Toxocara ORW Anju

8/17/2019 Toxocara ORW Anju

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Serological detection of Toxocara canis infection in dogs with recombinant

Cathepsin-L1, arginine kinase and TES-26 of T. canis

Otline of !esearch work "O!#$

for

%h&'& 'egree

()

*n+ arghese

!oll o& 12./

'i0ision of eterinar) %arasitolog)

To

Deemed University

INDIAN VATERINARY RESEARCH INSTITUTE

*T*3*! (*!ELL4-25122

1


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ntrodction

Toxocara canis is a common helminth parasite of dos! a ca"sative aent of canine

to#ocarosis $ith $orld$ide distri%"tion&The parasite is prevalent in all locations that har%o"r

domestic dos partic"larly the p"ppies and other canids '(lic)man and Schant*!+,-+.&

To#ocarosis is a serio"s condition and a lo%al *oonotic disease'(lic)man and /ana$al!

+,,0.& Toxocara canis is a common ro"nd $orm of dos and vario"s mammals incl"din

ra%%its! mice! pis! fo#es! %irds! etc& may act as paratenic hosts $hich may also play role in

its epidemioloy '1i and 2in! +,,-.&

Dos are the reservoir for T. canis %"t p"ppies pose the reatest ris) of spreadin theinfection to h"mans 'Cr"* et al., 344-.& Dos are associated $ith more than 54 *oonotic

diseases of $hich helminthic parasites in partic"lar pose serio"s p"%lic health concerns

'6hante et al &! 344,.& T. canis is considered as one of the main enteropathoens of dos!

especially in ne$ly $helped or neonates '7la%"rn et al.,+,,5. and is responsi%le for

important *oonotic disease! visceral larva mirans 'V8/.& Infection in ad"lt dos is

characteri*ed %y encysted second stae larvae& Ho$ever! these larvae can et activated in

prenant females and cross the placental %arrier to infect the p"ps! $ith vertical transmission

also occ"rrin thro"h mil) 'A)ao and 9hta! 344:. Infectio"s mothers and p"ppies "nder

five $ee)s old! pass es in their faeces& A close relationship %et$een h"mans and dos may

represent a potential p"%lic health ris)! since nat"ral transmission of parasitic infections from

dos to man may occ"r via environmental factors& Soil contamination of ardens and p"%lic

ro"nds %y infectio"s parasitic forms constit"tes a sinificant *oonotic ris) 'Ha%l"et*el et

al., 3440.&

H"man to#ocarosis ca"sed %y larvae of T. canis pose a reater ris) of infection to

yo"n children %eca"se of their ha%it of playin at p"%lic places and a tendency to place

contaminated o%;ects and dirt in their mo"th 'Naa)"ra et al.,+,,4.& Seroprevalence is hiher

in developin co"ntries! sometimes over


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ca"sed %y inestion of es $ith contaminated soil! ho$ever! a c"lt"ral dietary preference for

eatin ra$ or "ndercoo)ed animal tiss"es may also enhance the ris) of infection! as food

animals act as paratenic hosts 'Done et al., +,54? (alvin! +,5@? Naa)"ra et al &! +,-,.& In

h"man or other non>canid hosts! these larvae $ander a%errantly thro"ho"t the internal

orans vi*& eyes! l"ns! %rain! heart! m"scles! liver etc& These $anderin larvae do not

develop f"rther in these hosts %"t can ca"se severe local reactionsd"e 'Hel$ih et al&! +,,,?

1i and 2in! +,,-.& T.canis es re"ire several $ee)s of inc"%ation period o"tside the host

%efore %ecomin infective! so freshly laid "nem%ryonated es cannot ca"se to#ocarosis

'Amir et al &! +,,cpl>+.! arinine )inase!

s"rface associated lycoproteins of molec"lar $eihts 03! e#cretory>secretory antien 'TES>secretory antien

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http://animaldiversity.ummz.umich.edu/site/accounts/information/Toxocara_canis.html#a958ba27742deacdf297da4268ace4e1http://animaldiversity.ummz.umich.edu/site/accounts/information/Toxocara_canis.html#a958ba27742deacdf297da4268ace4e1http://animaldiversity.ummz.umich.edu/site/accounts/information/Toxocara_canis.html#a958ba27742deacdf297da4268ace4e1http://animaldiversity.ummz.umich.edu/site/accounts/information/Toxocara_canis.html#7fe09105b21c63d7852ec89ebe74a536http://animaldiversity.ummz.umich.edu/site/accounts/information/Toxocara_canis.html#a958ba27742deacdf297da4268ace4e1http://animaldiversity.ummz.umich.edu/site/accounts/information/Toxocara_canis.html#7fe09105b21c63d7852ec89ebe74a536


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'rTES>+34! rTES>35! rTES>04US/. 'S"harni et al &! 344,.& Amonst these dianostic

antiens! arinine )inase has %een identified in several parasites li)e Trypanosoma cruzi, T.

brucei 'ereira et al &! +,,,! 3444 3443. , Ctenocephalides felis 'Ferr et al &! 344,.

Cathepsin>8 en*ymes have %een identified as potential tarets for dr" or vaccine

development in many parasites! as their f"nctions are essential in a variety of important

%ioloical processes $ithin the host! s"ch as moltin! c"ticle remodellin! em%ryoenesis! feedin

and imm"ne evasion& Cathepsin>8! a ma;or cysteine proteinase secreted %y the parasite plays a

pivotal role in vario"s aspects of its pathoenecity& The en*yme ta)es part in n"trient ac"isition %y

cata%oli*in host proteins to a%sor%a%le peptides! facilitates the miration of the parasite thro"h

the host intestine and liver %y cleavin interstitial matri# proteins s"ch as fi%ronectin! laminin and

native collaen and is implicated in the inactivation of host imm"ne defences %y cleavin

imm"nolo%"lins& Accordinly! the protease has %een reconi*ed as an important taret at $hich

parasite intervention strateies sho"ld %e directed& Cathepsin>8 cysteine proteinases are also

reported as sensitive and specific mar)ers for the imm"nodianosis of vario"s parasitic diseases

