Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 109580 5 pageshttpdxdoiorg1011552013109580

Research ArticleA Comparative Histopathology Serology and Molecular Studyon Experimental Ocular Toxocariasis by Toxocara cati inMongolian Gerbils and Wistar Rats

Mohammad Zibaei1 Seyed Mahmoud Sadjjadi23 Mehdi Karamian4 Shoji Uga5

Ahmad Oryan6 and Seyed Hamidreza Jahadi-Hosseini7

1 Department of Parasitology and Mycology School of Medicine Alborz University of Medical Sciences PO Box 31485561 Karaj Iran2Department of Parasitology andMycology School of Medicine Shiraz University of Medical Sciences PO Box 713451735 Shiraz Iran3 Basic Sciences in Infectious Diseases Research Center School of Medicine Shiraz University of Medical SciencesPO Box 713451735 Shiraz Iran

4Department of Microbiology School of Medicine Birjand University of Medical Sciences PO Box 917751365 Birjand Iran5 Department of Parasitology Faculty of Health Sciences Kobe University Graduate School of Health SciencesPO Box 6540142 Kobe Japan

6Department of Pathology School of Veterinary Medicine Shiraz University PO Box 713451735 Shiraz Iran7Department of Ophthalmology Khalili Hospital Shiraz University of Medical Sciences PO Box 713451735 Shiraz Iran

Correspondence should be addressed to Seyed Mahmoud Sadjjadi smsadjjadisumsacir

Received 27 April 2013 Revised 22 July 2013 Accepted 29 July 2013

Academic Editor Christen Rune Stensvold

Copyright copy 2013 Mohammad Zibaei 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

The aim of this study was to compare the performance of three in-house diagnostic tests that is histopathology enzyme-linkedimmunosorbent assay (ELISA) and polymerase chain reaction (PCR) for the diagnosis after experimental infection with Toxocaracati Twenty Mongolian gerbils and Wistar rats were divided into ten groups (119899 = 2group) Toxocara cati infections wereestablished in Mongolian gerbils and Wistar rats by administering doses of 240 and 2500 embryonated Toxocara cati eggs bygavage respectively Tissue sections were stained with Haematoxylin and Eosin and observed under the light microscope Seraand vitreous fluid collected from separate infected groups were tested against Toxocara cati antigens for 92 days postinfectionGenomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) blocks and aqueous fluids belong to the animalsThe histopathology test gave negative results among the groups of animals examined between 5 and 92 days postinfection TheELISA results showed that anti-Toxocara antibodies have risen between 7 and 61 days postinfection in sera and vitreous fluid in theanimals infected respectively Analysis of PCR products revealed positive band (660 bp) in the orbital tissue infected Mongoliangerbils at 5 days postinfection Of the three evaluated methods the PCR could be recommended for scientific and laboratorydiagnoses of toxocariasis in experimentally infected animals

1 Introduction

Toxocariasis is one of the most commonly reported zoonotichelminthic infections in the world Both Toxocara canis (around worm of dogs) and Toxocara cati (common roundworm of cats) cause toxocariasis Ingestion of Toxocaraspecies eggs containing third-stage larvae due to geophagiapica and the consumption of contaminated raw meat or

liver is considered as possible cause of visceral larva migrans(VLM) or ocular larva migrans (OLM) [1 2]

Ocular infection with Toxocara larvae species is uncom-mon but of interest to ophthalmologists because it largelyaffects the young people in whom it can cause significantocular morbidity or even blindness The clinical sign ofocular toxocariasis in human often includes diminishedvision leukocoria red eye and strabismus The diagnosis of

2 BioMed Research International

suspected ocular toxocariasis is supported by the presenceof chorioretinal or focal lesions in posterior eye segmentin the presence of positive serology [3 4] Toxocara cati(T cati) is the common worm of cats and its third-stagelarvae are possible cause of OLM Several studies have beenreported that human can be infected by the ingestion of Tcati embryonated eggswith contaminated soil or eating larvaewithin paratenic hosts including birds and small rodentsThere is little finding on the location of T cati larvae inparatenic hosts including the eye as causative agents of humantoxocariasis [5 6]

In the last few years several authors have approacheddiagnosis through the specific detection of antigens or anti-bodies [7 8] Nevertheless the histopathological demonstra-tion of larvae is relatively intensive especially due to lowlarvae burden [9 10] Thus the development of new andmore sensitive diagnostic tools is needed Desirably theisnew approach should permit the diagnosis in early infectionto prevent the development of severe pathologies associatedwith later infection Application ofmolecularmethods for thediagnosis of Toxocara infections has been increasing Severalstudies have reported the usefulness of polymerase chainreaction (PCR) for the characterization and the diagnosis oftoxocariasis compared to other parasitic diseases in naturalor experimentally infected animals [11 12]

Based on the widespread use of polymerase chain reac-tion for the diagnosis of parasitic diseases and to the bestof our knowledge that there are no reports about of useof PCR in diagnosis of ocular toxocariasis this study wasdesigned to evaluate PCR for being used in the diagnosisafter experimental ocular infection with T cati third-stagelarvae compared to histopathology and Enzyme-LinkedImmunosorbent Assay (ELISA)

2 Materials and Methods

21 Animals and Experimental Infections Ten Mongoliangerbils (one-month-old males) and ten Wistar rats (one-month-old males) were assigned in ten experimental groups(119899 = 2group) each two animals of the same were infectedorally with approximately 240 and 2500 embryonated T catieggs respectively [6 13ndash15] The eggs were derived fromadult female worms and were embryonated according to ourprevious paper [16] The animals were maintained understandard conditions in an environment with controlledtemperature and humidity The inoculated animals wereeuthanized on days 5 30 49 70 and 92 postinfection (PI)The vitreous fluid which is a jelly compound was taken fromthe eyeballs of infected animals using a 22-gauge needlelinked to a powerful syringe and stored at minus30∘C untilusedThe experimental protocol was approved by the EthicalCommittee of the Shiraz University of Medical Sciences

22 Preparation of T cati Excretory-Secretory (ES) AntigensES antigens for ELISA assay were obtained from third-stage larvae (ESL

3) Adult worms of T cati were collected

from the intestines of infected stray cats and the eggs wereisolated from uteri of female worms were decoated by

sodium hypochlorite (7 wv) and were embryonated in25 formalinringer solution while being incubated at anatmosphere of 5 CO

2in 25∘C for 3 weeks [16] The larvae

were collected aseptically from a Baermanns apparatus ESantigens of T cati were prepared from third-stage larvae bythe method of de Savigny (1975) with modification [17]

23 Pathological Study Samples of eyeball from the infectedanimals were fixed in 10 neutral buffered formalin dehy-dratedwith graded ethanol and embedded in paraffin Tissuesections of 5 120583m thickness were stained with Haematoxylinand Eosin (H amp E) and after mounting were observed underthe light microscope

24 Immunological Study ELISA was carried out in flat-bottom96 microplates The ES antigen was collected fromculture supernatant of T cati third-stage larvae as describedThe plates were coated with 100120583L of ES antigen in coatingbuffer (005M carbonate bicarbonate-buffer pH 96) andincubated at 4∘C overnight Excess antigen was removed bywashing the plate three times in phosphate buffered saline-Tween 20 (PBST pH 74 containing 005 Tween 20) Excessbinding sites were blocked for 1 hr at 37∘C with 100120583L ofbovine serum albumin (1 dilution in PBS) The wells werewashed and then 100120583L of diluted serum (1 100 in PBS)and vitreous humor (1 2 in PBS) were added to each welland incubated for 1 hr at 37∘C The plates were washedas before and 100120583L of horseradish peroxidase-conjugatedsheep anti-mouse IgG at a 11000 dilution in PBST was addedand incubated for one hour at 37∘C After washing theplates were incubatedwith chromogensubstrate (100120583LwellABTS peroxidase substrate) and the reaction was terminatedwith peroxidase stop solution (50120583L diluted 1 5) after 5minThe absorbance at 405 nm was monitored with a MUNKmicroplate reader The cutoff point was set as 2SD above themean of control samples

25 DNA Isolation and PCR Analyses Extraction of DNAwas done from formalin-fixed paraffin-embedded (FFPE)blocks and aqueous fluids belong to gerbils and rats FFPEblocks were cut and deparaffinized using Xylol due tostandard protocols Genomic DNA was isolated from indi-vidual samples using QIAamp DNA Mini and Blood MiniHandbook Kit (Qiagen Hilden Germany) according to themanufacturerrsquos instructionThe specific forward primer JW4(51015840-ACTGTCGAGGATGAGCGTGA-31015840) [18] was used withNC2 primer (reverse 51015840-TTAGTTTCTTTTCCTCCGCT-31015840)[11] to amplify partial ITS-1 complete 58S and ITS-2 rDNAof T cati PCR reaction was performed in 10mM Tris-HCLpH 88 15mMMgCl

2 50mMKCl 01 Triton X-100 dNTP

200120583M primers 1120583M and 1U Taq polymerase per 25 120583LThe reaction was carried out under the following conditions94∘C for 45 s 58∘C for 45 s 72∘C for 90s and final extension72∘C for 10min Corresponding PCR products were elec-trophoresed in 14 agarose gel with 05 120583gmL ethidiumbromide and a 100-bp ladder was used as DNA sizemarkerfor estimating the size of the amplicons and photographedusing a gel documentation system (UV Transilluminator)

BioMed Research International 3

Figure 1 The negative reaction in the retina of the infected animals(paraffin section stained H with E)

3 Results

In an attempt to identify the optimal procedure for thediagnosis of ocular Toxocara infection the performanceand agreement among the three different methods namelyhistopathology ELISA and PCR methods were evaluated

31 Pathological Observation Figure 1 corresponds to imageof the ocular structure of an infected animal In none of thestudied animals we observed peripheral granuloma in theposterior pole or retrolental hyaline membranes

