Research note

Visceral larva migrans: migratory pattern of Toxocara canisin pigs

Anne B. Helwigha, *, Peter Lindb, Peter Nansena

aDanish Centre for Experimental Parasitology, The Royal Veterinary and Agricultural University, Ridebanevej 3, DK-1870

Frederiksberg C, DenmarkbDanish Veterinary Laboratory, Department of Immunology and Biochemistry, BuÈlowsvej 27, DK-1870 Frederiksberg C, Denmark

Received 24 September 1998; received in revised form 22 December 1998; accepted 22 December 1998

Abstract

The migratory pattern of Toxocara canis was investigated following infection of pigs with 60 000 infective eggs.Groups of six pigs were slaughtered at 7, 14 and 28 days after infection (p.i.), and the number of larvae in selectedorgans and muscles was determined by digestion. A group of uninfected pigs was used as negative controls for

blood parameters and weight gain. Toxocara canis migrated well in the pig, although the relative numbers of larvaerecovered decreased signi®cantly during the experiment. On day 7 p.i., high numbers of larvae were recovered fromthe lymph nodes around the small intestine and to some extent also from the lymph nodes around the large

intestine, and from the lungs and the liver. On day 14, the majority of larvae were recovered from the lungs and thelymph nodes around the small intestine, and by day 28 p.i. most larvae were found in the lungs. Larvae wererecovered from the brain on days 14 and 21, with a maximum on day 14 p.i. No larvae were found in the eyes.

Severe pathological changes were observed in the liver and lungs, especially on day 14 p.i.; also, development ofgranulomas was observed in the kidneys. Finally, a strong speci®c antibody response towards T. canis L2/L3 ESproducts was observed from day 14 p.i. until termination of the experiment, and the maximum eosinophil responsewas observed 14 days p.i. The pig is a useful non-primate model for human visceral larva migrans, since T. canis

migrate well and induce a strong immunological response in the pig. However, the importance of the pig as aparatenic host is probably minor, because of the relatively early death of most of the larvae. # 1999 AustralianSociety for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Migration pattern; Pig; Toxocara canis; Visceral larva migrans

1. Introduction

It is well known that Toxocara canis larvae

migrate in paratenic hosts, including humans [1, 2]

where it may cause visceral larva migrans [3] andocular larva migrans [4]. Treatment of toxocaria-sis in humans by use of anthelmintics is unsatis-factory, as symptoms may continue for longperiods after treatment [5] and, in the case ofocular larva migrans, the patient is at risk of los-ing the sight of the a�ected eye. Most serologicalstudies on T. canis in humans have shown that

International Journal for Parasitology 29 (1999) 559±565

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(99 )00007-7

* Corresponding author. Fax: +45-35-28-27-74; e-mail:

abh@kvl.dk

2±18% of the general population have positiveantibody reactions against T. canis antigen usingthe ELISA [6±8]. However, the number of casesdiagnosed as larva migrans is much lower thanexpected from these prevalences, indicating thatonly a fraction of infections leads to clinicalsigns, and that hitherto unknown aspects of bothparasite migratory behaviour and host responsemay determine the severity of symptoms. Thus, agood animal model is required for experimentalinvestigations on migrating larvae in humans.Until today, mice have been used most often as amodel for human visceral larvae migrans [9, 10].The larvae can survive for a long period in thisparatenic host [10±12], even for as long as1 year [13]. However, several authors havepointed out that the pig is probably the mostsuitable non-primate model to use in humanresearch because of the many immunological,physiological and biochemical similarities [14±16].Serological investigations in Britain have shownthat the pig serves as a paratenic host for T.canis, with 4.5% of the pigs having antibodiesagainst T. canis [17]. Therefore, it may be ofgreat importance to investigate the migratorybehaviour of T. canis larvae in the pig to eluci-date relevant of the host±parasite relationshipswith special reference to toxocariasis in humans.Hence, it was decided to investigate the mi-gratory behaviour of T. canis larvae in the earlyphase of the infection in pigs on a larger scaleafter a single medium-dose infection.

Twenty-four helminth-naive castrated maleDanish Landrace±Yorkshire±Duroc crossbredpigs were housed under parasite-free conditionsin elevated pens. The pigs had free access towater and were fed twice daily with a standarddiet consisting of ground barley with proteinsupplement [18]. At the start of the experimentthe pigs were 8±10-weeks old and weighed onaverage 21.824.8 kg.