'Varhese et al., 34+3.& Cathepsin>8 is secreted %y all staes of the developin parasites and is hihly

antienic in infected animals& The p"rified or recom%inant cysteine proteinase antiens have %een "sed

for the dianosis of h"man and Gor animal paraonimiosis! clonorchiosis! schistosomosis and

fasciolosis 'Raina et al.! 344


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$or) is therefore! envisaed to e#press some of the recom%inant antiens of this parasite that

have %een sho$n in earlier $or)s $ith a hih potential in the detection of occ"lt infection

d"e to tiss"e em%edded larvae in h"mans& Ho$ever! no st"dies have %een cond"cted on these

t$o antiens on their dianostic potential in canines& T. canis arinine )inase has %een

e#pressed and eval"ated in earlier st"dy '7ade et al., 34+0. for its potential in the detection

of T. canis infection in dos that has sho$n this antien detectin infection in canines $ith a

relia%le sensitivity& 6eepin this %ac)ro"nd in vie$! the present st"dy is aimed to e#press

the a%ove three antiens in a %acterial system and st"dy them for their "sef"lness in the

development of a relia%le dianostic assay for T. canis infection in dos&

The following ob+ecti0es of the research programme are en0isaged

E7pression of Toxocara canis cathepsin-L-1, TES-26 and arginine kinase

recombinant proteins in a prokar)otic s)stem&

E0alation of sensiti0it) and specificit) of these recombinant antigens for

detection of Toxocara canis infection in canines&

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!e0iew of Literatre

8man to7ocarosis

H&C& Filder $as the first researcher to descri%e to#ocarosis in h"mans! $hen he

p"%lished a paper in +,0:= in Spain! secretory antien %y E8ISA in s"%;ects residin in r"ral areas

near Chandiarh and in clinically s"spected patients $as st"died& A positive anti%ody response to TES

antien in 5&@= s"%;ects and 30&0= of s"spected patients indicated that h"man to#ocarosis

may %e endemic in certain reions of northern India '/alla et al., 3443.& A related factor for the

prevalence of Toxocara infection in h"mans $as the condition and stat"s of veeta%les ta)en %y the

s"%;ects& The children $ho $ere of the ha%it of eatin ra$ veeta%les $ere more prone to infection

'05&


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factor for the prevalence of to#ocarosis 'Dar et al., 344-.& Contact $ith dos or presence of pet in

ho"se also constit"ted a hih ris) factor for Toxocara infection& In their st"dy! the prevalence of

Toxocara infection $as more in people "sin $ater from streams! rivers! ponds and $ells than those

"sin tap $ater& In the 6ashmir valley a hiher rate of infection $as reported in the pop"lation than in

the s"%;ects residin in a r"ral area near Chandiarh& This may %e d"e to lo$ standards of hyiene!

fre"ent contact $ith the contaminated soil and lac) of ed"cation 'Ahmad et al., 3443.&

To7ocarosis in dogs

Recent st"dies have indicated the follo$in prevalence rates of to#ocarosis in dos in

different co"ntries! +0&:= in Tan*ania 'S$ai et al &! 34+4.! @4= in a)istan 'Chattha et al &! 344,.!

54= in Iran 'Daryani et al &! 344,.! +3&


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An estimation of T. canis prevalence in dos , in the /arche reion of Italy revealed the

hihest infection rate in r"ral h"ntin dos '5@&:=. and the lo$est in "r%an companion dos

'33&+=. 'Ha%l"et*el et al., 3440.& The 35&3= T & canis positivity estimated for "r%an dos in

this st"dy $as in areement $ith a previo"s investiation in the same area! $hich recorded

3-= e prevalence in faecal samples collected in p"%lic reen areas 'alliola et al., +,-5.&T$ice as many dos from r"ral areas '@-&@=. har%o"red e sheddin nematodes compared

to the "r%an '35&3=. ones& This difference may res"lt from more fre"ent antihelminthic

treatments of "r%an dos! d"e to a reater health conscio"sness of their o$ners 'olayen et

al., 3444.&

A st"dy on the role of dos in the transmission of astrointestinal parasites in Assam!

India! revealed that nearly all ',,=. dos har%ored one or more *oonotic species of

astrointestinal parasites! $ith a total prevalence of ++= for T . canis infection 'Tra"% et al &!

3443.& Baecal samples of 5@3 stray dos of 2odhp"r city revealed a total of --=

astrointestinal parasite infection $ith a prevalence of @&,= for Toxocara 'S"%hash and

Tan$ar! 344:.. In a st"dy at 7areilly! prevalence rate $as fo"nd hih in less than 5 months

old stray 'livin nematodes $ere commonly

enco"ntered in the soil 'Das et al &! 344,.&

To determine the prevalence of T. canis infection in do pop"lation in and

aro"nd 7areilly! Uttar radesh! India! a total of


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Soil contamination

For) done on the threshold recovery of T. canis es from soil s"ested that the

centrif"e>flotation techni"e is efficient for the detection of Toxocara es! even in samples

containin lo$ e n"m%ers '1avier et al &! 34+4.& /ohammad et al. '34+4. st"died the soil

contamination $ith Toxocara spp& es in the p"%lic par)s from three areas of 6horram A%ad!Iran& A total of 3-< soil samples from 2an"ary to /arch 344, in +- p"%lic par)s $ere

collected to test for soil contamination $ith Toxocara es "sin s"crose floatation method&

Distri%"tion of Toxocara spp& es in samples collected from p"%lic par)s $as 50&0=&

The hihest n"m%er of es recovered from 344 of soil $as +3-& Their investiation

sho$ed that p"%lic par)s $ere contaminated $ith Toxocara es in 6horram A%ad!

s"estin for e#ercisin care $hen "sin p"%lic par)s '/ohammad et al &! 34+4.&

In a st"dy cond"cted on the prevalence of parasites in soil and do faeces! soil samples

$ere collected from 3< p"%lic s"ares in Seropdica! 7ra*il& The techni"e descri%ed %y

D"nsmore et al & '+,-@. and an adaptation of the R"i* et al & '3444. method $ere "sed to

recover parasites in soil& Seven s"ares $ere fo"nd to %e contaminated and the most prevalent

parasites $ere Ancylostoma spp '+0&5=. and Toxocara spp '@&4=.& The prevalence of

*oonotic parasites in soil has implications for the spread of h"man disease in these areas

'/andarino>ereira et al &! 34+4.& A st"dy cond"cted %y Tei#eira et al. '344-. on prevalence

of T. canis infection in p"%lic s"ares of the ConcJrdia City! Santa Catarina! 7ra*il! reported

that the schools! s"ares and par)s $here sand is present can constit"te an important

transmission so"rce for several parasitic *oonoses! representin a potential ris)! mainly for

children of school ae playin at these places& Amonst several *oonoses! visceral larva

mirans 'V8/. is an important patholoy characteri*ed %y the miration of the larval

staes of Toxocara spp& in h"man tiss"es leadin to imm"no>alleric type of reactions& The

o%;ective of their $or) $as to determine the occ"rrence of parasite es in the samples of sand

from p"%lic s"ares of Concordia city? Centro! Na*are! Vista Alere and Ind"striarios& A

sinificantly hiher prevalence of T.canis es $as d"e to the hih n"m%er of canine

pop"lation in the m"nicipal district and their easy access to these places 'Tei#eira et al &!

