it wasnot identified in any of the dogs tested in the present studyUnambiguous identification of D repens and A reconditumin dogs from the study population by histochemical stainingand sequencing of PCR products was possible in this study

Recently A reconditum (93 per cent) was identified asthe most common species in North India [12] followed byD repens (67 per cent) and D immitis (15 per cent) Inthe present study the microfilariae with a new histochemicalstaining pattern (local staining at the anal pore and diffusestaining at the central body) were more common Based on


90 80 70 60 50 40 30 20 10 0904

B malayi like 2 (dog) JN601137


B pahangi dog JN601135

Brugia malayi human JN413104

Nucleotide substitutions (times100)

Figure 7 Phylogenetic tree constructed based on Hha1 sequence of different Brugia species

Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574

Figure 8 Distance matrix based on Hha1 sequence of different Brugia species

97 per cent identity of 58S-ITS2-28S region of rDNA gene[3] of this form of microfilariae with that of D repens thespecies was confirmed as D repens Therefore D repens isidentified as the most common species of filarial worm in thestudy area However 97 per cent identitymay also indicate aninterspecific difference and therefore the possibility of a newspecies could not be ruled out

Another important finding of the study was the presenceof Brugia species in the dogs of the area The most prevalentBrugia species identified in the study area was B pahangiOut of 164 dogs examined 18 (11 per cent) harboured micro-filariae with acid phosphatase staining pattern specific to thatparasite Histochemical staining pattern similar to B malayiwas observed in 6 out of 164 dogs examined PCR specificfor Brugia sp (Hha1) revealed the diagnostic 322 bp productin 23 samples that included B malayi like microfilariae andB pahangi as indicated by histochemical staining Hha1 PCRproducts amplified in samples of dogs showing only either ofthese parasites based on histochemical staining were selectedfor sequencing Similarly theHha1 fragment of the B malayi(human) was also sequenced Phylogenetic analysis of thesequences revealed that the suspected B malayi like parasitewas genetically closer to B pahangi (dogs) than to B malayi(human) Therefore the results of the present study did notindicate potential of dogs acting as reservoirs of B malayi

However a recent study conducted at Thrissur Kerala(100 km away from the study area) revealed that out of 100dogswith symptoms of filariosis (fever anorexia conjunctivi-tis oedema of limb and scrotum) circulating microfilariaewere detected in 80 cases of which all the 16 cases withsheathed microfilariae were identified as B malayi based onhistochemical staining and PCR [20] The primers specificfor amplification of 294 bp trans-spliced exon 1 (SLX) region(5S r RNA) of Brugia species [16] followed by sequenceanalysis (90 bases with query coverage of 32 per cent)was used for confirming the identification of B malayi indogs The authors concluded that the high prevalence of Bmalayi in Thrissur emphasized the possible role of dogs in

the transmission of humanfilariosisThe results of the presentstudy are contrary to the above report

Lymphatic filariosis is one of the major diseases ofmankind in tropical and subtropical countries and the globalburden of this type of disease is 1191 million cases [34] ofwhich 129 million are affected by Brugian filariosis In IndiaB malayiwas reported from a few foci only the largest singletract being Travancore-Cochin state (present study area) [17]B malayi is known to occur in two forms periodic andsubperiodic The disease caused by subperiodic B malayi inMalaysia and Indonesia is considered zoonotic due to theexistence of animal reservoir hosts like cats and dogs [16 35]Previously two out of 57 cats examined in Orissa state Indiawere found infected with B malayi like microfilariae [21]

Other epidemiological factors also should be consideredbefore confirming the absence of reservoir status in dogsfor B malayi in Cherthala The zoonotic subperiodic Bmalayi is transmitted in nature byMansonia bonnaewhileMdives M annulata and M uniformis are efficient laboratoryvectors The Indian situation is different Microfilariae ofB malayi in India are purely nocturnal in their periodicity[21] In Cherthala ldquoTalukrdquo (an administrative subdivision of adistrict) of Alappuzha district of Kerala Brugian filariosis istransmitted by M annulifera M uniformis and M Indiana[17ndash19] The efficient vector (M bonnae) for the transmissionof zoonotic Brugian filariosis is not reported from this areaAlso a 907 per cent decline in the trend of disease ratefor human filarial infections in Cherthala Taluk was alsoreported [36] If animal reservoirs like dogs were there inexistence there would have been further increase in thenumber of outbreaks of the disease because of the availabilityof reservoirs (including stray dog population) and absence ofmicrofilaricidal therapy in them Also when B malayi couldnot be detected from Cherthala the endemic hotspot it isdifficult to detect the parasite from a place which is 100 kmaway from the endemic area So the possibility for dogs actingas reservoirs of B malayi in these areas is very remote Inaddition the clinical symptoms reported in dogs of Thrissur