Toxocara canis eggs were isolated from stoolsof naturally infected dogs at a Danish kennel.The eggs were embryonated in 0.1 N-H2SO4 andkept in the dark at room temperature for3 months. The embryonation of the eggs was fol-lowed using a light microscope, and the infectiv-ity of the T. canis eggs was tested prior to the

experiments by inoculating two pigs with 50 000eggs each. The pigs were slaughtered 7 days laterand severe liver lesions were observed. Further,larvae were recovered from both liver and lungsby the agar-gel method described by Slotved etal. [19] and the Baermann method described byEriksen et al. [20]. Both methods involve anactive migration of the larvae.

Eighteen pigs were each inoculated with 60 000infective T. canis eggs by use of a stomach tube,and three pigs were not infected and served asnegative controls for blood tests. On days 7, 14and 28 p.i., groups of six infected pigs were killedwith a captive pistol and exsanguinated; the threeuninfected pigs were not slaughtered.

Faecal egg counts were estimated using a con-centration McMaster technique as described byRoepstor� and Nansen [21], with a sensitivitylevel of 20 eggs per gram of faeces (EPG). Atslaughter, the pigs were exsanguinated and thesmall intestine, liver, lungs, brain, kidney, dia-phragm, masseter, tongue, heart, eyes and thelymph nodes around the small and large intes-tines were removed for further investigation. Theliver, lungs and kidneys of all pigs were examinedfor macroscopic lesions, i.e. the degree of ®brosiswas recorded and the numbers of granulation-tis-sue type white spots [22] were recorded. On day21 p.i. the retinas of the eyes of the pigs wereexamined for lesions by using a ophthalmoscope.The pigs were weighed at the start of the exper-iment and at slaughter. Faecal egg count examin-ations were carried out at weekly intervals tocertify that no unintended parasite infection hadoccurred. Blood samples were taken once a weekto measure eosinophil counts and antibody re-sponse.

The samples, except for the lymph nodes andcontents of the small intestine, were cut intopieces of approximately 5 mm by use of a kitchenblender, and 50-g subsamples were digested. Ifthe sample weighed less than 50 g the totalsample was digested. Subsamples of 20 and 10 gwere collected from the lymph nodes around thesmall and large intestines, respectively, andcrushed through a garlic press before digestion.All samples were digested in a solution contain-ing 1% pepsin (1:10 000) and 1% HCl (37%) for

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2 h at 38±428C under constant stirring. The pro-portion between tissue (g) and ¯uid (ml) was ap-proximately 1:20. Following digestion, thesamples were neutralised with NaOH and left fora 4-h sedimentation, and the supernatant wasremoved. The remaining pellet with the larvaewas ®xed in an iodine solution (6.25% iodine,31.25% potassium and 62.5% distilledwater).The total content of the small intestinewas processed by a modi®ed agar-gel technique(G Jungersen, Experimental Ascaris suum infec-tion: manipulated infections and immune re-sponses to larval migration in pigs. PhD thesis,Royal Veterinary and Agricultural University,Copenhagen, 1998). Before counting in a lightmicroscope, the samples were decolourised usinga 3% thiosulphate solution.

The number of eosinophils in the peripheralblood was determined as described by Astrup etal. [23]. Swine sera were tested in duplicateagainst T. canis ES antigens by use of an indirectELISA, speci®c for IgG antibodies, modi®edafter Windelborg Nielsen et al. [24] using T. canisantigens (L2/L3-ES) obtained from in vitro culti-vation of 2nd- and 3rd-stage larvae. Antigencoating concentrations and dilutions of sera andconjugates were determined from checkerboardtitrations using pooled sera. The L2/L3-ES T.canis antigens were diluted 1:300, sera werediluted 1:1000 and the secondary antibody (HRPgoat-anti-swine IgG; Kierkegaard & Perry) wasdiluted 1:10 000. A dilution series of T. canis-positive sera and a negative control (Ascarissuum positive sera) was included on each plateand the O.D. values were corrected for plate-to-plate variation.

The calculated arithmetic mean2S.D. areused in the present experiment. The distributionof larvae per gram tissue of each sample was cal-culated for each group per slaughter day. TheMann±Whitney U-test was used to analyse di�er-ences in pig weights, eosinophil levels and T.canis antibody response (O.D. values) bysampling days and to analyse di�erences in thenumber of larvae per gram brain on days 14 and28 p.i. The numbers of larvae per gram liver,lungs and intestinal lymph nodes were analysedusing Kruskal±Wallis non-parametric analysis of

variance to test for di�erences between slaughterdays. Changes in the weight of the lungs wereanalysed by slaughter day using one-way analysisof variance. Pearson's correlation coe�cient wasused to analyse whether a linear relationshipexisted between number of larvae per gramsample, eosinophil level and antibody response atslaughter.