344-.&

In a st"dy on the contamination of soil $ith feline and canine ascarid es in p"%lic

par)s! %ac)yards and sand pits in ra"e! C*ech Rep"%lic! soil samples from shelters and

r"ral areas $ere analy*ed& The hihest rate of contamination '@


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inha%ited %y feral cats& The es of Toxocara spp& $ere fo"nd in 34&@= of par)s! +4= of

shelters and


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so"rce of soil contamination for transmission of to#ocarosis $hich is of p"%lic health

importance in this reion 'Sah" et al., 34+0.&

Characteri9ation of Toxocara canis antigens

Characteri*ation of the proteins that comprise ad"lt T. canis $orms $ill prove "sef"l

in the development of vaccines and dianosis of infection and in st"dies on the %ioloy of

and imm"nity to nematodes and other parasites& The identification of specific antiens of

T. canis is important in order to develop %etter dianostic techni"es The e#cretory>

secretory proteins and associated car%ohydrates of the third stae larvae of T. canis

'/ai*els et al &! 3445. and the proteins of ad"lt Ascaris suum 'Brontera et al &! 3440?

Ta)ashima et al &! 3440.! a relative of T. canis $ere e#amined in some detail %y these

a"thors&

/orales and co $or)ers identified T. canis antiens %y $estern %lot in e#perimentally

infected ra%%its& They infected ten ra%%its orally $ith a dose of 3- )Da antiens appeared later& All positive sera in the E8ISA $ere also positive in F7&

T$o antien %ands! ,3 and 0< )Da $ere identified since the %einnin and thro"ho"t the

co"rse of infection& They reported that these antiens merit f"rther eval"ation as candidates

for "se in dianosis '/orales et al &! 3443.&

S"n and co>$or)ers did a st"dy on comparison of the protein constit"ents of the

ma;or %ody compartments of T. canis& An analysis of the protein profiles of intact $orms and

isolated tiss"es of ad"lt male and female T. canis $orms $as cond"cted& Sol"%le proteins

recovered from homoeni*ed $hole specimens and dissected tiss"es '%ody $all! reprod"ctive

tract! oesopha"s and intestine. of T. canis ad"lts from several different canine hosts $ere

separated %y si*e "sin radient sodi"m dodecyl s"lphate electrophoresis 'SDS>A(E.& SDS>

A(E profiles of $orms from different hosts $ere fo"nd to %e virt"ally identical! irrespective

of se# or tiss"e type& Recovered proteins raned in si*e from 0&@ to 03< )Da& The larest

n"m%er of recovered proteins $as present in the female reprod"ctive tract e#tracts 'S"n et

al &! 344:.&

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The e#cretory>secretory antiens of the infective stae larvae of T. canis 'TES. have

%een e#amined in detail %y several $or)ers& These TES proteins have %een

characteri*ed as m"cins 'lycosylated proteins of hih molec"lar $eiht.! +34! @4K@<

)Da! lectins 's"ar>%indin proteins.! :4 and 03 )Da as $ell as several other proteins

at 35 and ES antien $ith ra%%it TC>ES antisera sho$ed 0

common reactive %ands '/ona et al &! 344@.& /orales! et al &! 3443 s"ested that ,3 and

0< )Da polypeptides of T. canis second stae larvae EGS antien are specific to Toxocara

infection $hile ES

antien! positive h"man and ra%%it TC>ES anti>sera sho$ed < common imm"no>reactive

%ands at @:&@! @4&5! 04&

al & 34+0%& The specific reactivity of the T. canis e#cretory> secretory 'TC>ES.

proteins $as chec)ed %y Festern %lottin& The imm"no>reactivity of the nat"rally

infected do sera $ith the TC>ES antiens sho$ed five %ands at @0! +:< )Da& The imm"no>reactivity of the hyper imm"ne ser"m raised in ra%%its aainst

TC>ES antiens $as o%served $ith ten polypeptides of 3+! 3

:: and +4< )Da& Common antien %ands $ere o%served at


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Serological characteri9ation

D"e to occ"lt nat"re of h"man to#ocarosis! serodianosis is reconi*ed as an effective

approach to the la%oratory dianosis of Toxocara infection& It is also "sef"l for epidemioloical

st"dies on the prevalence of the disease& Foodr"ff and 7radstreet '+,5-. first "sed s)in

testin to detect Toxocara specific IE anti%odies& Any pediatric patient $ith an"ne#plained fe%rile illness and eosinophilia sho"ld %e s"spected of havin V8/&

Hepatosplenomealy and evidence of m"ltisystem disease and history of pica ma)e

the dianosis of V8/ more li)ely& 9ther indicators of infection incl"de hyper>

ammalo%"linemia and an elevated isohemal"tinin titre& Th"s! a constellation of

clinical disease descri%ed a%ove! a history of pica! eosinophilia and positive seroloy

stronly point to the dianosis& 8iver %iopsy may reveal a ran"loma s"rro"ndin a

larva %"t a s"ccessf"l dianosis "sin this approach is fort"ito"s at %est and not

recommended& Dianostic tests for V8/ are primarily imm"noloical& The en*yme>

lin)ed imm"nosor%ent assay 'E8ISA.! $hich employs antiens secreted %y the

second>stae larvae! has s"fficient specificity to %e the %est indirect test for

dianosin this infection& Recom%inant antiens have %een prod"ced from the

second>stae larvae of T. canis that promises to add even reater specificity to an

already>relia%le test 'appro#imately ,3=. employin E8ISA& The E8ISA has a

reasona%ly hih deree of sensitivity&

De Saviny '+,::. reported dianosis of en"s specific Toxocara infection $ith a

passive haemal"tination test "sin Toxocara e#cretory>secretion antiens& The standard

seroloical test for confirmin to#ocarosis is an E8ISA& Festern %lot analysis! $hich is as

sensitive as E8ISA and relatively specific! sho"ld %e "sed as a confirmatory test after

screenin %y E8ISA '/anaval et al &! +,,+.& In a st"dy on eval"ation of dot>E8ISA in

comparison $ith standard E8ISA for the imm"nodianosis of h"man to#ocarosis! the

sensitivity and specificity of the dot>E8ISA $ere fo"nd to %e +44 and ,

TES antien of T. Canis larvae $hich may %e "sef"l for determinin the parasite %"rden in

early infection& El>/assry '+,,,. sho$ed that SDS>A(E of T. canis ad"lt somatic antiens

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had %ands at +3

An investiation of anti%odies aainst T. canis $as carried o"t in +< nat"rally

infected p"ppies %y E8ISA! "sin fo"r different antiens> es containin second

stae larvae! larvae! ad"lt $orms and e#cretory>secretory prod"cts of larvae& The

E8ISA val"es aainst fo"r different antiens increased $ith the ae of the p"ps& The

correlation %et$een ae and E8ISA val"es $as sinificant in the positive direction

%"t a sinificant neative co>relationship %et$een the n"m%er of $orms and the

E8ISA val"es $as o%served& The E8ISA val"es in the p"ppies havin "nfertili*ed

es $ere reater than those havin fertili*ed es& These findins s"est that in the

nat"rally infected p"ppies there are intensive relationships amon the anti%ody levels!

ae! n"m%er of $orms and the condition of e fertili*ation '/ats"m"ra and Endo!