32 ELISA for Anti-Toxocara Antibodies The ELISA resultswere positive in sera and aqueous fluid for Mongolian gerbilsand Wistar rats examined at 1 and 8 weeks PI respectivelySpecimens with an OD (optical density) reading greaterthan 0300 and less than 0260 can be interpreted withassurance as positive and negative respectively Caution maybe necessary with specimens with inter with mediate valuesAt the seventh day PI the test was positive in 15 out of 20animals (75 sensitivity) where the animals showed anti-Toxocara antibodies in sera The test was positive at an ODgreater than 0310 and higher in 10 Mongolian gerbils and5 Wistar rats At the sixty-one day PI the test was positivefor aqueous fluid in 11 out of 20 animals (55 sensitivity)where the test was positive at an OD of 0300 or more in nineMongolian gerbils and two Wistar rats (Figure 2)

33 PCR Analysis Analysis of PCR products revealed pos-itive band (660 bp) in the orbital tissue infected Mongoliangerbils at 5 days PI The DNA of T cati was found in the vit-reous fluid of experimentally infected Mongolian gerbils Nopositive band was shown in samples ofWistar rats (Figure 3)

4 Discussion

Toxocariasis is an important infection among socioeconom-ically disadvantaged young children and is low in adultsOcular larva migrans occurs when Toxocara larvae reach eyetissue where they induce inflammatory reaction frequentlywithout the sign or symptoms that accompany the visceraltoxocariasis [19ndash22] The diagnosis of ocular larva migrans is

0

02

04

06

08

1

12

14

5 30 49 70 92

SerumVitreous fluidCut off

Days post infection (DPI)

Opt

ical

den

sity

(OD

)

Figure 2 Development of specific Toxocara cati L2S ES antigens ininfectedMongolian gerbils andWistar rats with 240 and 2500 T catiembryonated eggs respectively OD values of uninfected animalswere lower than the negative cutoff 0260 and those of infectedanimals were higher than positive cutoff 0300

based on retinoscopic and retinographic studies and is con-firmed by the finding of larvae in lesions [23] The presenceof anti-Toxocara antibodies or antigens in vitreous humorhas been reported to be a sensitive method for detectingtoxocariasis in infections or human ocular toxocariasis [25]

In this study we compared the performance of threedifferent diagnostic techniques including histopathologyindirect ELISA and PCR on eyeball and retina tissue duringthe infection of Mongolian gerbils and Wistar rats from 5 to92 days PI

The infection with T cati in the animals not causedimportant histological alternative in cellular structures ofthe eye Histopathologic examination did not detect foreignbodies or tumors in the affected region and the ophthalmicexamination did not suggest an infectious process Serologi-cal testing is the primary diagnostic tool in patient suspectedfor ocular and visceral larvamigrans but these tests are insuf-ficient as cross-reactions may occuring For example cross-reactivity has been reported between T canis and Trichinellaspiralis and T canis and Ascaris lumbricoides [26] Sommer-felt et al (2006) detected specific antibodies against T caniswith ELISA in eighteen pigs at 7 days PI with high titerspersisting over an 84-day period [27] In current study animmunological response in sera was early 7 days PI and wasmaintained throughout the study (92 days) and these resultsare similar to those of other authors Beside from 61 days ofthe infection antibodies in vitreous fluid were detected

An alternative approach to detection of toxocariasis is touse a molecular-based-method such as PCR When PCR wasapplied to eye tissue and aqueous fluid of infectedMongoliangerbils detection of the specific amplification started at 5days PI This was not possible in other stage of infection(30 49 70 and 92 days PI) Our PCR approach in retinademonstrated the presence of the parasite genome beforeone week PI while the histological method was not usefulfor detection of parasite at the retinal and subretinal levels

4 BioMed Research International

660bp 1000 bp500bp

100 bp

(a)

660bp 1000 bp500bp

100 bp

(b)

Figure 3 (a) Analysis of PCR products (eye tissue of infected animals) by 14 agarose gel electrophoresis Lanes 1ndash5 days 5 to 92 PI ofMongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm as positive control lane 12 negativecontrol lane M 100ndash1500 bp DNA size marker (b) DNA amplification of vitreous fluid samples of infected animals by PCR on 14 agarosegel Lanes 1ndash5 days 5 to 92 PI of Mongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm aspositive control lane M 100ndash1500 bp DNA size marker

The PCR in vitreous humor could also be more sensitivethan the detection of antibodies by ELISA in the animalsin early infection since prominent positive serology wasdetectable from 61 days PI This period in animal challengealthough not strictly equivalent to human infection in clinicalcourse could be considered as an acute infection in patientsregarding the parasitic migration and development It seemsthat the transmission of larvae was first from intestine to theliver lung and eye and then 5 days PI they migrated to otherorgans Sometimes larvae return back to the eye to createlesions at the end of migration It has been demonstratedthat Toxocara larvae may migrate and persist in the eye ofexperimental infected animals This opinion is challenged byseveral authors who argued that Toxocara is as a causativeocular toxocariasis [28 29] In a relatively similar studyCho et al (2007) infected Mongolian gerbils with 1000Toxocara ova and reported that 10 of infective larvae werefound before day 5 in liver tissue and also showed thatophthalmoscopically a motile larva was observed in theretina at 14 days PI [30] In earlier studies investigatorsrevealed the parasite DNA by PCR during early infectionperiods in the animals model for Trypanosoma cruzi andToxoplasma gondii thus permitting the diagnosis of infectionearlier than when conventional diagnostic techniques such asmicroscopy or serology are being used [31 32]

5 Conclusion

Histopathology serology (ELISA) and molecular (PCR)methods tested for diagnosis of ocular toxocariasis causedby T cati in experimentally infected Mongolian gerbils andWistar rats revealed that molecular method was the most

sensitive and superior compared to both histopathology andELISA in the early diagnosis of ocular infection The PCRprovided a rapid reliable and sensitive means by which oneestablish the exact etiology of such parasitic infection sothat optimal therapy can be started promptly The superiorperformance of the PCR in detecting infected Mongoliangerbils andWistar rats in this small experimental populationwarrants further evaluation in a larger population of animalmodels and humans naturally infected with T cati

Conflict of Interests

The authors report no conflict of interest in this work

Acknowledgments

The technical assistance of Mrs S Kazemian and MrB Farhang Mehr of the Department of Parasitology andMycology School of Medicine Shiraz University of MedicalSciences Iran is acknowledged with pleasure The criticalcomments of Professor Akao from Section of EnvironmentalParasitology Graduate School of Tokyo Medical and DentalUniversity Japan are gratefully acknowledged This studywas funded by Shiraz University of Medical Sciences ShirazIran Grant no 4108

References

[1] S Azizi A Oryan S M Sadjjadi and M Zibaei ldquoHistopatho-logic changes and larval recovery ofToxocara cati in experimen-tally infected chickensrdquo Parasitology Research vol 102 no 1 pp47ndash52 2007

BioMed Research International 5

[2] D Despommier ldquoToxocariasis clinical aspects epidemiologymedical ecology and molecular aspectsrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 265ndash272 2003

[3] R S Clemett H J Williamson R R Hidajat R A Allardyceand A C Stewart ldquoOcular Toxocara canis infections diagnosisby enzyme immunoassayrdquo Australian and New Zealand Journalof Ophthalmology vol 15 no 2 pp 145ndash150 1987

[4] L deVisser A Rothova J H de Boer et al ldquoDiagnosis of oculartoxocariasis by establishing intraocular antibody productionrdquoAmerican Journal of Ophthalmology vol 145 no 2 pp 369ndash3742008

[5] N Akao and N Ohta ldquoToxocariasis in Japanrdquo ParasitologyInternational vol 56 no 2 pp 87ndash93 2007

[6] M Zibaei S M Sadjjadi and S Uga ldquoExperimental Toxocaracati infection in gerbils and ratsrdquoKorean Journal of Parasitologyvol 48 no 4 pp 331ndash333 2010

[7] T H Dzbenski W Hautz and E Bitkowska ldquoExperimentaltoxocariasis in rabbits immunological markers of ocular infec-tionsrdquoWiadomosci Parazytologiczne vol 47 no 4 pp 591ndash5962001

[8] R Sakai H Kawashima H Shibui K Kamata C Kambaraand H Matsuoka ldquoToxocara cati-induced ocular toxocariasisrdquoArchives of Ophthalmology vol 116 no 12 pp 1686ndash1687 1998

[9] N Cardillo A Rosa M Ribicich C Lopez and I SommerfeltldquoExperimental infection with Toxocara cati in BALB-c micemigratory behaviour and pathological changesrdquo Zoonoses andPublic Health vol 56 no 4 pp 198ndash205 2009

[10] K Taira Y Saitoh and C M O Kapel ldquoToxocara cati larvaepersist and retain high infectivity in muscles of experimentallyinfected chickensrdquo Veterinary Parasitology vol 180 no 3-4 pp287ndash291 2011

[11] X Q Zhu S DrsquoAmelio H W Palm L Paggi M George-Nascimento and R B Gasser ldquoSSCP-based identificationof members within the Pseudoterranova decipiens complex(Nematoda Ascaridoidea Anisakidae) using genetic markersin the internal transcribed spacers of ribosomal DNArdquo Para-sitology vol 124 no 6 pp 615ndash623 2002

[12] A Borecka J Gawor M Niedworok and B Sordyl ldquoDetec-tion of Toxocara canis larvae by PCR in the liver of experi-mentally infected Mongolian gerbils (Meriones unguiculatus)rdquoHelminthologia vol 45 no 3 pp 147ndash149 2008

[13] NAkao TH Takayanagi R Suzuki S Tsukidate andK FujitaldquoOcular larva migrans caused by Toxocara cati in Mongoliangerbils and a comparison of ophthalmologic findings with thoseproduced by T canisrdquo Journal of Parasitology vol 86 no 5 pp1133ndash1135 2000

[14] E Hayashi N Akao and K Fujita ldquoEvidence for the involve-ment of the optic nerve as a migration route for larvae in oculartoxocariasis of Mongolian gerbilsrdquo Journal of Helminthologyvol 77 no 4 pp 311ndash315 2003