Kerala [20] were similar to that of dogs infected with Bpahangi [37] which include varying levels of microfilaraemiaepisodic lymphadenopathy lymphangitis and limb oedemaFor these reasons the detection of B malayi in dogs ofThrissur might be a false detection

The microfilariae of three species namely B tupiae(reported previously fromMalaysiaThailand and Vietnam)B ceylonensis and B pahangi are liable to be confused withthose of B malayi [38] B ceylonensis was first describedin lymphatics of dogs in 1962 from Sri Lanka [39] Thisparasite which is transmitted by Aedes aegypti was reportedfrom Kerala too as early in 1974 but there is no recentdocumentation of this parasite from the state By contrast Bceylonensis was recently reported from the conjunctiva of ahuman patient in Sri Lanka [40] Also a Sri Lankan survey of65 dogs revealed 7 per cent prevalence of single infectionwithB ceylonensis [41] The present study could not differentiatetheBmalayi likemicrofilariae of dogs fromB ceylonensisdueto the absence of reports on the typical histochemical stainingreaction ofB ceylonensismicrofilariae or gene accessionsTheB malayi like microfilariae observed in dogs of the study areacould be a new Brugia species B ceylonensis or a geneticvariant of B pahangi

5 Conclusion

Microfilariae of D repens A reconditum and B pahangioccur in dogs of Cherthala Taluk Alappuzha district Keralathe human B malayi endemic area of south India Sequenceanalysis of 58S-ITS2-28S fragment of rDNA gene of micro-filariae with the acid phosphatase staining pattern of localstaining at the anal pore and diffuse staining in the centralbody revealed 97 per cent homology with D repens The Bmalayi like microfilariae observed in dogs of the study areacould be a new Brugia species B ceylonensis or a geneticvariant of B pahangi

Abbreviations

DNA Deoxyribonucleic acidPCR Polymerase chain reactionrDNA Ribosomal DNADPX Distyrene plasticiser (dibutyl phthalate)

and xylene

Conflict of Interests

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

Acknowledgments

Financial support from Kerala State Council for ScienceTechnology and Environment through the research projectentitled ldquoInvestigation on the role of dogs in the transmissionof Brugian filarial infections to humans and molecular epi-demiology of filarial infections (020SRSAGR2006CSTE)rdquo

is thankfully acknowledged The help rendered by veteri-narians working in Alapuzha district especially Dr RaniBharathan Dr Sangeeth Narayanan and Dr JayachandraKamath is also thankfully acknowledged

References

[1] P J Irwin ldquoCompanion animal parasitology a clinical perspec-tiverdquo International Journal for Parasitology vol 32 no 5 pp581ndash593 2002

[2] J D Kelly ldquoCanine heart worm diseaserdquo in Current VeterinaryTherapy VII pp 326ndash335 W B Saunders Philadelphia PaUSA 1979

[3] M Rishniw S C Barr K W Simpson M F Frongillo MFranz and J L Dominguez Alpizar ldquoDiscrimination betweensix species of canine microfilariae by a single polymerase chainreactionrdquo Veterinary Parasitology vol 135 no 3-4 pp 303ndash3142006

[4] F Simon L H Kramer A Roman et al ldquoImmunopathology ofDirofilaria immitis infectionrdquo Veterinary Research Communica-tions vol 31 no 2 pp 161ndash171 2007

[5] K V Valsala and R Bhaskaran ldquoDirofilariosis in dogsrdquo KeralaJournal of Veterinary Science vol 5 pp 74ndash77 1974