+,-3.&

/ats"m"ra et al & '+,-0a. st"died correlation of anti%ody prod"ction and ae

of the dos $ith T. canis infection& In the sera of do fet"ses and ne$%orn p"ppies! no

anti%ody to T. canis $as fo"nd %"t an elevation of the circ"latin to#ocaral antiens

'CTA. $as detected& In the mother dos! detecta%le I( and I/ anti%odies $ere

fo"nd& The I( anti%odies in the ne$ %orn p"ppies rad"ally increased in the co"rse

of this o%servation& The most elevated levels of IA anti%odies $ere o%served one

month after %irth& The same $as tr"e for the CTA levels& There $as no placental

transfer of the anti%odies nor co"ld they %e transferred from the mother do to the

offsprin via the colostr"m&

In order to interpret the relation %et$een the I/ anti%ody activities to T.

canis and the parasitoloical stat"s! sera from p"ppies and ad"lt dos $ere meas"red

%y E8ISA& A"topsy $as performed on these dos for co"ntin the n"m%er of T. canis

$orms and stool e#amination $as carried o"t for o%servin the fertili*ation of es&

The rates of infection %y T. canis in the dos decreased $ith ae& The I/ anti%ody

activities rad"ally increased "ntil 0 months of ae& Thereafter! the sinificant levels

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$ere maintained even at the ad"lt ae& Ho$ever! the I/ anti%ody levels $ere not

associated $ith the ae and the n"m%er of $orms 'M4&4

vitro. Analysis $ith monoclonal anti%odies '/A%s. confirmed the identity of s"rface and

ES molec"les and these /A%s sho$ed differin patterns of %indin to the epic"ticle! the c"tic"lar

matri# and to the oral orifice '/ai*els and ae! +,,4.&

8o")as and co>$or)ers! +,,- characteri*ed cathepsin 8>li)e cysteine protease

from T. canis infective larvae& Cysteine proteases play vital %ioloical roles in %oth

intracell"lar and e#tracell"lar environments& A cysteine protease miratin at 04 )Da $as

identified in somatic e#tracts of T. canis larvae 'TE1. %y its %indin to the %iotinylated

inhi%itor he>Ala>CH3B& TE1 proteases readily cleaved the cathepsin>8 and 7>specific

peptide s"%strate >he>Ar>A/C and to a lesser e#tent! the cathepsin 7>specific peptide

>Ar>Ar>A/C& E#cretory G secretory 'TES. prod"cts of T. canis larvae did not cleave

either s"%strate& artial se"ence encodin the li)e en*yme from Brugia pahangi and a rane of parasite and plant papain>li)e proteases&

The recom%inant protease 'rTc>C8>+. $as e#pressed in %acteria for imm"ni*ation of mice

and the s"%se"ent antiser"m sho$n to specifically react $ith the 04 )Da native protease in

TE1& Sera from mice infected $ith the parasite also contained anti%odies to rTc>C8>+! as

did sera from nine patients $ith proven to#ocarosis '8o")as et al., +,,-.&

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Cathepsin>8! a lysosomal endopeptidase e#pressed in most e")aryotic cells! is a

mem%er of the papain>li)e family of cysteine proteinases '7arrett and 6irsch)e! +,-+? Roth!

3444.& Cathepsin>8 plays a ma;or role in antien processin! t"mor invasion and metastasis!

%one resorption and t"rnover of intracell"lar and secreted proteins involved in ro$th re"lation

'6ane and (ottesman! +,,4.& Altho"h commonly reconi*ed as a lysosomal protease!

cathepsin>8 is also secreted& This %road>spectr"m protease is potent in deradin several

e#tracell"lar proteins 'laminins! fi%ronectin! collaens I and IV! elastin! and other str"ct"ral

proteins of %asement mem%ranes. as $ell as ser"m proteins and cytoplasmic and n"clear

proteins '8o"ise et al., 344,.&

Cathepsin>8 proteases are secreted %y several parasites incl"din T. solium and

constit"te important antiens for imm"nodianosis& A protein fraction $ith cathepsin>8 activity

$as p"rified from the cysticercus fl"id %y si*e e#cl"sion and ion>e#chane chromatoraphy&

The p"rified protein fraction incl"ded antiens of +. from T. solium

metacestode $as identified and characteri*ed for the %iochemical properties of the

recom%inant en*yme& The cloned cDNA of +3+5 %p encoded 00, amino acids $ith an

appro#imate molec"lar $eiht of 0:&5 )Da& Se"ence alinments of TsC8>+ sho$ed lo$

se"ence similarity of 3:&0> @@&5= to cathepsin 8>li)e cysteine proteases from other

helminth parasites '8ia et al., 3445.&

Cathepsin>8! a ma;or cysteine proteinase secreted %y asciola species are also

reported as sensitive and specific mar)ers for the imm"nodianosis of fasciolosis in r"minants&

A dianostic E8ISA $ith recom%inant . hepatica and . gigantica cathepsin 8>li)e protease as antien $as developed to detect anti%odies aainst . hepatica in sheep and

cattle ' Raina et al &! 344


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Trypanosoma carassii $as carried o"t& A cathepsin 8>li)e proteinase from T. carassii

$as cloned and the n"cleotide se"ence of +0:+ %p translated into a preproprotein of @a%"ndance transcripts incl"de a m"cin and a C>type lectin! $hich are %oth ma;or

e#cretory>secretory antiens released %y parasites& Bo"r hihly e#pressed novel ene

transcripts! termed ant 'a%"ndant novel transcript. enes $ere fo"nd& Toether! these fo"r

enes comprised +-= of all cDNA clones isolated %"t no similar se"ences occ"r in the

C. elegans enome& The discovery of these a%"ndant! parasite>specific enes of ne$ly

identified lectins and m"cins! as $ell as a rane of conserved and novel proteins provided

defined candidates for f"t"re analysis of the molec"lar %asis of imm"ne evasion %y T. canis

'Tetteh et al., +,,,.&

8o")as and co>$or)ers '3444. st"died novel C>type lectin secreted %y a tiss"e>

d$ellin parasitic nematode& /any parasitic nematodes live for s"rprisinly lon periods in

the tiss"es of their hosts! implyin sophisticated mechanisms for evadin the host imm"ne

system& The nematode T. canis s"rvives for years in mammalian tiss"es and $hen

c"ltivated in vitro! secretes antiens s"ch as TES>03& Brom the peptide se"ence! they

cloned TES>03 cDNA! $hich encodes a 3+, amino>acid protein that has a domain

characteristic of host calci"m>dependent 'C>type. lectins! a family of proteins associated

$ith imm"ne defence& Homoloy modelin predicted that TES>03 %ears remar)a%le str"ct"ral

similarity to mammalian imm"ne>system lectins& Native TES>03 acted as a f"nctional lectin in

affinity chromatoraphy& Un"s"ally! it %o"nd %oth mannose and alactose>type

monosaccharides! a pattern precl"ded in mammalian lectins %y a constrainin loop ad;acent to

the car%ohydrate>%indin site& In TES>03! this loop appeared to %e less o%tr"sive! permittin a

%roader rane of liand %indin& The similarity of TES>03 to host imm"ne cell receptors

17


18/36

s"ests a hitherto "ns"spected stratey for parasite imm"ne evasion '8o")as et al &!