[15] S Z Lescano M L Queiroz and P P Chieffi ldquoLarval recoveryof Toxocara canis in organs and tissues of experimentallyinfected Rattus norvegicusrdquo Memorias do Instituto OswaldoCruz vol 99 no 6 pp 627ndash628 2004

[16] M Zibaei S M Sadjjadi S H Jahadi Hosseini and B SarkarildquoA method for accelerating the maturation of Toxocara catieggsrdquo Iranian Journal of Parasitology vol 2 no 1 pp 39ndash422007

[17] D H De Savigny ldquoIn vitro maintenance of Toxocara canislarvae and a simple method for the production of ToxocaraES antigen for use in serodiagnostic tests for visceral larvamigransrdquo Journal of Parasitology vol 61 no 4 pp 781ndash782 1975

[18] M W Li R Q Lin H H Chen R A Sani H Q Song and XQ Zhu ldquoPCR tools for the verification of the specific identityof ascaridoid nematodes from dogs and catsrdquo Molecular andCellular Probes vol 21 no 5-6 pp 349ndash354 2007

[19] R Molk ldquoOcular toxocariasis a review of the literaturerdquoAnnalsof Ophthalmology vol 15 no 3 pp 216ndash231 1983

[20] J A Shields ldquoOcular toxocariasis A reviewrdquo Survey of Ophthal-mology vol 28 no 5 pp 361ndash381 1984

[21] S H Gillespie W J Dinning A Voller and N S CrowcroftldquoThe spectrum of ocular toxocariasisrdquo Eye vol 7 no 3 pp 415ndash418 1993

[22] B Good C V Holland M R H Taylor J Larragy P Moriartyand M OrsquoRegan ldquoOcular toxocariasis in schoolchildrenrdquo Clin-ical Infectious Diseases vol 39 no 2 pp 173ndash178 2004

[23] L I D C Zanandrea G M Oliveira A S Abreu and F EL Pereira ldquoOcular lesions in gerbils (Meriones unguiculatus)infected with low larval burden of Toxocara canis observationsusing indirect binocular ophthalmoscopyrdquoRevista da SociedadeBrasileira deMedicina Tropical vol 41 no 6 pp 570ndash574 2008

[24] J-F Magnaval L Malard B Morassin and R Fabre ldquoImmun-odiagnosis of ocular toxocariasis using Western-blot for thedetection of specific anti-Toxocara IgG and CAPŮ for the mea-surement of specific anti-Toxocara IgErdquo Journal of Helminthol-ogy vol 76 no 4 pp 335ndash339 2002

[25] E Bertelmann K-H Velhagen U Pleyer and C HartmannldquoOcular toxocariasis Diagnostic and therapeutic optionsrdquoOph-thalmologe vol 100 no 11 pp 950ndash954 2003

[26] H Zarnowska and M Jastrzębska ldquoExcretory-secretory larvalantigens of Toxocara canis physico-chemical characteristicsand specificity assayed by Western blot techniquerdquo Acta Para-sitologica vol 39 no 3 pp 41ndash45 1994

[27] I E Sommerfelt G Santillan C Lopez M Ribicich and A JFranco ldquoImmunological and hematological response in exper-imental Toxocara canis-infected pigsrdquo Veterinary Parasitologyvol 96 no 2 pp 127ndash134 2001

[28] T H Takayanagi N Akao R Suzuki M Tomoda S Tsuki-date and K Fujita ldquoNew animal model for human oculartoxocariasis ophthalmoscopic observationrdquo British Journal ofOphthalmology vol 83 no 8 pp 967ndash972 1999

[29] S V Santos S Z Lescano J M Castro and P P Chieffi ldquoLarvalrecovery of Toxocara cati in experimentally infected Rattusnorvegicus and analysis of the rat as potential reservoir for thisascaridrdquoMemorias do InstitutoOswaldo Cruz vol 104 no 6 pp933ndash934 2009

[30] S Cho M Egami H Ohnuki et al ldquoMigration behaviour andpathogenesis of five ascarid nematode species in theMongoliangerbilMeriones unguiculatusrdquo Journal of Helminthology vol 81no 1 pp 43ndash47 2007

[31] L M Weiss S A Udem M Salgo H B Tanowitz and MWittner ldquoSensitive and specific detection of toxoplasma DNAin an experimental murine model use of Toxoplasma gondii-specific cDNA and the polymerase chain reactionrdquo Journal ofInfectious Diseases vol 163 no 1 pp 180ndash186 1991

[32] L V Kirchhoff J R Votava D E Ochs and D R MoserldquoComparison of PCR and microscopic methods for detectingTrypanosoma cruzirdquo Journal of ClinicalMicrobiology vol 34 no5 pp 1171ndash1175 1996

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International Journal of

Microbiology

2 BioMed Research International

suspected ocular toxocariasis is supported by the presenceof chorioretinal or focal lesions in posterior eye segmentin the presence of positive serology [3 4] Toxocara cati(T cati) is the common worm of cats and its third-stagelarvae are possible cause of OLM Several studies have beenreported that human can be infected by the ingestion of Tcati embryonated eggswith contaminated soil or eating larvaewithin paratenic hosts including birds and small rodentsThere is little finding on the location of T cati larvae inparatenic hosts including the eye as causative agents of humantoxocariasis [5 6]

In the last few years several authors have approacheddiagnosis through the specific detection of antigens or anti-bodies [7 8] Nevertheless the histopathological demonstra-tion of larvae is relatively intensive especially due to lowlarvae burden [9 10] Thus the development of new andmore sensitive diagnostic tools is needed Desirably theisnew approach should permit the diagnosis in early infectionto prevent the development of severe pathologies associatedwith later infection Application ofmolecularmethods for thediagnosis of Toxocara infections has been increasing Severalstudies have reported the usefulness of polymerase chainreaction (PCR) for the characterization and the diagnosis oftoxocariasis compared to other parasitic diseases in naturalor experimentally infected animals [11 12]

Based on the widespread use of polymerase chain reac-tion for the diagnosis of parasitic diseases and to the bestof our knowledge that there are no reports about of useof PCR in diagnosis of ocular toxocariasis this study wasdesigned to evaluate PCR for being used in the diagnosisafter experimental ocular infection with T cati third-stagelarvae compared to histopathology and Enzyme-LinkedImmunosorbent Assay (ELISA)

2 Materials and Methods

21 Animals and Experimental Infections Ten Mongoliangerbils (one-month-old males) and ten Wistar rats (one-month-old males) were assigned in ten experimental groups(119899 = 2group) each two animals of the same were infectedorally with approximately 240 and 2500 embryonated T catieggs respectively [6 13ndash15] The eggs were derived fromadult female worms and were embryonated according to ourprevious paper [16] The animals were maintained understandard conditions in an environment with controlledtemperature and humidity The inoculated animals wereeuthanized on days 5 30 49 70 and 92 postinfection (PI)The vitreous fluid which is a jelly compound was taken fromthe eyeballs of infected animals using a 22-gauge needlelinked to a powerful syringe and stored at minus30∘C untilusedThe experimental protocol was approved by the EthicalCommittee of the Shiraz University of Medical Sciences

22 Preparation of T cati Excretory-Secretory (ES) AntigensES antigens for ELISA assay were obtained from third-stage larvae (ESL

3) Adult worms of T cati were collected

from the intestines of infected stray cats and the eggs wereisolated from uteri of female worms were decoated by

sodium hypochlorite (7 wv) and were embryonated in25 formalinringer solution while being incubated at anatmosphere of 5 CO

2in 25∘C for 3 weeks [16] The larvae

were collected aseptically from a Baermanns apparatus ESantigens of T cati were prepared from third-stage larvae bythe method of de Savigny (1975) with modification [17]

23 Pathological Study Samples of eyeball from the infectedanimals were fixed in 10 neutral buffered formalin dehy-dratedwith graded ethanol and embedded in paraffin Tissuesections of 5 120583m thickness were stained with Haematoxylinand Eosin (H amp E) and after mounting were observed underthe light microscope

24 Immunological Study ELISA was carried out in flat-bottom96 microplates The ES antigen was collected fromculture supernatant of T cati third-stage larvae as describedThe plates were coated with 100120583L of ES antigen in coatingbuffer (005M carbonate bicarbonate-buffer pH 96) andincubated at 4∘C overnight Excess antigen was removed bywashing the plate three times in phosphate buffered saline-Tween 20 (PBST pH 74 containing 005 Tween 20) Excessbinding sites were blocked for 1 hr at 37∘C with 100120583L ofbovine serum albumin (1 dilution in PBS) The wells werewashed and then 100120583L of diluted serum (1 100 in PBS)and vitreous humor (1 2 in PBS) were added to each welland incubated for 1 hr at 37∘C The plates were washedas before and 100120583L of horseradish peroxidase-conjugatedsheep anti-mouse IgG at a 11000 dilution in PBST was addedand incubated for one hour at 37∘C After washing theplates were incubatedwith chromogensubstrate (100120583LwellABTS peroxidase substrate) and the reaction was terminatedwith peroxidase stop solution (50120583L diluted 1 5) after 5minThe absorbance at 405 nm was monitored with a MUNKmicroplate reader The cutoff point was set as 2SD above themean of control samples

25 DNA Isolation and PCR Analyses Extraction of DNAwas done from formalin-fixed paraffin-embedded (FFPE)blocks and aqueous fluids belong to gerbils and rats FFPEblocks were cut and deparaffinized using Xylol due tostandard protocols Genomic DNA was isolated from indi-vidual samples using QIAamp DNA Mini and Blood MiniHandbook Kit (Qiagen Hilden Germany) according to themanufacturerrsquos instructionThe specific forward primer JW4(51015840-ACTGTCGAGGATGAGCGTGA-31015840) [18] was used withNC2 primer (reverse 51015840-TTAGTTTCTTTTCCTCCGCT-31015840)[11] to amplify partial ITS-1 complete 58S and ITS-2 rDNAof T cati PCR reaction was performed in 10mM Tris-HCLpH 88 15mMMgCl