[6] G Balasubramaniam R Anandan and V S Alwar ldquoOn theoccurrence of Dipetalonema grassi (Noe 1907) from dogs inIndiardquo Indian Veterinary Journal vol 52 pp 513ndash516 1975

[7] P Sarkar D K Barak and H M Bhattarchajee ldquoPathology ofDirofilaria immitis infection in dogsrdquo Indian Veterinary Journalvol 53 pp 55ndash57 1976

[8] A Chakrabarthi and M N Choudhury ldquoStudies on caninefilariasis in West Bengalrdquo Indian Journal of Animal Health vol22 pp 151ndash155 1983

[9] M M Patnaik ldquoOn filarial nematodes in domestic animals inOrissardquo Indian Veterinary Journal vol 66 pp 573ndash574 1989

[10] R Radhika Prevalence clinical pathology and treatment ofmicrofilariasis in dogs [MS thesis] Kerala Agricultural Univer-sity Thrissur India 1997

[11] K J Ananda Studies on microfilariosis in dogs of Mangaloreand Bangalore regions in Karnataka [MS thesis] University ofAgricultural Sciences Bangalore India 2003

[12] P A Megat Abd Rani P J Irwin M Gatne G T Coleman LMMcInnes and R J Traub ldquoA survey of canine filarial diseasesof veterinary and public health significance in Indiardquo Parasitesamp Vectors vol 3 no 1 p 30 2010

[13] S K Bortharkur K Sarmah T K Rajakhowa M R Das andS Rahman ldquoDirofilaria immitis infection in a dogrdquo Journal ofVeterinary Parasitology vol 20 pp 167ndash169 2006

[14] K J Ananda P E DrsquoSouza and M S Jaganath ldquoMethodsfor identification of microfilaria of Dirofilaria repens andDipetalonema reconditumrdquo Journal of Veterinary Parasitologyvol 20 no 1 pp 45ndash47 2006

[15] L Sabu K Devada andH Subramanian ldquoDirofilariosis in dogsand humans in Keralardquo Indian Journal of Medical Research vol121 no 5 pp 691ndash693 2005

[16] K Chansiri T Tejangkura P Kwaosak N Sarataphan S Phan-tana and W Sukhumsirichart ldquoPCR based method for iden-tification of zoonostic Brugia malayi microfilariae in domesticcatsrdquo Molecular and Cellular Probes vol 16 no 2 pp 129ndash1352002


[17] M O T Iyengar ldquoStudies on the epidemiology of filariasis inTravancorerdquo Indian Medical Research Memoirs vol 30 pp 1ndash179 1938

[18] P K Rajagopalan K N Panicker and S P Pani ldquoImpact of50 years of vector control on the prevalence of Brugia malayiin Shertallai area of Kerala staterdquo Indian Journal of MedicalResearch AInfectious Diseases vol 89 pp 418ndash425 1989

[19] S SabesanN P Kumar K Krishnamoorthy andKN PanickerldquoSeasonal abundance and biting behaviour ofMansonia annulif-era M uniformis and M indiana and their relative role in thetransmission of malayan filariasis in Shertallai (Kerala state)rdquoIndian Journal of Medical Research A Infectious Diseases vol93 pp 253ndash258 1991

[20] V R Ambily U N Pillai R Arun S Pramod and K MJayakumar ldquoDetection of human filarial parasite Brugia malayiin dogs by histochemical staining and molecular techniquesrdquoVeterinary Parasitology vol 181 no 2ndash4 pp 210ndash214 2011

[21] J F B Edeson ldquoThe epidemiology and treatment of infectiondue to Brugia malayirdquo Bulletin of World Health Organizationvol 27 pp 529ndash541 1962

[22] L Chalifoux and R D Hunt ldquoHistochemical differentiation ofDirofilaria immitis and Dipetalonema reconditumrdquo Journal ofthe American Veterinary Medical Association vol 158 no 5 pp601ndash605 1971

[23] R A Acevedo J H Theis J F Kraus and W M LonghurstldquoCombination of filtration and histochemical stain for detectionand differentiation of Dirofilaria immitis and Dipetalonemareconditum in the dogrdquo American Journal of VeterinaryResearch vol 42 no 3 pp 537ndash540 1981