3444.&

(ems and /ai*els '+,,5. $or)ed on an a%"ndantly e#pressed m"cin>li)e

protein from T.canis infective larvae! the prec"rsor of the larval s"rface coat

lycoproteins& They reported that evasion of host imm"nity %y T. canis infective larvae ismediated %y the nematode s"rface coat! $hich is shed in response to %indin %y host anti%ody

molec"les or effector cells& The ma;or constit"ent of the coat is the TES>+34 lycoprotein

series& They isolated a :04>%p cDNA from the ene encodin the apoprotein prec"rsor of

TES>+34& The mRNA is a%sent from T. canis ad"lts %"t hyper a%"ndant in larvae! ma)in

"p appro#imately +4= of total mRNA and is trans>spliced $ith the nematode terminal

end of the p"tative protein are t$o 05>amino acid repeats containin si# Cys resid"es! in a

motif that can also %e identified in several enes in C. elegans& Altho"h TES>+34 displays

si*e and chare heteroeneity! there is a sinle copy ene and a homoeneo"s si*e of

mRNA& The association of over e#pression of some mem%rane>associated m"cins $ith

imm"nos"ppression and t"mor metastasis s"ests a possi%le model for the role of the

s"rface coat in imm"ne evasion %y parasitic nematodes '(ems and /ai*els! +,,5.&

In a st"dy cond"cted %y /ai*els and co $or)ers '3444. to "nderstand the a%ility of

T. canis larvae to s"rvive in the imm"no>competent host! they "ndertoo) molec"lar analyses

of the ma;or enes e#pressed at this stae& 7y a com%ination of protein se"encin! ene

identification and e#pressed se"ence ta 'EST. analysis they characteri*ed a rane of

potentially important ene prod"cts from this parasite& Some of these are homolo"es of

prominent mammalian proteins s"ch as C>type lectins 'represented %y the secreted

prod"cts TES>03 and TES>:4. and m"cins 'TES>+34. and additional prod"cts sho$

stron similarities to )no$n cysteine proteases! phosphatidyl ethanolamine>%indin

proteins and other liands& A n"m%er of these proteins incl"de a conspic"o"s 05>amino

acid motif containin si# cysteines& This domain 'termed NC5 or S1C. appears to %e an

evol"tionarily mo%ile mod"le! $hich in T. canis is com%ined $ith a spectr"m of diverse

f"nctional domains in different enes& In addition! they identified a set of novel ene

se"ences that sho$ no resem%lance to any enes encoded %y the free>livin nematode C.

18


19/36

elegans& Bo"r of these are desinated a%"ndant novel transcripts and collectively these

acco"nt for nearly 34= of the cDNA isolated from the arrested infective stae& S"ch parasite>

specific enes e#pressed at a hih level %y a stae that sho$s remar)a%le end"rance may

represent critical prod"cts necessary for the s"ccess of the parasitic mode of life '/ai*els

et al &! 3444.&

Yamasa)i et al. '3444. developed a recom%inant T.canis second>stae larva antien

correspondin to the 04>)Da protein of the TES and tested its sensitivity and specificity&

The specificity of the recom%inant T.canis antien developed for the

imm"nodianosis of h"man to#ocarosis $as compared $ith that of the e#cretory>

secretory antien from T. canis second>stae larvae %y E8ISA& A total of +reactions $ere o%served in :, 'reaction rate decreased sharply to

only 3&+=! correspondin to 0 of +@3 cases& The cross>reactions occ"rred $ith one

case each of nathostomosis! paraonimosis $ith "aragonimus miyaza#ii! and

spirometriosis! in $hich hih anti%ody titres $ere detected& In addition! the

recom%inant antien sho$ed neative reactions $ith ser"m samples from patients

infected $ith Ascaris and hoo)$orms! the most common h"man parasites& These

findins $ere also s"pported %y e#periments $ith animals infected $ith Ascaris and

hoo)$orm& The a"thors s"ested the recom%inant antien is hihly specific for

T.canis infection and may provide more relia%le dianostic res"lts than other

methods'Yamasa)i et al., 3444.

Fic)ramasinhe et al. '344:. reported molec"lar clonin! characteri*ation!

e#pression and comparison of the )inetics of cDNA>derived arinine )inase of T. canis&

Arinine )inase 'A6. is a mem%er of a hihly conserved family of phosphaen )inases&

Alinment of the cDNA>derived amino acid se"ence of T. canis A6 $ith other

phosphaen )inase se"ences sho$ed hih amino acid identity $ith other nematode A6s and

phyloenetic analysis placed it as a distinct %ranch $ithin a nematode A6 cl"ster& Analysis of

19


20/36

the N>termin"s se"ence of T. canis A6 revealed the presence of a sinal taretin peptide

pres"ma%ly taretin this protein to cytosol or endoplasmic retic"l"m& This $as the first st"dy

$hich reported description of arinine )inase in a *oonotic nematode& The determination of T.

canis A6 and its phosphaen %iosynthetic path$ay! $hich is completely different from those

in mammalian host tiss"es! s"ests this en*yme as a possi%le novel chemotherapy taret for

V8/ syndrome in h"mans 'Fic)ramasinhe et al &! 344:.&

The same a"thors in the s"%se"ent year '344-. reported e#pression of the

arinine )inase recom%inant protein in a pro)aryotic system& A recom%inant arinine

)inase from the infective second>stae larvae $as enerated and a hihly sensitive '+44= at

+O+44 and +OE8ISA for antien capt"re in the

mo"se model $as developed 'Fic)ramasinhe et al., 344-.& This $as the first

demonstration of dianostic potential of T.canis arinine )inase in to#ocarosis& The

a"thors s"ested that the recom%inant> A6 %ased I(>E8ISA co"ld %e applied for

imm"nodianosis of h"man to#ocarosis& Ho$ever! it is necessary to eval"ate the

specificity of this recom%inant antien $ith similar eohelminth infections&

9>/ethylated lycans from Toxocara are specific tarets for anti%ody %indin in

h"man and animal infections& Anti%odies enerated to helminth infections other than T. canis

$ere "nreactive $ith the lycans! e#cept anti%odies to other mem%ers of the Toxocara en"s&

Hence! the car%ohydrate str"ct"res represent imm"noenic! en"s>specific antiens

'Scha%"ssova et al., 344:.&

The "se of recom%inant antiens is promisin for improvin the specificity of the dianosis

of to#ocarosis& To$ard this oal! an I(@ E8ISA involvin three recom%inant antiensO rTES>