2 50mMKCl 01 Triton X-100 dNTP

200120583M primers 1120583M and 1U Taq polymerase per 25 120583LThe reaction was carried out under the following conditions94∘C for 45 s 58∘C for 45 s 72∘C for 90s and final extension72∘C for 10min Corresponding PCR products were elec-trophoresed in 14 agarose gel with 05 120583gmL ethidiumbromide and a 100-bp ladder was used as DNA sizemarkerfor estimating the size of the amplicons and photographedusing a gel documentation system (UV Transilluminator)

BioMed Research International 3

Figure 1 The negative reaction in the retina of the infected animals(paraffin section stained H with E)

3 Results

In an attempt to identify the optimal procedure for thediagnosis of ocular Toxocara infection the performanceand agreement among the three different methods namelyhistopathology ELISA and PCR methods were evaluated

31 Pathological Observation Figure 1 corresponds to imageof the ocular structure of an infected animal In none of thestudied animals we observed peripheral granuloma in theposterior pole or retrolental hyaline membranes

32 ELISA for Anti-Toxocara Antibodies The ELISA resultswere positive in sera and aqueous fluid for Mongolian gerbilsand Wistar rats examined at 1 and 8 weeks PI respectivelySpecimens with an OD (optical density) reading greaterthan 0300 and less than 0260 can be interpreted withassurance as positive and negative respectively Caution maybe necessary with specimens with inter with mediate valuesAt the seventh day PI the test was positive in 15 out of 20animals (75 sensitivity) where the animals showed anti-Toxocara antibodies in sera The test was positive at an ODgreater than 0310 and higher in 10 Mongolian gerbils and5 Wistar rats At the sixty-one day PI the test was positivefor aqueous fluid in 11 out of 20 animals (55 sensitivity)where the test was positive at an OD of 0300 or more in nineMongolian gerbils and two Wistar rats (Figure 2)

33 PCR Analysis Analysis of PCR products revealed pos-itive band (660 bp) in the orbital tissue infected Mongoliangerbils at 5 days PI The DNA of T cati was found in the vit-reous fluid of experimentally infected Mongolian gerbils Nopositive band was shown in samples ofWistar rats (Figure 3)

4 Discussion

Toxocariasis is an important infection among socioeconom-ically disadvantaged young children and is low in adultsOcular larva migrans occurs when Toxocara larvae reach eyetissue where they induce inflammatory reaction frequentlywithout the sign or symptoms that accompany the visceraltoxocariasis [19ndash22] The diagnosis of ocular larva migrans is

0

02

04

06

08

1

12

14

5 30 49 70 92

SerumVitreous fluidCut off

Days post infection (DPI)

Opt

ical

den

sity

(OD

)

Figure 2 Development of specific Toxocara cati L2S ES antigens ininfectedMongolian gerbils andWistar rats with 240 and 2500 T catiembryonated eggs respectively OD values of uninfected animalswere lower than the negative cutoff 0260 and those of infectedanimals were higher than positive cutoff 0300

based on retinoscopic and retinographic studies and is con-firmed by the finding of larvae in lesions [23] The presenceof anti-Toxocara antibodies or antigens in vitreous humorhas been reported to be a sensitive method for detectingtoxocariasis in infections or human ocular toxocariasis [25]

In this study we compared the performance of threedifferent diagnostic techniques including histopathologyindirect ELISA and PCR on eyeball and retina tissue duringthe infection of Mongolian gerbils and Wistar rats from 5 to92 days PI

The infection with T cati in the animals not causedimportant histological alternative in cellular structures ofthe eye Histopathologic examination did not detect foreignbodies or tumors in the affected region and the ophthalmicexamination did not suggest an infectious process Serologi-cal testing is the primary diagnostic tool in patient suspectedfor ocular and visceral larvamigrans but these tests are insuf-ficient as cross-reactions may occuring For example cross-reactivity has been reported between T canis and Trichinellaspiralis and T canis and Ascaris lumbricoides [26] Sommer-felt et al (2006) detected specific antibodies against T caniswith ELISA in eighteen pigs at 7 days PI with high titerspersisting over an 84-day period [27] In current study animmunological response in sera was early 7 days PI and wasmaintained throughout the study (92 days) and these resultsare similar to those of other authors Beside from 61 days ofthe infection antibodies in vitreous fluid were detected

An alternative approach to detection of toxocariasis is touse a molecular-based-method such as PCR When PCR wasapplied to eye tissue and aqueous fluid of infectedMongoliangerbils detection of the specific amplification started at 5days PI This was not possible in other stage of infection(30 49 70 and 92 days PI) Our PCR approach in retinademonstrated the presence of the parasite genome beforeone week PI while the histological method was not usefulfor detection of parasite at the retinal and subretinal levels

4 BioMed Research International

660bp 1000 bp500bp

100 bp

(a)

660bp 1000 bp500bp

100 bp

(b)

Figure 3 (a) Analysis of PCR products (eye tissue of infected animals) by 14 agarose gel electrophoresis Lanes 1ndash5 days 5 to 92 PI ofMongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm as positive control lane 12 negativecontrol lane M 100ndash1500 bp DNA size marker (b) DNA amplification of vitreous fluid samples of infected animals by PCR on 14 agarosegel Lanes 1ndash5 days 5 to 92 PI of Mongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm aspositive control lane M 100ndash1500 bp DNA size marker

The PCR in vitreous humor could also be more sensitivethan the detection of antibodies by ELISA in the animalsin early infection since prominent positive serology wasdetectable from 61 days PI This period in animal challengealthough not strictly equivalent to human infection in clinicalcourse could be considered as an acute infection in patientsregarding the parasitic migration and development It seemsthat the transmission of larvae was first from intestine to theliver lung and eye and then 5 days PI they migrated to otherorgans Sometimes larvae return back to the eye to createlesions at the end of migration It has been demonstratedthat Toxocara larvae may migrate and persist in the eye ofexperimental infected animals This opinion is challenged byseveral authors who argued that Toxocara is as a causativeocular toxocariasis [28 29] In a relatively similar studyCho et al (2007) infected Mongolian gerbils with 1000Toxocara ova and reported that 10 of infective larvae werefound before day 5 in liver tissue and also showed thatophthalmoscopically a motile larva was observed in theretina at 14 days PI [30] In earlier studies investigatorsrevealed the parasite DNA by PCR during early infectionperiods in the animals model for Trypanosoma cruzi andToxoplasma gondii thus permitting the diagnosis of infectionearlier than when conventional diagnostic techniques such asmicroscopy or serology are being used [31 32]

5 Conclusion

Histopathology serology (ELISA) and molecular (PCR)methods tested for diagnosis of ocular toxocariasis causedby T cati in experimentally infected Mongolian gerbils andWistar rats revealed that molecular method was the most

sensitive and superior compared to both histopathology andELISA in the early diagnosis of ocular infection The PCRprovided a rapid reliable and sensitive means by which oneestablish the exact etiology of such parasitic infection sothat optimal therapy can be started promptly The superiorperformance of the PCR in detecting infected Mongoliangerbils andWistar rats in this small experimental populationwarrants further evaluation in a larger population of animalmodels and humans naturally infected with T cati

Conflict of Interests

The authors report no conflict of interest in this work

Acknowledgments

The technical assistance of Mrs S Kazemian and MrB Farhang Mehr of the Department of Parasitology andMycology School of Medicine Shiraz University of MedicalSciences Iran is acknowledged with pleasure The criticalcomments of Professor Akao from Section of EnvironmentalParasitology Graduate School of Tokyo Medical and DentalUniversity Japan are gratefully acknowledged This studywas funded by Shiraz University of Medical Sciences ShirazIran Grant no 4108

References

[1] S Azizi A Oryan S M Sadjjadi and M Zibaei ldquoHistopatho-logic changes and larval recovery ofToxocara cati in experimen-tally infected chickensrdquo Parasitology Research vol 102 no 1 pp47ndash52 2007

BioMed Research International 5

[2] D Despommier ldquoToxocariasis clinical aspects epidemiologymedical ecology and molecular aspectsrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 265ndash272 2003

[3] R S Clemett H J Williamson R R Hidajat R A Allardyceand A C Stewart ldquoOcular Toxocara canis infections diagnosisby enzyme immunoassayrdquo Australian and New Zealand Journalof Ophthalmology vol 15 no 2 pp 145ndash150 1987

[4] L deVisser A Rothova J H de Boer et al ldquoDiagnosis of oculartoxocariasis by establishing intraocular antibody productionrdquoAmerican Journal of Ophthalmology vol 145 no 2 pp 369ndash3742008

[5] N Akao and N Ohta ldquoToxocariasis in Japanrdquo ParasitologyInternational vol 56 no 2 pp 87ndash93 2007

[6] M Zibaei S M Sadjjadi and S Uga ldquoExperimental Toxocaracati infection in gerbils and ratsrdquoKorean Journal of Parasitologyvol 48 no 4 pp 331ndash333 2010

[7] T H Dzbenski W Hautz and E Bitkowska ldquoExperimentaltoxocariasis in rabbits immunological markers of ocular infec-tionsrdquoWiadomosci Parazytologiczne vol 47 no 4 pp 591ndash5962001

[8] R Sakai H Kawashima H Shibui K Kamata C Kambaraand H Matsuoka ldquoToxocara cati-induced ocular toxocariasisrdquoArchives of Ophthalmology vol 116 no 12 pp 1686ndash1687 1998

[9] N Cardillo A Rosa M Ribicich C Lopez and I SommerfeltldquoExperimental infection with Toxocara cati in BALB-c micemigratory behaviour and pathological changesrdquo Zoonoses andPublic Health vol 56 no 4 pp 198ndash205 2009

[10] K Taira Y Saitoh and C M O Kapel ldquoToxocara cati larvaepersist and retain high infectivity in muscles of experimentallyinfected chickensrdquo Veterinary Parasitology vol 180 no 3-4 pp287ndash291 2011