During the experiment, no clinical signs ofparasitism were observed in the pigs and theresults of the faecal examinations were negativethroughout the experiment. From week 14, theinfected pigs gained less weight than the unin-fected control pigs, and by day 28 p.i. theweights were 27.126.6 kg and 33.321.1 kg, re-spectively. However, there was no signi®cantdi�erence between the groups (P>0.10).

Larvae were recovered from all 18 inoculatedpigs. The recovery of T. canis larvae per gramtissue from the selected organs and muscles ofthe pig is presented in Table 1, except for thecontents of the small intestine where the totalnumbers of larvae are presented. The total loadof larvae in a pig was not calculated, as it wasnot possible to estimate the total weight of thelymph nodes or the muscle tissue, and becausethe larvae are probably unevenly distributed inthe di�erent muscles as well as in the lymphnodes. Throughout the study the majority of thelarvae were recovered from the lymph nodesaround the small and large intestines, the liverand lungs, although more than a 90% reductionin the recovery of larvae per gram tissue fromthese organs was observed from day 7 to days 14and 21 p.i. This reduction over time was signi®-cant in all four organs (P<0.0001). On day 7p.i. the majority of the larvae were recoveredfrom the lymph nodes around the small intestine.The nodes were enlarged to a 1.5 cm ``ribbon''along the intestine and contained, on average,86.6 larvae per gram tissue. High recoveries werealso observed in the lymph nodes around thelarge intestine (10.1 larvae per g). On day 14 p.i.most of the larvae were recovered from the lungsand the lymph nodes around the small intestine.Five out of six pigs had larvae in the brain onday 14 p.i. Twenty-one days p.i. most of the lar-vae were recovered from the lungs and only two

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pigs had larvae in the brain. However, because ofthe large variation within groups, there was nosigni®cant reduction in the number of larvaefound in the brain from days 14 and 21 p.i.(P=0.80). On all three slaughter days few larvaewere recovered from the kidneys. Generally,almost no larvae were found in the tongue andmasseter, and on all slaughter days at least 50%of the pigs had no larvae in these muscles. Twolarvae were recovered from the heart of one pigon day 14 p.i. only and no larvae were found inthe eyes on any occasion. On day 7 p.i. a few lar-vae were recovered from the contents of thesmall intestine, and on day 21 p.i. two larvaewere found in the contents of the small intestineof one pig. On all the slaughter days the numbersof larvae recovered from the di�erent organs andmuscles were normally distributed.

On days 7, 14 and 28 p.i. the mean numbers ofwhite spots on the liver surface were 10082312,250289 and 902116, respectively. There was asigni®cant reduction in the number of whitespots over time (P<0.0001). However, the sizeof the white spots and the development of ®bro-sis also changed with time. On day 7 p.i. thewhite spots were approximately 3 mm in diameterand the livers had some development of ®brotic

tissue By day 14 the damage in the liver wassevere: the white spots (probably to some extentcon¯uent smaller spots) were up to 1 cm in size,and some were yellowish white, had a haemor-rhage centre and were swollen. The livers werewhite and very ®brotic. On day 28 the whitespots had started to heal, the size was approxi-mately 3.5 mm and the livers were less white andmuch less ®brotic.

The lungs had many granulomas with a centralknot surrounded by haemorrhage on day 7 p.i.On day 14 p.i. the granulomas were numerouswith a central knot surrounded by haemorrhage,and the lungs felt very gritty and oedematous.The lungs were signi®cantly heavier on day 14 p.i.compared with the weights on days 7 and 28 p.i.(P=0.01). By day 28 p.i. the majority of thegranulomas had healed and the lungs feltnormal.

The kidneys had an average of 20217 whitespots on the surface on day 7 p.i., 1 mm in size.On day 14 p.i. there was an average of 158251white spots, 1±6 mm in size and some had haem-orrhage in the middle. By day 28 p.i. the spotswere in the process of healing and there were onaverage 19220 spots on the surface of thekidneys with a size of approximately 3 mm. The

Table 1

Mean number of Toxocara canis larvae per g tissue in selected organs/muscles of pigs orally inoculated with 60 000 T. canis eggs

Day 7 p.i. Day 14 p.i. Day 28 p.i.