04US/! rTES>35! and rTES>+34 $ere developed& The dianostic potential of each p"rified

recom%inant antien $as tested $ith vario"s imm"nolo%"lin classes 'I(! I/! and IE. and

I( s"%classes& The I(@ E8ISA $as descri%ed to have the hihest specificity and $as

f"rther eval"ated "sin a panel of ser"m samples& The rTES>35 I(@ E8ISA sho$ed -4&4='3@G04 samples positive. sensitivity and %oth the rTES>04US/ I(@ E8ISA and rTES>+34

I(@ E8ISA had ,0&4= '3-G04. sensitivity& Com%ined "se of rTES>+34 and rTES>04 I(@

E8ISA for the dianosis of to#ocarosis provided +44= sensitivity& The specificities of rTES>

35! rTES>04US/! and rTES>+34 antiens $ere ,5&3=! ,0&,=! and ,3&4=! respectively&

These res"lts indicate that a dianostic test %ased on these three recom%inant antiens $ill

allo$ for more>acc"rate detection of to#ocarosis 'S"harni et al., 344,.&

20


21/36

Ishiyama et al & '344,. attempted to esta%lish a dianostic method for detectin

circ"latin Toxocara canis antien "sin a sand$ich>E8ISA& /onoclonal anti%odies

$ere prod"ced aainst the e#cretory>secretory 'ES. antien of second stae T. canis

larvae. The cross>reactivity of the sand$ich>E8ISA aainst thirteen different )inds of

parasite antiens $as e#amined& The res"lts revealed that the anti%ody reacted $ith T.

canis ES antien! T & canis female antien and T. canis second>stae larvae antien %"t

did not react $ith any other antiens& The method $as applied to s"spected

to#ocarosis patients and e#amined the circ"latin antien in their sera& 9"t of nine

ser"m samples collected from patients $ith s"spected to#ocarosis %ased on %oth their

clinical symptoms and hih anti%ody titers! five sera sho$ed antien>positive

reactions $hile the remainin fo"r $ere neative& These res"lts indicated that a%o"t

@@&4= of the anti%ody>positive patients $ere antien>neative $ith no active

infection 'Ishiyama et al &! 344,.&

2in et al & '34+0. investiated the serodianostic efficacy of E8ISA "sin

somatic larval antien of T. canis larvae 'TC8A.& Ser"m specimens of 5@ clinically

confirmed to#ocarosis! ++< healthy controls and ++, other tiss"e>invadin helminthic

infections $ere screened %y E8ISA "sin TC8A that sho$ed ,3&3= sensitivity and

-5&5= specificity& Its positive dianostic predictivity $as :-&:= and neative

predictivity ,:&-=& Ser"m samples from patients $ith Anisa#is spp& '@reacted& Imm"no%lot analysis on TC8A

reconi*ed antienic proteins of 3-> and 04>)Da %ands $ith a stron %lottin

reactivity& The a"thors o%served that the E8ISA $ith TC8A antien $as relatively

accepta%le for ma)in differential dianosis for patients $ith any sin of oran

infiltration and eosinophilia&

The sensitivity and specificity of the seroloical tests developed so far have

%een compromised d"e to the iss"es of cross>reactivity of different antiens of

helminths parasiti*in a do& Therefore! eval"atin ne$ taret molec"les for their

hiher sensitivity and specificity is re"ired for developin a relia%le seroloical test&

resent $or) is therefore! envisaed to e#press some ne$ taret antiens of this

parasite and e#ploit them for achievin hiher sensitivity and specificity for detectin

T. canis infection in canines and s"%se"ently in h"mans&

21


22/36

22


23/36

Technical %rogramme

E7pression of Toxocara canis recombinant cathepsin-L1, arginine kinase and TES-26 proteins

"ps nat"rally infected $ith Toxocara canis ad"lt $orms $ill %e treated $ith a s"ita%leanthelminthic for e#p"lsion of the $orms from the host&

Ad"lt $orms $ill %e retrieved! $ashed and teased for release of the es from ravid "ter"s

in sterile tap $ater& A c"lt"re of the es $ill %e set "p for the development of 3nd stae '83.

larvae in sterile $ater at 3-oC in a 79D inc"%ator&

Development of the larvae to 83 stae $ill %e follo$ed microscopically& Hatchin of the

infective 83 larvae from the em%ryonated es $ill %e carried o"t %y treatment of these es$ith 3= sodi"m hypochlorite! follo$ed %y mechanical disr"ption of these es& 8ive

infective larvae $ill %e isolated from the "nem%ryonated es! dead and "nhatched larvae %y

7aermann techni"e&

Total RNA from the 83 larvae $ill %e isolated "sin Tri*ol reaent as per the standard

protocols and reverse transcri%ed to cDNA "sin olio>dT primer&

cDNA synthesi*ed from the total RNA $ill %e CR amplified for cathepsin>8+ cysteine

proteinase! arinine )inase and TES>35 enes "sin ene specific primers&

A"thenticity of the a%ove three enes $ill %e confirmed %y se"encin and cloned in s"ita%le

pro)aryotic e#pression vectors for e#pression of recom%inant proteins in &scherichia coli&

The recom%inant proteins $ill %e p"rified "sin Nic)el affinity chromatoraphy&

Sero-diagnostic assa)s

Sera from Toxocara canis p"ps positive for faecal es $ill %e retrieved and "sed as positive

control for eval"atin the sensitivity of the three recom%inant antiens& "ps free from T.

canis infection as determined %y their faecal e#amination and E8ISA for anti%odies $ill %e

"sed as healthy control animals for standardi*ation of the dianostic assay&

23


24/36

/ice $ill %e infected $ith varyin doses of T. canis infective '83. es "nder la%oratory

conditions and infection allo$ed to develop into visceral larva mirans& The three antiens

$ill %e screened for their detection sensitivity in this la%oratory model&

8i)e$ise! five p"ps! : month old! free from T. canis infection $ill %e e#perimentally

infected $ith varyin doses of T. canis infective '83. es "nder la%oratory conditions andallo$ed to develop into tiss"e d$ellin larvae& The three antiens $ill %e screened for their

dianostic sensitivity in these e#perimentally infected p"ps&

Sera from ad"lt dos %ro"ht to the IVRI polyclinic for ro"tine health chec) "p and also

from stray dos $ill %e screened for T. canis anti%odies "sin these recom%inant proteins&

The st"dy $ill %e e#tended to eval"atin the "tility of these three antiens in the sero>

detection of T. canis infection in h"mans& H"man sera $ill %e o%tained from theopthalmoloy department! AII/S! Ne$ Delhi from T. canis s"spected patients $ith oc"lar

ran"loma for detection of anti>T. canis anti%odies&

Comparative dianostic sensitivity of these antiens $ill %e eval"ated in E8ISA! D9T>