[11] X Q Zhu S DrsquoAmelio H W Palm L Paggi M George-Nascimento and R B Gasser ldquoSSCP-based identificationof members within the Pseudoterranova decipiens complex(Nematoda Ascaridoidea Anisakidae) using genetic markersin the internal transcribed spacers of ribosomal DNArdquo Para-sitology vol 124 no 6 pp 615ndash623 2002

[12] A Borecka J Gawor M Niedworok and B Sordyl ldquoDetec-tion of Toxocara canis larvae by PCR in the liver of experi-mentally infected Mongolian gerbils (Meriones unguiculatus)rdquoHelminthologia vol 45 no 3 pp 147ndash149 2008

[13] NAkao TH Takayanagi R Suzuki S Tsukidate andK FujitaldquoOcular larva migrans caused by Toxocara cati in Mongoliangerbils and a comparison of ophthalmologic findings with thoseproduced by T canisrdquo Journal of Parasitology vol 86 no 5 pp1133ndash1135 2000

[14] E Hayashi N Akao and K Fujita ldquoEvidence for the involve-ment of the optic nerve as a migration route for larvae in oculartoxocariasis of Mongolian gerbilsrdquo Journal of Helminthologyvol 77 no 4 pp 311ndash315 2003

[15] S Z Lescano M L Queiroz and P P Chieffi ldquoLarval recoveryof Toxocara canis in organs and tissues of experimentallyinfected Rattus norvegicusrdquo Memorias do Instituto OswaldoCruz vol 99 no 6 pp 627ndash628 2004

[16] M Zibaei S M Sadjjadi S H Jahadi Hosseini and B SarkarildquoA method for accelerating the maturation of Toxocara catieggsrdquo Iranian Journal of Parasitology vol 2 no 1 pp 39ndash422007

[17] D H De Savigny ldquoIn vitro maintenance of Toxocara canislarvae and a simple method for the production of ToxocaraES antigen for use in serodiagnostic tests for visceral larvamigransrdquo Journal of Parasitology vol 61 no 4 pp 781ndash782 1975

[18] M W Li R Q Lin H H Chen R A Sani H Q Song and XQ Zhu ldquoPCR tools for the verification of the specific identityof ascaridoid nematodes from dogs and catsrdquo Molecular andCellular Probes vol 21 no 5-6 pp 349ndash354 2007

[19] R Molk ldquoOcular toxocariasis a review of the literaturerdquoAnnalsof Ophthalmology vol 15 no 3 pp 216ndash231 1983

[20] J A Shields ldquoOcular toxocariasis A reviewrdquo Survey of Ophthal-mology vol 28 no 5 pp 361ndash381 1984

[21] S H Gillespie W J Dinning A Voller and N S CrowcroftldquoThe spectrum of ocular toxocariasisrdquo Eye vol 7 no 3 pp 415ndash418 1993

[22] B Good C V Holland M R H Taylor J Larragy P Moriartyand M OrsquoRegan ldquoOcular toxocariasis in schoolchildrenrdquo Clin-ical Infectious Diseases vol 39 no 2 pp 173ndash178 2004

[23] L I D C Zanandrea G M Oliveira A S Abreu and F EL Pereira ldquoOcular lesions in gerbils (Meriones unguiculatus)infected with low larval burden of Toxocara canis observationsusing indirect binocular ophthalmoscopyrdquoRevista da SociedadeBrasileira deMedicina Tropical vol 41 no 6 pp 570ndash574 2008

[24] J-F Magnaval L Malard B Morassin and R Fabre ldquoImmun-odiagnosis of ocular toxocariasis using Western-blot for thedetection of specific anti-Toxocara IgG and CAPŮ for the mea-surement of specific anti-Toxocara IgErdquo Journal of Helminthol-ogy vol 76 no 4 pp 335ndash339 2002

[25] E Bertelmann K-H Velhagen U Pleyer and C HartmannldquoOcular toxocariasis Diagnostic and therapeutic optionsrdquoOph-thalmologe vol 100 no 11 pp 950ndash954 2003

[26] H Zarnowska and M Jastrzębska ldquoExcretory-secretory larvalantigens of Toxocara canis physico-chemical characteristicsand specificity assayed by Western blot techniquerdquo Acta Para-sitologica vol 39 no 3 pp 41ndash45 1994

[27] I E Sommerfelt G Santillan C Lopez M Ribicich and A JFranco ldquoImmunological and hematological response in exper-imental Toxocara canis-infected pigsrdquo Veterinary Parasitologyvol 96 no 2 pp 127ndash134 2001

[28] T H Takayanagi N Akao R Suzuki M Tomoda S Tsuki-date and K Fujita ldquoNew animal model for human oculartoxocariasis ophthalmoscopic observationrdquo British Journal ofOphthalmology vol 83 no 8 pp 967ndash972 1999

[29] S V Santos S Z Lescano J M Castro and P P Chieffi ldquoLarvalrecovery of Toxocara cati in experimentally infected Rattusnorvegicus and analysis of the rat as potential reservoir for thisascaridrdquoMemorias do InstitutoOswaldo Cruz vol 104 no 6 pp933ndash934 2009

[30] S Cho M Egami H Ohnuki et al ldquoMigration behaviour andpathogenesis of five ascarid nematode species in theMongoliangerbilMeriones unguiculatusrdquo Journal of Helminthology vol 81no 1 pp 43ndash47 2007

[31] L M Weiss S A Udem M Salgo H B Tanowitz and MWittner ldquoSensitive and specific detection of toxoplasma DNAin an experimental murine model use of Toxoplasma gondii-specific cDNA and the polymerase chain reactionrdquo Journal ofInfectious Diseases vol 163 no 1 pp 180ndash186 1991

[32] L V Kirchhoff J R Votava D E Ochs and D R MoserldquoComparison of PCR and microscopic methods for detectingTrypanosoma cruzirdquo Journal of ClinicalMicrobiology vol 34 no5 pp 1171ndash1175 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

Figure 1 The negative reaction in the retina of the infected animals(paraffin section stained H with E)

3 Results

In an attempt to identify the optimal procedure for thediagnosis of ocular Toxocara infection the performanceand agreement among the three different methods namelyhistopathology ELISA and PCR methods were evaluated

31 Pathological Observation Figure 1 corresponds to imageof the ocular structure of an infected animal In none of thestudied animals we observed peripheral granuloma in theposterior pole or retrolental hyaline membranes

32 ELISA for Anti-Toxocara Antibodies The ELISA resultswere positive in sera and aqueous fluid for Mongolian gerbilsand Wistar rats examined at 1 and 8 weeks PI respectivelySpecimens with an OD (optical density) reading greaterthan 0300 and less than 0260 can be interpreted withassurance as positive and negative respectively Caution maybe necessary with specimens with inter with mediate valuesAt the seventh day PI the test was positive in 15 out of 20animals (75 sensitivity) where the animals showed anti-Toxocara antibodies in sera The test was positive at an ODgreater than 0310 and higher in 10 Mongolian gerbils and5 Wistar rats At the sixty-one day PI the test was positivefor aqueous fluid in 11 out of 20 animals (55 sensitivity)where the test was positive at an OD of 0300 or more in nineMongolian gerbils and two Wistar rats (Figure 2)

33 PCR Analysis Analysis of PCR products revealed pos-itive band (660 bp) in the orbital tissue infected Mongoliangerbils at 5 days PI The DNA of T cati was found in the vit-reous fluid of experimentally infected Mongolian gerbils Nopositive band was shown in samples ofWistar rats (Figure 3)

4 Discussion

Toxocariasis is an important infection among socioeconom-ically disadvantaged young children and is low in adultsOcular larva migrans occurs when Toxocara larvae reach eyetissue where they induce inflammatory reaction frequentlywithout the sign or symptoms that accompany the visceraltoxocariasis [19ndash22] The diagnosis of ocular larva migrans is

0

02

04

06

08

1

12

14

5 30 49 70 92

SerumVitreous fluidCut off

Days post infection (DPI)

Opt

ical

den

sity

(OD

)

Figure 2 Development of specific Toxocara cati L2S ES antigens ininfectedMongolian gerbils andWistar rats with 240 and 2500 T catiembryonated eggs respectively OD values of uninfected animalswere lower than the negative cutoff 0260 and those of infectedanimals were higher than positive cutoff 0300

based on retinoscopic and retinographic studies and is con-firmed by the finding of larvae in lesions [23] The presenceof anti-Toxocara antibodies or antigens in vitreous humorhas been reported to be a sensitive method for detectingtoxocariasis in infections or human ocular toxocariasis [25]

In this study we compared the performance of threedifferent diagnostic techniques including histopathologyindirect ELISA and PCR on eyeball and retina tissue duringthe infection of Mongolian gerbils and Wistar rats from 5 to92 days PI

The infection with T cati in the animals not causedimportant histological alternative in cellular structures ofthe eye Histopathologic examination did not detect foreignbodies or tumors in the affected region and the ophthalmicexamination did not suggest an infectious process Serologi-cal testing is the primary diagnostic tool in patient suspectedfor ocular and visceral larvamigrans but these tests are insuf-ficient as cross-reactions may occuring For example cross-reactivity has been reported between T canis and Trichinellaspiralis and T canis and Ascaris lumbricoides [26] Sommer-felt et al (2006) detected specific antibodies against T caniswith ELISA in eighteen pigs at 7 days PI with high titerspersisting over an 84-day period [27] In current study animmunological response in sera was early 7 days PI and wasmaintained throughout the study (92 days) and these resultsare similar to those of other authors Beside from 61 days ofthe infection antibodies in vitreous fluid were detected

An alternative approach to detection of toxocariasis is touse a molecular-based-method such as PCR When PCR wasapplied to eye tissue and aqueous fluid of infectedMongoliangerbils detection of the specific amplification started at 5days PI This was not possible in other stage of infection(30 49 70 and 92 days PI) Our PCR approach in retinademonstrated the presence of the parasite genome beforeone week PI while the histological method was not usefulfor detection of parasite at the retinal and subretinal levels

4 BioMed Research International

660bp 1000 bp500bp

100 bp

(a)

660bp 1000 bp500bp

100 bp

(b)