Weight Larvae/g tissue Weight Larvae/g tissue Weight Larvae/g tissue

Organ/muscle (g) (range) (g) (range) (g) (range)

Small intestine (total contents) Ð 8.33 (0±17) Ð 0 Ð 0.33 (0±2)

Lymph nodes (SI) Ð 86.62 (39.6±126.4) Ð 4.57 (0.9±9.6) Ð 0.11 (0.0±0.4)

Lymph nodes (LI) Ð 10.13 (1.4±20.2) Ð 0.83 (0.0±2.1) Ð 0.30 (0.0±1.4)

Liver 554 0.80 (0.2±1.8) 656 0.01 (0.0±0.03) 653 0.01 (0.0±0.01)

Lungs 297 3.33 (1.3±5.1) 425 6.58 (0.8±15.9) 296 1.33 (0.6±2.5)

Brain 46 0 75 0.26 (0.0±0.7) 55 0.07 (0.0±0.4)

Kidney 117 0.04 (0.0±0.1) 137 0.09 (0.0±0.4) 138 0.01 (0.0±0.02)

Diaphragm 84 0.15 (0.1±0.3) 79 0.02 (0.0±0.04) 106 0.04 (0.0±0.1)

Masseter 36 0.03 (0.0±0.1) 58 0.03 (0.0±0.08) 60 0.01 (0.0±0.06)

Tongue 81 0.03 (0.0±0.1) 95 0.01 (0.0±0.02) 84 0.01 (0.0±0.03)

Heart 110 0 76 0.01 (0.0±0.3) 111 0

Eyes 10 0 11 0 11 0

SI, Small intestine; LI, large intestine; Ð, not weighed.

A.B. Helwigh et al. / International Journal for Parasitology 29 (1999) 559±565562

texture of the kidneys felt normal on all slaughterdays.

From days 7±21 p.i. eosinophil levels were sig-ni®cantly higher in the immunised pigs comparedwith the uninfected control pigs (P<0.03). Themean level of eosinophils reached a maximum of3431 per ml blood on day 14 p.i. The antibody re-sponse against T. canis L2/L3 ES-antigen is pre-sented in Fig. 1. Seroconversion took placebetween days 7 and 14 p.i., and antibody activityremained high during the experimental period(P<0.03).

The relationships among number of larvae pergram tissue sample, antibody response, eosino-phil counts and number of white spots were cal-culated. Only few signi®cant relationship werefound. On day 14 p.i. a positive correlation wasobserved between eosinophil counts and numberof larvae in the lymph nodes around both thesmall and large intestines (P<0.05). Twenty-eight days p.i. a positive correlation was foundbetween eosinophil counts and number of larvaein the kidney (P=0.02), between the antibodyresponse and number of larvae in the lungs(P=0.01) and between the antibody responseand number of larvae in the lymph nodes aroundthe large intestine (P=0.05).

The migratory pattern observed in the presentstudy is generally in accordance with investi-gations in mice [12, 13]. Abo-Shehada andHerbert [12] divided the migration of the larvae

in the mouse into two phases, the ®rst periodcovering the visceral phase and the second themyotropic±neurotropic phase, where the changefrom the visceral phase to the myotropic±neuro-tropic phase took place at around day 7 p.i. Themigration pattern of the larvae in the pig seemsto be di�erent, as high numbers of larvae wereobserved in both the lungs and brain on day 14p.i. in the present study, thereby having bothphases of migration represented in the pig at alater stage of infection. Done et al. [25] found asimilar pattern in their pig experiment, althoughthey infected their pigs with very high numbersof eggs in relation to the host size and at eachslaughter only one pig was killed.

Only a few studies have examined the lymphnodes for migrating T. canis larvae and generallyrecovery was low [2, 13, 25]. This is in contrast tothe present investigation, where very high num-bers of larvae were recovered from the lymphnodes, especially on day 7 p.i. Burren [13]suggested that hatched larvae might arrive in theliver from the intestine via the lymphatic vessels.Our observations support this hypothesis, as themajority of the larvae were recovered from thelymph nodes on day 7 p.i. The penetration areaseems to be less restricted than that of A.suum [26] as larvae were located in lymph nodesaround both the small and large intestines. Thisis in line with Done et al. [25], who observedlesions in the wall of both small and large intes-tines after inoculation of pigs with 1 250 000 T.canis eggs.

The maximum number of larvae in the brainwas observed on day 14 p.i., when ®ve of the sixpigs investigated had larvae in the brain. On day28 p.i. larvae were found in the brain of two pigsonly. This correlates well with the observationsby Done et al. [25], who found larvae in thebrain of pigs 8 and 16 days after infection with250 000 T. canis eggs and with Prokopic andFigallova [2], who found a maximum number oflarvae in the brain 14 days after infecting micewith 2100 eggs. Skerrett and Holland [9]observed decreasing numbers of larvae in thebrains of mice infected with 100 eggs and increas-ing numbers of larvae in the brains of miceinfected with 3000 eggs from day 5 to day 26

Fig. 1. Speci®c IgG antibody response against T. canis L2/L3

ES antigen. O.D. values: corrected optical density values;

infected group: inoculated with 60 000 T. canis eggs.