[24] S E Lee K H Song J Liu et al ldquoComparison of theacid-phosphatase staining and polymerase chain reaction fordetection of Dirofilaria repens infection in dogs in KoreardquoJournal of Veterinary Medical Science vol 66 no 9 pp 1087ndash1089 2004

[25] L M Ortega-Mora M Gomez-Bautista and F A Rojo-Vazquez ldquoThe acid phosphatase activity and morphologicalcharacteristics of Dipetalonema dracunculoides (cobbold 1870)microfilariaerdquoVeterinary Parasitology vol 33 no 2 pp 187ndash1901989

[26] B C Redington C A Montgomery H R Jervis and W THockmeyer ldquoHistochemical differentiation of the microfilariaeof Brugia pahangi and sub periodic Brugia malayirdquo Annals ofTropical Medicine and Parasitology vol 69 no 4 pp 489ndash4921975

[27] J Sambrook and D W Russell ldquoMolecular cloningrdquo in A Lab-oratory Manual pp 61ndash612 Cold Spring Harbor LaboratoryPress Cold Spring Harbor NY USA 3rd edition 2001

[28] N Vakalis G Spanakos E Patsoula andN C VamvakopoulosldquoImproved detection of Dirofilaria repens DNA by direct poly-merase chain reactionrdquo Parasitology International vol 48 no 2pp 145ndash150 1999

[29] P H Mar I Yang G N Chang and A C Y Fei ldquoSpecific poly-merase chain reaction for differential diagnosis of Dirofilariaimmitis and Dipetalonema reconditum using primers derivedfrom internal transcribed spacer region 2 (ITS2)rdquo VeterinaryParasitology vol 106 no 3 pp 243ndash252 2002

[30] M R Lizotte T Supall F Partono and S A Williams ldquoApolymerase chain reaction assay for the detection of Brugiamalayi in bloodrdquo American Journal of Tropical Medicine andHygiene vol 51 no 3 pp 314ndash321 1994

[31] M Casiraghi C Bazzocchi M Mortarino E Ottina and CGenchi ldquoA simple molecular method for discriminating com-mon filarial nematodes of dogs (Canis familiaris)rdquo VeterinaryParasitology vol 141 no 3-4 pp 368ndash372 2006

[32] S Nuchprayoon A Junpee Y Poovorawan and A L ScottldquoDetection and differentiation of filarial parasites by universalprimers and polymerase chain reaction-restriction fragmentlength polymorphism analysisrdquo American Journal of TropicalMedicine and Hygiene vol 73 no 5 pp 895ndash900 2005

[33] M R Saseendranath C G Varghese and K M JayakumarldquolsquoIncidence of canine dirofilariosisrsquo in Trichur Keralardquo IndianJournal of Veterinary Medicine vol 6 p 139 1986

[34] World Health Organization ldquoEpidemiologic approaches tolymphatic filariasis eliminationrdquo in Initial Assessment Monitor-ing and Certification vol 99 pp 1ndash35 WHO Atlanta Ga USA1998

[35] A B C Laing J F B Edeson and R H Wharton ldquoStudieson filariasis inMalaya further experiments on the transmissionof Brugia malayi and Wuchereria bancroftirdquo Annals of TropicalMedicine and Parasitology vol 55 pp 86ndash92 1961

[36] K Regu R Rajendran M K S Ali S M Koya A C Dhariwaland S Lal ldquoDecline of Brugian filariasis in Cherthala talukAlappuzha district Keralardquo Journal of Communicable Diseasesvol 37 no 3 pp 209ndash218 2005

[37] K F Snowden and B Hammerberg ldquoThe lymphatic pathologyof chronic Brugia pahangi infection in the dogrdquo Transactions ofthe Royal Society of Tropical Medicine and Hygiene vol 83 no5 pp 670ndash678 1989

[38] World Health Organization ldquoLymphatic filariasis the diseaseand its controlrdquo WHO Technical Report Series 821 WHOGeneva Switzerland 1992

[39] L G Jayewardene ldquoOn two filarial parasites from dogs inCeylon Brugia ceylonensis n sp and Dipetalonema spinqrdquoJournal of Helminthology vol 36 pp 269ndash280 1962