Figure 3 (a) Analysis of PCR products (eye tissue of infected animals) by 14 agarose gel electrophoresis Lanes 1ndash5 days 5 to 92 PI ofMongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm as positive control lane 12 negativecontrol lane M 100ndash1500 bp DNA size marker (b) DNA amplification of vitreous fluid samples of infected animals by PCR on 14 agarosegel Lanes 1ndash5 days 5 to 92 PI of Mongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm aspositive control lane M 100ndash1500 bp DNA size marker

The PCR in vitreous humor could also be more sensitivethan the detection of antibodies by ELISA in the animalsin early infection since prominent positive serology wasdetectable from 61 days PI This period in animal challengealthough not strictly equivalent to human infection in clinicalcourse could be considered as an acute infection in patientsregarding the parasitic migration and development It seemsthat the transmission of larvae was first from intestine to theliver lung and eye and then 5 days PI they migrated to otherorgans Sometimes larvae return back to the eye to createlesions at the end of migration It has been demonstratedthat Toxocara larvae may migrate and persist in the eye ofexperimental infected animals This opinion is challenged byseveral authors who argued that Toxocara is as a causativeocular toxocariasis [28 29] In a relatively similar studyCho et al (2007) infected Mongolian gerbils with 1000Toxocara ova and reported that 10 of infective larvae werefound before day 5 in liver tissue and also showed thatophthalmoscopically a motile larva was observed in theretina at 14 days PI [30] In earlier studies investigatorsrevealed the parasite DNA by PCR during early infectionperiods in the animals model for Trypanosoma cruzi andToxoplasma gondii thus permitting the diagnosis of infectionearlier than when conventional diagnostic techniques such asmicroscopy or serology are being used [31 32]

5 Conclusion

Histopathology serology (ELISA) and molecular (PCR)methods tested for diagnosis of ocular toxocariasis causedby T cati in experimentally infected Mongolian gerbils andWistar rats revealed that molecular method was the most

sensitive and superior compared to both histopathology andELISA in the early diagnosis of ocular infection The PCRprovided a rapid reliable and sensitive means by which oneestablish the exact etiology of such parasitic infection sothat optimal therapy can be started promptly The superiorperformance of the PCR in detecting infected Mongoliangerbils andWistar rats in this small experimental populationwarrants further evaluation in a larger population of animalmodels and humans naturally infected with T cati

Conflict of Interests

The authors report no conflict of interest in this work

Acknowledgments

The technical assistance of Mrs S Kazemian and MrB Farhang Mehr of the Department of Parasitology andMycology School of Medicine Shiraz University of MedicalSciences Iran is acknowledged with pleasure The criticalcomments of Professor Akao from Section of EnvironmentalParasitology Graduate School of Tokyo Medical and DentalUniversity Japan are gratefully acknowledged This studywas funded by Shiraz University of Medical Sciences ShirazIran Grant no 4108

References

[1] S Azizi A Oryan S M Sadjjadi and M Zibaei ldquoHistopatho-logic changes and larval recovery ofToxocara cati in experimen-tally infected chickensrdquo Parasitology Research vol 102 no 1 pp47ndash52 2007

BioMed Research International 5

[2] D Despommier ldquoToxocariasis clinical aspects epidemiologymedical ecology and molecular aspectsrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 265ndash272 2003

[3] R S Clemett H J Williamson R R Hidajat R A Allardyceand A C Stewart ldquoOcular Toxocara canis infections diagnosisby enzyme immunoassayrdquo Australian and New Zealand Journalof Ophthalmology vol 15 no 2 pp 145ndash150 1987

[4] L deVisser A Rothova J H de Boer et al ldquoDiagnosis of oculartoxocariasis by establishing intraocular antibody productionrdquoAmerican Journal of Ophthalmology vol 145 no 2 pp 369ndash3742008

[5] N Akao and N Ohta ldquoToxocariasis in Japanrdquo ParasitologyInternational vol 56 no 2 pp 87ndash93 2007

[6] M Zibaei S M Sadjjadi and S Uga ldquoExperimental Toxocaracati infection in gerbils and ratsrdquoKorean Journal of Parasitologyvol 48 no 4 pp 331ndash333 2010

[7] T H Dzbenski W Hautz and E Bitkowska ldquoExperimentaltoxocariasis in rabbits immunological markers of ocular infec-tionsrdquoWiadomosci Parazytologiczne vol 47 no 4 pp 591ndash5962001

[8] R Sakai H Kawashima H Shibui K Kamata C Kambaraand H Matsuoka ldquoToxocara cati-induced ocular toxocariasisrdquoArchives of Ophthalmology vol 116 no 12 pp 1686ndash1687 1998

[9] N Cardillo A Rosa M Ribicich C Lopez and I SommerfeltldquoExperimental infection with Toxocara cati in BALB-c micemigratory behaviour and pathological changesrdquo Zoonoses andPublic Health vol 56 no 4 pp 198ndash205 2009

[10] K Taira Y Saitoh and C M O Kapel ldquoToxocara cati larvaepersist and retain high infectivity in muscles of experimentallyinfected chickensrdquo Veterinary Parasitology vol 180 no 3-4 pp287ndash291 2011

[11] X Q Zhu S DrsquoAmelio H W Palm L Paggi M George-Nascimento and R B Gasser ldquoSSCP-based identificationof members within the Pseudoterranova decipiens complex(Nematoda Ascaridoidea Anisakidae) using genetic markersin the internal transcribed spacers of ribosomal DNArdquo Para-sitology vol 124 no 6 pp 615ndash623 2002

[12] A Borecka J Gawor M Niedworok and B Sordyl ldquoDetec-tion of Toxocara canis larvae by PCR in the liver of experi-mentally infected Mongolian gerbils (Meriones unguiculatus)rdquoHelminthologia vol 45 no 3 pp 147ndash149 2008

[13] NAkao TH Takayanagi R Suzuki S Tsukidate andK FujitaldquoOcular larva migrans caused by Toxocara cati in Mongoliangerbils and a comparison of ophthalmologic findings with thoseproduced by T canisrdquo Journal of Parasitology vol 86 no 5 pp1133ndash1135 2000

[14] E Hayashi N Akao and K Fujita ldquoEvidence for the involve-ment of the optic nerve as a migration route for larvae in oculartoxocariasis of Mongolian gerbilsrdquo Journal of Helminthologyvol 77 no 4 pp 311ndash315 2003

[15] S Z Lescano M L Queiroz and P P Chieffi ldquoLarval recoveryof Toxocara canis in organs and tissues of experimentallyinfected Rattus norvegicusrdquo Memorias do Instituto OswaldoCruz vol 99 no 6 pp 627ndash628 2004

[16] M Zibaei S M Sadjjadi S H Jahadi Hosseini and B SarkarildquoA method for accelerating the maturation of Toxocara catieggsrdquo Iranian Journal of Parasitology vol 2 no 1 pp 39ndash422007

[17] D H De Savigny ldquoIn vitro maintenance of Toxocara canislarvae and a simple method for the production of ToxocaraES antigen for use in serodiagnostic tests for visceral larvamigransrdquo Journal of Parasitology vol 61 no 4 pp 781ndash782 1975

[18] M W Li R Q Lin H H Chen R A Sani H Q Song and XQ Zhu ldquoPCR tools for the verification of the specific identityof ascaridoid nematodes from dogs and catsrdquo Molecular andCellular Probes vol 21 no 5-6 pp 349ndash354 2007

[19] R Molk ldquoOcular toxocariasis a review of the literaturerdquoAnnalsof Ophthalmology vol 15 no 3 pp 216ndash231 1983

[20] J A Shields ldquoOcular toxocariasis A reviewrdquo Survey of Ophthal-mology vol 28 no 5 pp 361ndash381 1984

[21] S H Gillespie W J Dinning A Voller and N S CrowcroftldquoThe spectrum of ocular toxocariasisrdquo Eye vol 7 no 3 pp 415ndash418 1993

[22] B Good C V Holland M R H Taylor J Larragy P Moriartyand M OrsquoRegan ldquoOcular toxocariasis in schoolchildrenrdquo Clin-ical Infectious Diseases vol 39 no 2 pp 173ndash178 2004

[23] L I D C Zanandrea G M Oliveira A S Abreu and F EL Pereira ldquoOcular lesions in gerbils (Meriones unguiculatus)infected with low larval burden of Toxocara canis observationsusing indirect binocular ophthalmoscopyrdquoRevista da SociedadeBrasileira deMedicina Tropical vol 41 no 6 pp 570ndash574 2008

[24] J-F Magnaval L Malard B Morassin and R Fabre ldquoImmun-odiagnosis of ocular toxocariasis using Western-blot for thedetection of specific anti-Toxocara IgG and CAPŮ for the mea-surement of specific anti-Toxocara IgErdquo Journal of Helminthol-ogy vol 76 no 4 pp 335ndash339 2002

[25] E Bertelmann K-H Velhagen U Pleyer and C HartmannldquoOcular toxocariasis Diagnostic and therapeutic optionsrdquoOph-thalmologe vol 100 no 11 pp 950ndash954 2003

[26] H Zarnowska and M Jastrzębska ldquoExcretory-secretory larvalantigens of Toxocara canis physico-chemical characteristicsand specificity assayed by Western blot techniquerdquo Acta Para-sitologica vol 39 no 3 pp 41ndash45 1994

[27] I E Sommerfelt G Santillan C Lopez M Ribicich and A JFranco ldquoImmunological and hematological response in exper-imental Toxocara canis-infected pigsrdquo Veterinary Parasitologyvol 96 no 2 pp 127ndash134 2001

[28] T H Takayanagi N Akao R Suzuki M Tomoda S Tsuki-date and K Fujita ldquoNew animal model for human oculartoxocariasis ophthalmoscopic observationrdquo British Journal ofOphthalmology vol 83 no 8 pp 967ndash972 1999

[29] S V Santos S Z Lescano J M Castro and P P Chieffi ldquoLarvalrecovery of Toxocara cati in experimentally infected Rattusnorvegicus and analysis of the rat as potential reservoir for thisascaridrdquoMemorias do InstitutoOswaldo Cruz vol 104 no 6 pp933ndash934 2009