E8ISA and late# al"tination test&

Cross>reactivity of these antiens $ith common canine nematodes viz & Ancylostoma

caninum and 'irofilaria immitis $ill %e st"died to determine specificity&

Data $ill %e analy*ed for comparative sensitivity and specificity of these three recom%inant

antiens in the dianosis of canine to#ocarosis&

24


25/36

!eferences

Ahmad! 7&! 7hatti! (&! Tho)ar! /&A& and /all! N& 3443& H"man to#ocariasis and ascariasisO

concomitant parasitism in Srinaar! 6ashmir! India& Indian 2& athol& /icro%iol& 5:O

0+0+-&

A)ao! N& and 9hta! N& 344:& To#ocariasis in 2apan& arasitol& Int& :6O -:>,0&

Ale)sandra! R&! /aria B&! H""%! B&2&! Savel)o"l! & and (eert! B& 344- /olec"lar clonin and

f"nctional characteri*ation of a cathepsin 8>li)e proteinase from the fish )inetoplastid

parasite Trypanosoma carassii. Imm"noloy& 25O 343+@&

Amir! 2&! Harel! 8&! Eidlit*>/ar)"s! T& and Varsano! I& +,,,4&

7achmeyer! C&! 8amar"e! (& and /orari"! R& 3440& Visceral larva mirans mimic)in lymphoma&

Chest& 12O +3,5>+3,:&

7adley! 2&E&! (rieve! R&7&! 7o$man! D&D&! (lic)man! 8&T& and Roc)ey! 2&H&+,-:& Analysis of

Toxocara canis larval e#cretoryKsecretory antiensO physicochemical characteri*ation

and anti%ody reconition& 2& arasitol& ;O


26/36

7la%"rn! 7&8&! 8indsay! D&S&! Va"han! 2&8&! Rippey! N&S&! Friht! 2&C&! 8ynn! R&C&! 6elch! F&2&!

Ritchie! (&C& and Hepler! D&I& +,,5& revalence of canine parasites %ased on faecal

flotation& Comp& Contin& Ed"c& Vet& ract. 1.O @-0K+5&

De Saviny! D&H& and Ti**ard! I&R& +,::& Toxocara larva miransO the "se of larval secretory

antiens in haemal"tination and sol"%le antien fl"orescent anti%ody tests& Trans& R&

Soc& Trop& /ed& Hy& ;1O


27/36

D"%na! S&! 8anrova! I&! Napravni)! 2&! 2an)ovs)a! I&! Vadle;ch! 2&! e)ar! S& and Bechtner! 2& 344:&

The prevalence of intestinal parasites in dos from ra"e! r"ral areas and shelters of

the C*ech Rep"%lic& Vet& arasitol& 15:O +34>+3-&

D"nsmore! 2&D&! Thompson! R&C&A& and 7ates! I&A& +,-@& revalence and s"rvival of Toxocara canis

es in the "r%an environment of erth A"stralia& Vet& arasitol& 16O 040>0++&

El>/assry! A& A&+,,,& Characteri*ation of antienic property of Toxocara canis and Toxascaris

ad"lts and larvae thro"h imm"nodianostic electrophoresis 'SDS>A(E. and $estern

%lot techni"e& 2& Eypt& Soc& arasitol& 2


28/36

Ha%l"et*el! A&! Traldi! (&! R"ieri! S&! Attili! A& and Sc"ppa! & 3440& An estimation of Toxocara

canis prevalence in dos! environmental e contamination and ris) of h"man infection

in the /arche reion of Italy& Vet& arasitol& 1O 3@0>303&

Hel$ih! A&! 8ind! & and Nansen! D& +,,,& Visceral larva miransO miratory pattern of Toxocara

canis in pis& Int& 2& arasitol& 2@0,&

6ane! S& and (ottesman! /& +,,4& The role of cathepsin 8 in malinant transformation& Semin&

Cancer 7iol& 1O +3:> +05&

6ayes! S& +,,:& H"man to#ocariasis and the visceral larva mirans syndromeO correlative

imm"nopatholoy&InO Breedman! D&9& 'Ed&.! Imm"nopathoenetic aspects of disease

ind"ced %y helminth parasites& Chem& Imm"nol& 7& ,,K+3@&

6hante! (&! 6han! 8&! 7od)he! A&! S"rya$anshi! &! /a;ed! /&! S"rad)ar! U& and (ai)$ad! S& 344,&

Epidemioloical s"rvey of astro>intestinal parasites of non>descript dos in Nap"r

City& Vet&Forld& 2O 33>30&

6im!Y& and H"h! S& 344


29/36

6na"s! 7&U& and 7et)e! & +,-5& 8arva mirans visceralis occ"rrence of Toxocara canis in dos in

the East (erman district of Cott%"s& Ane$& arasitol& 2;O +5,K+:0&

8ia! A&H&! /oon! S&U&! ar! Y&6&! Na! 7&6&! H$an! /& (&! 9h! C&/&! Cho! S&H&! 6on! Y&! 6im! T&S& and

Ch"n! &R& 3445& Identification and characteri*ation of a cathepsin 8>li)e cysteine protease

from Taenia solium metacestode& Vet& arasitol& 151O 330-4&

8o")as! A&! Sel*er! &/& and /ai*els! R&/& +,,-& Characteri*ation of Tc>cpl >+! a cathepsin 8>li)e

cysteine protease from Toxocara canis infective larvae+& /ol& 7iochem& arasitol& :43&

/anaval! 2&B& and (lic)man! 8&T& 3445& /anaement and treatment options for h"man to#ocariasis&

InO Holland CV! Smith HV! editors& Toxocara the enimatic parasite& 9#fordshireO

CA7I "%lishin& ++0>+35&

/anaval! 2&B&!(lic)man! 8&T&! Dorchies! & and /orassin! 7& 344+& Hihlihts of h"man

to#ocariasis& 6or& 2& arasitol& ++&

/ai*els! R&/& and ae! A&& +,,4& S"rface associated lycoproteins from Toxocara canis larval

parasites& Acta Tropica& 5;O 0


30/36

/ai*els! R&/&! Tetteh! 6&6& and 8o")as! A& 3444& Toxocara canisO enes e#pressed %y the arrested

infective larval stae of a parasitic nematode& Int& 2& arasitol& /O@,+0&

/ats"m"ra! 6& and Endo! R& +,-3& Investiation of anti%odies aainst Toxocara canis in nat"rally

infected p"ppies& %l& 7a)t& Hy& A& 2:O+0,>+@+&

/ats"m"ra! 6&! 6a*"ta!Y&! Endo! R& and Tana)a! 6& +,-0a& The I/ anti%ody activities in relation to

the parasitoloic stat"s of Toxocara canis in dos& %l& 7a)t& Hy& A& 2::O @43>@4


31/36

/ohamad! &! A%dollhpo"r! B&! 7ira;andi! /& and Biroo*eh B& 34+4& Soil contamination $ith