A.B. Helwigh et al. / International Journal for Parasitology 29 (1999) 559±565 563

after infection. Neurological disease was observedby Done et al. [25], who found paresis in somepigs as early as 28 days p.i. with 250 000 T. caniseggs, with severe pathological changes in theCNS observed at autopsy.

No larvae were recovered from the eyes of anyof the slaughtered pigs in the present study, andno changes of the retina were observed on day 28p.i. This is in contrast to Burren [13], whoobserved larvae in the eyes of mice 5±15 daysafter infection with 2000 eggs, and Prokopic andFigallova [2], who found larvae in the eyes ofmice 4 and 21 days p.i. However, in both ex-periments the inoculation doses were very largerelative to the size of the experimental animalcompared with the present experiment.Unfortunately, Done et al. [25] did not investigatethe eyes of the pigs in their study and, althoughthe present study did not recover larvae from theeyes of the pig, the possibility of ®nding larvae inthe pig eye after trickle infections or higher infec-tion doses cannot be excluded. Glickman andSchantz [1] put forward the hypothesis that veryhigh infection levels would increase the prob-ability of both ocular larva migrans and viscerallarva migrans, because high numbers of larvaewould be distributed in the body.

On day 7 p.i. few larvae were recovered fromthe contents of the small intestine, probably orig-inating from eggs that had taken a long time tohatch. This is in accordance with Done et al. [25],who observed eggs in the faeces of pigs 1 weekp.i. However, the two larvae found in the con-tents of the small intestine of one pig 21 days p.i.could also accidentally have penetrated back intothe lumen of the intestine. Abo-Shehada andHerbert [12] discussed the possibility of larvaemigrating back into the lumen of the intestine inmice, because they recovered larvae in the lumenuntil 6 days p.i. During the present experiment,no larval growth was observed (based on visualvaluation when counting the larvae), whichagrees with observations made by Sprent [27] andby Done et al. [25], who found living larvae inthe liver 64 days p.i., but no increase in larvalsize.

The macroscopic pathological changes in theliver, lungs and the kidneys were in agreement

with Done et al. [25], who observed increasingseverity of the pathological changes in pigs until2 weeks p.i. However, in the present experimentthe pathological changes started to heal there-after, whereas Done et al. [25] observed numer-ous white spots in the liver and kidneys, as wellas oedema in the lungs, at termination of the ex-periment on day 64 p.i. with 250 000 eggs. Thesedi�erences are probably a result of lower infec-tion doses and larger animals used in the presentexperiment. By day 28 p.i. the white spotsshowed close resemblance to A. suum-inducedwhite spots. The increase in pathological changesfrom day 7 to day 14 p.i. was associated with astrong increase in eosinophil level and antibodyresponse. The eosinophil level had almostdeclined to normal by day 28. Bisseru [28]observed elevated eosinophil levels 1 week afterinfecting mice with 200±500 T. canis eggs, wherethe level had decreased by 3 weeks p.i. Lukes [29]also observed maximum eosinophil response 2±3 weeks after infecting rabbits with 5000 T. caniseggs. The decline in eosinophil levels to normalafter a short period is in contrast to the descrip-tion of visceral larva migrans [1], where persist-ently elevated eosinophil levels are often the case.This di�erence might be explained by the factthat only one inoculation is given in these exper-iments, whereas under natural conditions thehost may be continuously re-infected in smallerdoses, thereby constantly having migration of lar-vae in the body. However, di�erences betweenhosts could also be important, as Bisseru [28]found chronic eosinophilia in a monkey, Macacairus, infected once with 2500 T. canis eggs. In ad-dition, the pig is a useful model for human visc-eral larva migrans, since T. canis migrate welland induce strong immunological response. Thepig may also act as a paratenic host for T. canis,although the relatively early death of the ma-jority of the larvae appears to minimise the im-portance in this respect.

Acknowledgements

H. Mejer, L.E. Thomsen and L. Frùlundare kindly acknowledged for skilled technical

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assistance, H. Talvik for providing the T. canismaterial, C.M. Kapel for helping out with thedigestion method, A. Flagstad for examining theeyes of the pigs and T. Olsen, Federation ofDanish Slaughterhouses and Pig Producers(Sjñlland II), for taking good care of the ani-mals. The study was supported by the DanishNational Research Foundation. The pigs used inthis experiment were treated in accordance withthe Danish Animal Ethics Law.

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