[30] S Cho M Egami H Ohnuki et al ldquoMigration behaviour andpathogenesis of five ascarid nematode species in theMongoliangerbilMeriones unguiculatusrdquo Journal of Helminthology vol 81no 1 pp 43ndash47 2007

[31] L M Weiss S A Udem M Salgo H B Tanowitz and MWittner ldquoSensitive and specific detection of toxoplasma DNAin an experimental murine model use of Toxoplasma gondii-specific cDNA and the polymerase chain reactionrdquo Journal ofInfectious Diseases vol 163 no 1 pp 180ndash186 1991

[32] L V Kirchhoff J R Votava D E Ochs and D R MoserldquoComparison of PCR and microscopic methods for detectingTrypanosoma cruzirdquo Journal of ClinicalMicrobiology vol 34 no5 pp 1171ndash1175 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

660bp 1000 bp500bp

100 bp

(a)

660bp 1000 bp500bp

100 bp

(b)

Figure 3 (a) Analysis of PCR products (eye tissue of infected animals) by 14 agarose gel electrophoresis Lanes 1ndash5 days 5 to 92 PI ofMongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm as positive control lane 12 negativecontrol lane M 100ndash1500 bp DNA size marker (b) DNA amplification of vitreous fluid samples of infected animals by PCR on 14 agarosegel Lanes 1ndash5 days 5 to 92 PI of Mongolian gerbils lanes 6ndash10 days 5 to 92 PI of Wistar rats lane 11 DNA of Toxocara cati adult worm aspositive control lane M 100ndash1500 bp DNA size marker

The PCR in vitreous humor could also be more sensitivethan the detection of antibodies by ELISA in the animalsin early infection since prominent positive serology wasdetectable from 61 days PI This period in animal challengealthough not strictly equivalent to human infection in clinicalcourse could be considered as an acute infection in patientsregarding the parasitic migration and development It seemsthat the transmission of larvae was first from intestine to theliver lung and eye and then 5 days PI they migrated to otherorgans Sometimes larvae return back to the eye to createlesions at the end of migration It has been demonstratedthat Toxocara larvae may migrate and persist in the eye ofexperimental infected animals This opinion is challenged byseveral authors who argued that Toxocara is as a causativeocular toxocariasis [28 29] In a relatively similar studyCho et al (2007) infected Mongolian gerbils with 1000Toxocara ova and reported that 10 of infective larvae werefound before day 5 in liver tissue and also showed thatophthalmoscopically a motile larva was observed in theretina at 14 days PI [30] In earlier studies investigatorsrevealed the parasite DNA by PCR during early infectionperiods in the animals model for Trypanosoma cruzi andToxoplasma gondii thus permitting the diagnosis of infectionearlier than when conventional diagnostic techniques such asmicroscopy or serology are being used [31 32]

5 Conclusion

Histopathology serology (ELISA) and molecular (PCR)methods tested for diagnosis of ocular toxocariasis causedby T cati in experimentally infected Mongolian gerbils andWistar rats revealed that molecular method was the most

sensitive and superior compared to both histopathology andELISA in the early diagnosis of ocular infection The PCRprovided a rapid reliable and sensitive means by which oneestablish the exact etiology of such parasitic infection sothat optimal therapy can be started promptly The superiorperformance of the PCR in detecting infected Mongoliangerbils andWistar rats in this small experimental populationwarrants further evaluation in a larger population of animalmodels and humans naturally infected with T cati

Conflict of Interests

The authors report no conflict of interest in this work

Acknowledgments

The technical assistance of Mrs S Kazemian and MrB Farhang Mehr of the Department of Parasitology andMycology School of Medicine Shiraz University of MedicalSciences Iran is acknowledged with pleasure The criticalcomments of Professor Akao from Section of EnvironmentalParasitology Graduate School of Tokyo Medical and DentalUniversity Japan are gratefully acknowledged This studywas funded by Shiraz University of Medical Sciences ShirazIran Grant no 4108

References

[1] S Azizi A Oryan S M Sadjjadi and M Zibaei ldquoHistopatho-logic changes and larval recovery ofToxocara cati in experimen-tally infected chickensrdquo Parasitology Research vol 102 no 1 pp47ndash52 2007

BioMed Research International 5

[2] D Despommier ldquoToxocariasis clinical aspects epidemiologymedical ecology and molecular aspectsrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 265ndash272 2003

[3] R S Clemett H J Williamson R R Hidajat R A Allardyceand A C Stewart ldquoOcular Toxocara canis infections diagnosisby enzyme immunoassayrdquo Australian and New Zealand Journalof Ophthalmology vol 15 no 2 pp 145ndash150 1987

[4] L deVisser A Rothova J H de Boer et al ldquoDiagnosis of oculartoxocariasis by establishing intraocular antibody productionrdquoAmerican Journal of Ophthalmology vol 145 no 2 pp 369ndash3742008

[5] N Akao and N Ohta ldquoToxocariasis in Japanrdquo ParasitologyInternational vol 56 no 2 pp 87ndash93 2007

[6] M Zibaei S M Sadjjadi and S Uga ldquoExperimental Toxocaracati infection in gerbils and ratsrdquoKorean Journal of Parasitologyvol 48 no 4 pp 331ndash333 2010

[7] T H Dzbenski W Hautz and E Bitkowska ldquoExperimentaltoxocariasis in rabbits immunological markers of ocular infec-tionsrdquoWiadomosci Parazytologiczne vol 47 no 4 pp 591ndash5962001

[8] R Sakai H Kawashima H Shibui K Kamata C Kambaraand H Matsuoka ldquoToxocara cati-induced ocular toxocariasisrdquoArchives of Ophthalmology vol 116 no 12 pp 1686ndash1687 1998

[9] N Cardillo A Rosa M Ribicich C Lopez and I SommerfeltldquoExperimental infection with Toxocara cati in BALB-c micemigratory behaviour and pathological changesrdquo Zoonoses andPublic Health vol 56 no 4 pp 198ndash205 2009

[10] K Taira Y Saitoh and C M O Kapel ldquoToxocara cati larvaepersist and retain high infectivity in muscles of experimentallyinfected chickensrdquo Veterinary Parasitology vol 180 no 3-4 pp287ndash291 2011

[11] X Q Zhu S DrsquoAmelio H W Palm L Paggi M George-Nascimento and R B Gasser ldquoSSCP-based identificationof members within the Pseudoterranova decipiens complex(Nematoda Ascaridoidea Anisakidae) using genetic markersin the internal transcribed spacers of ribosomal DNArdquo Para-sitology vol 124 no 6 pp 615ndash623 2002

[12] A Borecka J Gawor M Niedworok and B Sordyl ldquoDetec-tion of Toxocara canis larvae by PCR in the liver of experi-mentally infected Mongolian gerbils (Meriones unguiculatus)rdquoHelminthologia vol 45 no 3 pp 147ndash149 2008

[13] NAkao TH Takayanagi R Suzuki S Tsukidate andK FujitaldquoOcular larva migrans caused by Toxocara cati in Mongoliangerbils and a comparison of ophthalmologic findings with thoseproduced by T canisrdquo Journal of Parasitology vol 86 no 5 pp1133ndash1135 2000

[14] E Hayashi N Akao and K Fujita ldquoEvidence for the involve-ment of the optic nerve as a migration route for larvae in oculartoxocariasis of Mongolian gerbilsrdquo Journal of Helminthologyvol 77 no 4 pp 311ndash315 2003

[15] S Z Lescano M L Queiroz and P P Chieffi ldquoLarval recoveryof Toxocara canis in organs and tissues of experimentallyinfected Rattus norvegicusrdquo Memorias do Instituto OswaldoCruz vol 99 no 6 pp 627ndash628 2004

[16] M Zibaei S M Sadjjadi S H Jahadi Hosseini and B SarkarildquoA method for accelerating the maturation of Toxocara catieggsrdquo Iranian Journal of Parasitology vol 2 no 1 pp 39ndash422007

[17] D H De Savigny ldquoIn vitro maintenance of Toxocara canislarvae and a simple method for the production of ToxocaraES antigen for use in serodiagnostic tests for visceral larvamigransrdquo Journal of Parasitology vol 61 no 4 pp 781ndash782 1975

[18] M W Li R Q Lin H H Chen R A Sani H Q Song and XQ Zhu ldquoPCR tools for the verification of the specific identityof ascaridoid nematodes from dogs and catsrdquo Molecular andCellular Probes vol 21 no 5-6 pp 349ndash354 2007

[19] R Molk ldquoOcular toxocariasis a review of the literaturerdquoAnnalsof Ophthalmology vol 15 no 3 pp 216ndash231 1983

[20] J A Shields ldquoOcular toxocariasis A reviewrdquo Survey of Ophthal-mology vol 28 no 5 pp 361ndash381 1984

[21] S H Gillespie W J Dinning A Voller and N S CrowcroftldquoThe spectrum of ocular toxocariasisrdquo Eye vol 7 no 3 pp 415ndash418 1993

[22] B Good C V Holland M R H Taylor J Larragy P Moriartyand M OrsquoRegan ldquoOcular toxocariasis in schoolchildrenrdquo Clin-ical Infectious Diseases vol 39 no 2 pp 173ndash178 2004

[23] L I D C Zanandrea G M Oliveira A S Abreu and F EL Pereira ldquoOcular lesions in gerbils (Meriones unguiculatus)infected with low larval burden of Toxocara canis observationsusing indirect binocular ophthalmoscopyrdquoRevista da SociedadeBrasileira deMedicina Tropical vol 41 no 6 pp 570ndash574 2008

[24] J-F Magnaval L Malard B Morassin and R Fabre ldquoImmun-odiagnosis of ocular toxocariasis using Western-blot for thedetection of specific anti-Toxocara IgG and CAPŮ for the mea-surement of specific anti-Toxocara IgErdquo Journal of Helminthol-ogy vol 76 no 4 pp 335ndash339 2002