Toxocara spp& es in the p"%lic par)s from three areas of 6horram A%ad! Iran&

N&/ed&C& 2& 12O50>53+5

Naa)"ra! 6&! Tachi%ana! H&! 6aneda! Y& and 6ato! Y& +,-,& To#ocariasis possi%ly ca"sed %y

inestin ra$ chic)en& 2& Infect& Dis& 16/O :0+@+,&

Na$al! A&H& and /ona! S&/& 344-& Serodianosis of h"man to#ocarosis "sin ad"lt antiens of

Toxocara canis. Res& 2& arasitol& O-,+&

N"nes! C&/&! T"ndisi! R&N&! Heinemann! /&7&! 9assa$ara! S& and Richt*enhain! 8&2& +,,,&

To#ocariasisO seroloical dianosis %y indirect anti%ody competition E8ISA& RevistaInst& /ed& Trop& Sao a"lo& 51O ,+44&

alliola! E&! Ci"chini! B&and /orelli! 8&+,-5& Diff"sione delle parassitosi intestinali nei cani di Roma

'triennio +,-3K+,-@.& 9%iettivi e doc"menti veterinary& 6O :0K:+:0&

ereira! C& A&! Alonso! (& D&! aveto! /& C&! Bla$ia! /& /& and Torres! H& N& +,,,& Q8>arinine

"pta)e and 8>phosphoarinine synthesis in Trypanosoma cruzi& 2& E")& /icro%iol& 56O


32/36

ereira! C&A&! Alonso! (&D&! aveto! /&C&! Iri%arren! A&! Ca%anas! /&8&! Torres! H&N& and Bla$ia!

/&/& 3444& Trypanosoma cruzi arinine )inase characteri*ation and clonin& A novel

eneretic path$ay in proto*oan parasites& 2& 7iol& Chem& 2;:O +@,035&

Rai! S&6&! Ua! S&! F"! &! Ta)ahashi! Y& and /ats"m"ra!T& +,,:& Use of polymerase chain reaction

in the dianosis of to#ocariasisO an e#perimental st"dy& So"theast Asian 2& Trop& /ed&

"%& Hlth& 2.O dianosis of %"%aline

fasciolosis $ith asciola gigantica cathepsin>8 recom%inant cathepsin>8+D

proteases& Acta Trop&


33/36

Sah"! S&! Samanta! S&! S"dha)ar! N&R&! Raina! 9&6&! ("pta! S&C&! /a"rya! &S&! a$de! A&/& and

6"mar! A& 34+0& revalence of canine to#ocariasis in 7areilly! Uttar radesh! India&2&

arasitic Diseases& D9I +4&+44:Gs+350,>4+3>434:>&

Samanta! S&! Ansari! /&& and Sahai! 7&N& +,-,& revalence of Toxocara canis in dos in and aro"nd

Ranchi! 7ihar& 7AU Res& 2& 1O 34+>340&

Samanta! S& and (hor"i! S&6& +,,-& revalence of Toxocara canis in dos in and aro"nd 7areilly

'U&.& Indian 2& Anim& Hlth& ;O /ethylated lycans

from Toxocara are specific tarets for anti%ody %indin in h"man and animal infections&

Int& 2& arasitol. ;O ,:K+4,&

S"%hash! 6& and Tan$ar! R&6& 344:& revalence of $orm infestation in stray dos in and aro"nd

2odhp"r& 2& Vet&arasitol. 21O+:+>+:3&

S"dha)ar! N&R&! Samanta! S&! Sah"! S&! Raina! 9&6&! ("pta! S&C&! /adh"! D&N& and 6"mar! A& 34+0a&revalence of Toxocara species es in soil samples of p"%lic health importance in

aro"nd 7areilly! Uttar radesh! India! and Vet& Forld& 6O -:>,4&

S"dha)ar! N&R&! Samanta! S&! Sah"! S&! Raina! 9&6&! ("pta! S&C&! (os$ami! T&6&! 8o)esh! 6&/& and

6"mar! A& 34+0%& Characteri*ation of e#cretory secretory antiens of ad"lt Toxocara

canis %y Festern %lottin& 2& arasitic Diseases& D9I +4&+44:Gs+350,>4+3>434,>#&

S"harni! /&! Norhaida! C& A& and Noordin!R& 344,& Development and eval"ation of a sensitive and

specific assay for dianosis of h"man to#ocariasis %y "se of three recom%inant antiens

'TES>35! TES>04US/! and TES>+34 ). 2& Clin /icro%iol& 5;O +:+3K+:+:&

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S"n!T&! 7ellosa! /& 8&! 8"cio>Borster! A&! Ho! D& S& and 7o$man! D& D& 344:& A comparison of the

protein constit"ents of the ma;or %ody compartments of the do ro"nd$orm! Toxocara

canis. Vet& arasitol& 1:/O +++K++5:&

Ta)ashima! /&! 9hmi! H&! Fatana%e! T&! 9)amoto! 6&! 6anoe! /& and Naai! S&! 3440& Attempts to

separate female Ascaris suum antien and to investiate its partial characteri*ation& Vet&

2& 16: O+5@K+5-&

Tei#eira! /&8&! 8iliana! R&! 8"ana De! B& and NatLlia! (& 344-& revalence of Toxocara canis infection

in p"%lic s"ares of the ConcJrdia City! Santa Catarina! 7ra*il& arasitol& 8atinoam& 6O 5,>

:+&

Tetteh! 6&6&! 8o")as! A&! Tripp! C& and /ai*els! R& /& +,,,& Identification of a%"ndantly e#pressed

novel and conserved enes from the infective larval stae of Toxocara canis %y an

e#pressed se"ence ta stratey& Infect& Imm"nol& 6;O@::+>@::,&

Tra"%! R&2&! Ro%ertson! I&D&! Ir$in! &! /enc)e! N& and Thompson! A&R&C& 3443& The role of dos in

transmission of astrointestinal parasites in a remote tea>ro$in comm"nity in

northeastern India& Am& 2& Trop& /ed& Hy& 6;O 8 cysteine protease %ased Dot>en*yme>lin)ed

imm"nosor%ent assay for the dianosis of asciola gigantica infection in %"ffaloes&

Vet& arasitol& 1.O 0-3>0-


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e#pression and comparison of the )inetics of cDNA>derived arinine )inase& E#p&

arasitol& 11;O +3@K+03&

Fic)ramasinhe! S&! Yata$ara! 8&! Naata)i! /&! Ta)amoto! /&! Fatana%e! Y&! Ra;apa)se! R& & V& 2&!

Uda! 6&! S"*")i! T& and Aats"ma! T& 344-& Development of a hihly sensitive I(>

E8ISA %ased on recom%inant arinine )inase of Toxocara canis for serodianosis of visceral larva mirans in the m"rine model& arasitol& Res&1/O -0:&

h"! 1&&! (asser! R&7&! Chilton! N&7& and 2aco%s! D&E& 344+& /olec"lar approaches for st"dyin

ascaridoid nematodes $ith *oonotic potential! $ith emphasis on Toxocara species& 2&

Helminthol& ;:O +4+>+4-&

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Toxocara ORW Anju