[40] A S Dissanaike C D Jayaweera Bandara H H PadminiR L Ihalamulla and T D S Naotunne ldquoRecovery of aspecies of Brugia probably B ceylonensis from the conjunctivaof a patient in Sri Lankardquo Annals of Tropical Medicine andParasitology vol 94 no 1 pp 83ndash86 2000

[41] R Rajapakshe W S R Perera R L Ihalamulla et al ldquoStudy ofdirofilariasis in a selected area in theWestern Provincerdquo CeylonMedical Journal vol 50 no 2 pp 58ndash61 2005


the existence of animal reservoir hosts like cats and dogs [16]Brugian filariosis is endemic in Alappuzha district of KeralaState [17ndash19] and thus the presence of B malayi in dogs isalways suspected Even though B malayi like microfilariae[20 21] were detected from cats and dogs from India theywere not confirmed asBmalayiThe present study focuses onthe detection and differentiation of microfilariae of domesticcanines inhabiting aBmalayi endemic area of southern India(Alappuzha district of Kerala state) based on histochemicalstaining and molecular techniques

2 Materials and Methods

21 Ethics Statement All study procedures and protocolswere approved by the institutional animal ethics committee ofCollege of Veterinary and Animal Sciences Pookode Kerala(which follows committee for the purpose of control andsupervision of experiments on animals (CPCSEA) guide-lines Ministry of Environment and Forest Government ofIndia) The committee did not deem it necessary for thisresearch group to obtain formal approval to conduct thisstudy National Centre for Diseases Control directly underDirectorate General of Health Services Ministry of Healthand Family Welfare Government of India is authorized tocollect microfilaria positive human blood samples Writteninformed consent was obtained from the human patients andowners of dogs before collection of samples

22 Preliminary Screening andCollection of Samples Sampleswere collected from dogs by collaborating veterinariansat ldquoprophylactic antirabies vaccination and blood parasitedetection campsrdquo in the south Indian panchayats of Keralastate namely Kuthiathode (KUT) Kadakkarappally (KAD)and Pattanakkad (PKT) Brugian filariosis is endemic in thisarea [17ndash19] Before sample collection data regarding the agesex and breed of these dogs were recorded

Blood samples were collected from the saphenous veinA drop of blood was examined by wet film techniqueat the camp site itself Thick smears were prepared fixedwith methanol stained using Giemsa and examined forthe presence of microfilariae with or without sheath Wholenonheparinized (1mL) andheparinized (1mL) blood sampleswere also collected from the saphenous vein of all these dogsA total of 164 blood samples of dogs (37 from Kadakkarap-pally 57 from Kuthiathode and 70 from Pattanakkad) werecollected

Blood sample of a known B malayi infected humanpatient from Alappuzha the endemic district of Kerala wasalso collected to serve as positive control for histochemicalstaining and PCR techniques

23 Histochemical Differentiation The nonheparinizedblood was allowed to clot and the serum fraction wascentrifuged in an Eppendorf tube for 5 minutes at 1000 rpmAfter discarding the supernatant the sediment containingthe microfilariae was resuspended in the remaining serumand a drop of this fluid was used for preparation of smearsThey were air dried fixed in absolute chilled acetone for

1 minute further air dried and stored in minus20∘C until usedThese smears were used for histochemical staining withintwo weeks after preparation

Histochemical staining was performed [22] to studythe differences in the acid phosphatase enzyme activity inmicrofilariae for identification of the species [22ndash26] Briefly20mL of solution I (Michaelis Veronal Acetate Buffer pH100) was mixed with 50mL of distilled water and 4mL ofsolution II (005 g Naphthol AS-TR phosphate sodium salt(Sigma) in 5mL of N N-dimethyl formamide) was added toit in a beaker In another beaker 32mL each of solutionsIII (10 g Pararosaniline hydrochloride 50mL concentratedhydrochloric acid and 200mL distilled water) and IV (4 percent sodiumnitrite) wasmixed and then added to themixturein the first beaker The pH of the mixture was adjustedto 50 with 01 N sodium hydroxide solution The finalsolution was prepared fresh every time before the stainingprocedure

The air dried smears were incubated in the substrate for1 hour at 37∘C rinsed in distilled water counterstained insolution V (mixture of 771mL of 02M sodium phosphate1229mL 01M nitric acid and 2 g methyl green) for 5minutes and rinsed in distilled water The slides were thendehydrated in 95 per cent and absolute ethyl alcohol respec-tively rinsed in xylene and mounted using DPXThe smearswere examined for the precipitated red azo dye indicating acidphosphatase activity

24 Polymerase Chain Reaction DNA isolation from theheparinised whole blood samples of dogs and human wasbased on phenol-chloroform isoamyl alcohol (PCI) method[27] The leukocyte DNA from a three-day-old pup servedas negative control For the Brugia specific PCR the DNAisolated from a known B malayi infected human patientrsquosblood served as positive control Canine blood samples withheavy (one microfilaria in every low power field) monoin-fection with D repens and A reconditum (confirmed byhistochemical staining) served as positive controls for thesespecies The reactions were carried out in a thermal cycler(Eppendorf Germany) the products were visualized (AlphaInnotech USA) after electrophoresis on a 15 per cent agarosegel and documented

241 D repens Specific PCR PCR assay for the amplificationof 246 bp direct tandem repeats of D repens using specificprimers [28] 51015840-CCGGTAGACCATGGCATTAT-31015840 (For-ward) and 51015840-CGGTCTTGGACGTTTGGTTA-31015840 (Reverse)custom synthesized from IDT USA was standardized ThePCR amplification was performed in 25 120583L reaction volumecontaining 25 120583L 10x PCR buffer 1 120583L (025mM) dNTP and30 pmol of each primer 15 U Taq polymerase and 5 120583L oftemplate DNA Reaction conditions were as follows after theinitial denaturation at 94∘C for 5 minutes 40 cycles eachwith 94∘C for 30 seconds 55∘C for 30 seconds and 72∘Cfor 25 seconds were run A final extension at 72∘C for 5minutes was also given One product of D repens specificPCR was directly sent to Chromos Biotech Bangalore forsequencing





Drepens

Arecondi-tum

Bpahangi

Newreaction

Bmalayilike

Drepens



Kuthiathode 57 23 29 18 2 3 28 0 28 2 7

Pattanakkad 70 17 25 16 0 12 19 3 26 0 10








3 Results





An


An

E






Am

AnE


An








different

breedsage

grou

psand

sexeso

fdogsinaB

malayiend

emicarea

ofKe

ralaInd

iabased

onGiemsa

staining

techniqu

e

Bloo

dsm

ears

Area

Breed

Age

grou

pSex

Total

ND

Exotic

Spitz

GSD

Dach

Dob

lt2years

gt2years

Male

Female

Exam

ined

Kadakkarappally

296

20

014

2333

437

Kuthiathod

e36

105

42

1344

3918

57Patta

nakk

ad49

172

11

3139

5614

70To

tal

114

339

53

58106

128

36164

Positive

Kadakkarappally

113

20

04

1216

016

(4324)

Kuthiathod

e16

62

32

425

245

29(5087)

Patta

nakk

ad17

61

01

322

205

25(3571)

Total

44(386

)15

(4546

)

5(5556

)

3(60

)3(100

)

11(1897

)

59(5566

)

60(4688

)

10(277

8)

70(4268

)

NDN

ondescrip

tGSDG

erman

shepherdD

achDachshu

ndD

obD

oberman


PAm

AnE






4 Discussion



P

AnAm

E







90 80 70 60 50 40 30 20 10 0904







Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574











5 Conclusion


Abbreviations


and xylene



Acknowledgments



References















































Drepens

Arecondi-tum

Bpahangi

Newreaction

Bmalayilike

Drepens



Kuthiathode 57 23 29 18 2 3 28 0 28 2 7

Pattanakkad 70 17 25 16 0 12 19 3 26 0 10








3 Results





An


An

E






Am

AnE


An








different

breedsage

grou

psand

sexeso

fdogsinaB

malayiend

emicarea

ofKe

ralaInd

iabased

onGiemsa

staining

techniqu

e

Bloo

dsm

ears

Area

Breed

Age

grou

pSex

Total

ND

Exotic

Spitz

GSD

Dach

Dob

lt2years

gt2years

Male

Female

Exam

ined

Kadakkarappally

296

20

014

2333

437

Kuthiathod

e36

105

42

1344

3918

57Patta

nakk

ad49

172

11

3139

5614

70To

tal

114

339

53

58106

128

36164

Positive

Kadakkarappally

113

20

04

1216

016

(4324)

Kuthiathod

e16

62

32

425

245

29(5087)

Patta

nakk

ad17

61

01

322

205

25(3571)

Total

44(386

)15

(4546

)

5(5556

)

3(60

)3(100

)

11(1897

)

59(5566

)

60(4688

)

10(277

8)

70(4268

)

NDN

ondescrip

tGSDG

erman

shepherdD

achDachshu

ndD

obD

oberman


PAm

AnE






4 Discussion



P

AnAm

E







90 80 70 60 50 40 30 20 10 0904







Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574











5 Conclusion


Abbreviations


and xylene



Acknowledgments



References












































An


An

E






Am

AnE


An








different

breedsage

grou

psand

sexeso

fdogsinaB

malayiend

emicarea

ofKe

ralaInd

iabased

onGiemsa

staining

techniqu

e

Bloo

dsm

ears

Area

Breed

Age

grou

pSex

Total

ND

Exotic

Spitz

GSD

Dach

Dob

lt2years

gt2years

Male

Female

Exam

ined

Kadakkarappally

296

20

014

2333

437

Kuthiathod

e36

105

42

1344

3918

57Patta

nakk

ad49

172

11

3139

5614

70To

tal

114

339

53

58106

128

36164

Positive

Kadakkarappally

113

20

04

1216

016

(4324)

Kuthiathod

e16

62

32

425

245

29(5087)

Patta

nakk

ad17

61

01

322

205

25(3571)

Total

44(386

)15

(4546

)

5(5556

)

3(60

)3(100

)

11(1897

)

59(5566

)

60(4688

)

10(277

8)

70(4268

)

NDN

ondescrip

tGSDG

erman

shepherdD

achDachshu

ndD

obD

oberman


PAm

AnE






4 Discussion



P

AnAm

E







90 80 70 60 50 40 30 20 10 0904







Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574











5 Conclusion


Abbreviations


and xylene



Acknowledgments



References













































different

breedsage

grou

psand

sexeso

fdogsinaB

malayiend

emicarea

ofKe

ralaInd

iabased

onGiemsa

staining

techniqu

e

Bloo

dsm

ears

Area

Breed

Age

grou

pSex

Total

ND

Exotic

Spitz

GSD

Dach

Dob

lt2years

gt2years

Male

Female

Exam

ined

Kadakkarappally

296

20

014

2333

437

Kuthiathod

e36

105

42

1344

3918

57Patta

nakk

ad49

172

11

3139

5614

70To

tal

114

339

53

58106

128

36164

Positive

Kadakkarappally

113

20

04

1216

016

(4324)

Kuthiathod

e16

62

32

425

245

29(5087)

Patta

nakk

ad17

61

01

322

205

25(3571)

Total

44(386

)15

(4546

)

5(5556

)

3(60

)3(100

)

11(1897

)

59(5566

)

60(4688

)

10(277

8)

70(4268

)

NDN

ondescrip

tGSDG

erman

shepherdD

achDachshu

ndD

obD

oberman


PAm

AnE






4 Discussion



P

AnAm

E







90 80 70 60 50 40 30 20 10 0904







Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574











5 Conclusion


Abbreviations


and xylene



Acknowledgments



References












































PAm

AnE






4 Discussion



P

AnAm

E







90 80 70 60 50 40 30 20 10 0904







Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574











5 Conclusion


Abbreviations


and xylene



Acknowledgments



References












































90 80 70 60 50 40 30 20 10 0904







Identity ()

Div

erge

nce 1

234

1234

21 3 4

21 3 4





359

758

268

368

295384

1419

15251977

2108

1574











5 Conclusion


Abbreviations


and xylene



Acknowledgments



References














































5 Conclusion


Abbreviations


and xylene



Acknowledgments



References











































canine filarial infections in a human brugia malayi

Documents