[25] E Bertelmann K-H Velhagen U Pleyer and C HartmannldquoOcular toxocariasis Diagnostic and therapeutic optionsrdquoOph-thalmologe vol 100 no 11 pp 950ndash954 2003

[26] H Zarnowska and M Jastrzębska ldquoExcretory-secretory larvalantigens of Toxocara canis physico-chemical characteristicsand specificity assayed by Western blot techniquerdquo Acta Para-sitologica vol 39 no 3 pp 41ndash45 1994

[27] I E Sommerfelt G Santillan C Lopez M Ribicich and A JFranco ldquoImmunological and hematological response in exper-imental Toxocara canis-infected pigsrdquo Veterinary Parasitologyvol 96 no 2 pp 127ndash134 2001

[28] T H Takayanagi N Akao R Suzuki M Tomoda S Tsuki-date and K Fujita ldquoNew animal model for human oculartoxocariasis ophthalmoscopic observationrdquo British Journal ofOphthalmology vol 83 no 8 pp 967ndash972 1999

[29] S V Santos S Z Lescano J M Castro and P P Chieffi ldquoLarvalrecovery of Toxocara cati in experimentally infected Rattusnorvegicus and analysis of the rat as potential reservoir for thisascaridrdquoMemorias do InstitutoOswaldo Cruz vol 104 no 6 pp933ndash934 2009

[30] S Cho M Egami H Ohnuki et al ldquoMigration behaviour andpathogenesis of five ascarid nematode species in theMongoliangerbilMeriones unguiculatusrdquo Journal of Helminthology vol 81no 1 pp 43ndash47 2007

[31] L M Weiss S A Udem M Salgo H B Tanowitz and MWittner ldquoSensitive and specific detection of toxoplasma DNAin an experimental murine model use of Toxoplasma gondii-specific cDNA and the polymerase chain reactionrdquo Journal ofInfectious Diseases vol 163 no 1 pp 180ndash186 1991

[32] L V Kirchhoff J R Votava D E Ochs and D R MoserldquoComparison of PCR and microscopic methods for detectingTrypanosoma cruzirdquo Journal of ClinicalMicrobiology vol 34 no5 pp 1171ndash1175 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

[2] D Despommier ldquoToxocariasis clinical aspects epidemiologymedical ecology and molecular aspectsrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 265ndash272 2003

[3] R S Clemett H J Williamson R R Hidajat R A Allardyceand A C Stewart ldquoOcular Toxocara canis infections diagnosisby enzyme immunoassayrdquo Australian and New Zealand Journalof Ophthalmology vol 15 no 2 pp 145ndash150 1987

[4] L deVisser A Rothova J H de Boer et al ldquoDiagnosis of oculartoxocariasis by establishing intraocular antibody productionrdquoAmerican Journal of Ophthalmology vol 145 no 2 pp 369ndash3742008

[5] N Akao and N Ohta ldquoToxocariasis in Japanrdquo ParasitologyInternational vol 56 no 2 pp 87ndash93 2007

[6] M Zibaei S M Sadjjadi and S Uga ldquoExperimental Toxocaracati infection in gerbils and ratsrdquoKorean Journal of Parasitologyvol 48 no 4 pp 331ndash333 2010

[7] T H Dzbenski W Hautz and E Bitkowska ldquoExperimentaltoxocariasis in rabbits immunological markers of ocular infec-tionsrdquoWiadomosci Parazytologiczne vol 47 no 4 pp 591ndash5962001

[8] R Sakai H Kawashima H Shibui K Kamata C Kambaraand H Matsuoka ldquoToxocara cati-induced ocular toxocariasisrdquoArchives of Ophthalmology vol 116 no 12 pp 1686ndash1687 1998

[9] N Cardillo A Rosa M Ribicich C Lopez and I SommerfeltldquoExperimental infection with Toxocara cati in BALB-c micemigratory behaviour and pathological changesrdquo Zoonoses andPublic Health vol 56 no 4 pp 198ndash205 2009

[10] K Taira Y Saitoh and C M O Kapel ldquoToxocara cati larvaepersist and retain high infectivity in muscles of experimentallyinfected chickensrdquo Veterinary Parasitology vol 180 no 3-4 pp287ndash291 2011

[11] X Q Zhu S DrsquoAmelio H W Palm L Paggi M George-Nascimento and R B Gasser ldquoSSCP-based identificationof members within the Pseudoterranova decipiens complex(Nematoda Ascaridoidea Anisakidae) using genetic markersin the internal transcribed spacers of ribosomal DNArdquo Para-sitology vol 124 no 6 pp 615ndash623 2002

[12] A Borecka J Gawor M Niedworok and B Sordyl ldquoDetec-tion of Toxocara canis larvae by PCR in the liver of experi-mentally infected Mongolian gerbils (Meriones unguiculatus)rdquoHelminthologia vol 45 no 3 pp 147ndash149 2008

[13] NAkao TH Takayanagi R Suzuki S Tsukidate andK FujitaldquoOcular larva migrans caused by Toxocara cati in Mongoliangerbils and a comparison of ophthalmologic findings with thoseproduced by T canisrdquo Journal of Parasitology vol 86 no 5 pp1133ndash1135 2000

[14] E Hayashi N Akao and K Fujita ldquoEvidence for the involve-ment of the optic nerve as a migration route for larvae in oculartoxocariasis of Mongolian gerbilsrdquo Journal of Helminthologyvol 77 no 4 pp 311ndash315 2003

[15] S Z Lescano M L Queiroz and P P Chieffi ldquoLarval recoveryof Toxocara canis in organs and tissues of experimentallyinfected Rattus norvegicusrdquo Memorias do Instituto OswaldoCruz vol 99 no 6 pp 627ndash628 2004

[16] M Zibaei S M Sadjjadi S H Jahadi Hosseini and B SarkarildquoA method for accelerating the maturation of Toxocara catieggsrdquo Iranian Journal of Parasitology vol 2 no 1 pp 39ndash422007

[17] D H De Savigny ldquoIn vitro maintenance of Toxocara canislarvae and a simple method for the production of ToxocaraES antigen for use in serodiagnostic tests for visceral larvamigransrdquo Journal of Parasitology vol 61 no 4 pp 781ndash782 1975

[18] M W Li R Q Lin H H Chen R A Sani H Q Song and XQ Zhu ldquoPCR tools for the verification of the specific identityof ascaridoid nematodes from dogs and catsrdquo Molecular andCellular Probes vol 21 no 5-6 pp 349ndash354 2007

[19] R Molk ldquoOcular toxocariasis a review of the literaturerdquoAnnalsof Ophthalmology vol 15 no 3 pp 216ndash231 1983

[20] J A Shields ldquoOcular toxocariasis A reviewrdquo Survey of Ophthal-mology vol 28 no 5 pp 361ndash381 1984

[21] S H Gillespie W J Dinning A Voller and N S CrowcroftldquoThe spectrum of ocular toxocariasisrdquo Eye vol 7 no 3 pp 415ndash418 1993

[22] B Good C V Holland M R H Taylor J Larragy P Moriartyand M OrsquoRegan ldquoOcular toxocariasis in schoolchildrenrdquo Clin-ical Infectious Diseases vol 39 no 2 pp 173ndash178 2004

[23] L I D C Zanandrea G M Oliveira A S Abreu and F EL Pereira ldquoOcular lesions in gerbils (Meriones unguiculatus)infected with low larval burden of Toxocara canis observationsusing indirect binocular ophthalmoscopyrdquoRevista da SociedadeBrasileira deMedicina Tropical vol 41 no 6 pp 570ndash574 2008

[24] J-F Magnaval L Malard B Morassin and R Fabre ldquoImmun-odiagnosis of ocular toxocariasis using Western-blot for thedetection of specific anti-Toxocara IgG and CAPŮ for the mea-surement of specific anti-Toxocara IgErdquo Journal of Helminthol-ogy vol 76 no 4 pp 335ndash339 2002

[25] E Bertelmann K-H Velhagen U Pleyer and C HartmannldquoOcular toxocariasis Diagnostic and therapeutic optionsrdquoOph-thalmologe vol 100 no 11 pp 950ndash954 2003

[26] H Zarnowska and M Jastrzębska ldquoExcretory-secretory larvalantigens of Toxocara canis physico-chemical characteristicsand specificity assayed by Western blot techniquerdquo Acta Para-sitologica vol 39 no 3 pp 41ndash45 1994

[27] I E Sommerfelt G Santillan C Lopez M Ribicich and A JFranco ldquoImmunological and hematological response in exper-imental Toxocara canis-infected pigsrdquo Veterinary Parasitologyvol 96 no 2 pp 127ndash134 2001

[28] T H Takayanagi N Akao R Suzuki M Tomoda S Tsuki-date and K Fujita ldquoNew animal model for human oculartoxocariasis ophthalmoscopic observationrdquo British Journal ofOphthalmology vol 83 no 8 pp 967ndash972 1999

[29] S V Santos S Z Lescano J M Castro and P P Chieffi ldquoLarvalrecovery of Toxocara cati in experimentally infected Rattusnorvegicus and analysis of the rat as potential reservoir for thisascaridrdquoMemorias do InstitutoOswaldo Cruz vol 104 no 6 pp933ndash934 2009

[30] S Cho M Egami H Ohnuki et al ldquoMigration behaviour andpathogenesis of five ascarid nematode species in theMongoliangerbilMeriones unguiculatusrdquo Journal of Helminthology vol 81no 1 pp 43ndash47 2007

[31] L M Weiss S A Udem M Salgo H B Tanowitz and MWittner ldquoSensitive and specific detection of toxoplasma DNAin an experimental murine model use of Toxoplasma gondii-specific cDNA and the polymerase chain reactionrdquo Journal ofInfectious Diseases vol 163 no 1 pp 180ndash186 1991

[32] L V Kirchhoff J R Votava D E Ochs and D R MoserldquoComparison of PCR and microscopic methods for detectingTrypanosoma cruzirdquo Journal of ClinicalMicrobiology vol 34 no5 pp 1171ndash1175 1996

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology