ORIGINAL PAPER

N. Coati Æ T. Schnieder Æ C. Epe

Vertical transmission of Toxocara cati Schrank 1788 (Anisakidae)in the cat

Received: 11 August 2003 / Accepted: 6 October 2003 / Published online: 3 December 2003� Springer-Verlag 2003

Abstract In eight cats and their offspring the mode oftransmission of Toxocara cati following natural andexperimental infection was investigated in three experi-ments. In experiments 1 and 2 the kittens of four cats witha chronic natural infection and of four cats with an acuteexperimental infection, respectively, were examined. Inexperiment 3 two queens of experiment 2 were matedagain to examine whether in the adult cat ‘‘dormant’’larvae exist in the tissue, that can be reactivated duringpregnancy or lactation to infect the offspring. Addition-ally, the muscle tissue and organs of two adult cats, onewith chronic one with acute infection, were examined forhypobiotic larvae. Pre-natal infections withT. cati did notoccur in experiments 1 or 2. In none of the kittens thatwere examined directly after birth were larvae found. Inthe offspring of experiment 1 one single larva of T. catiwas found 28 days post-partum.Whereas in the kittens ofexperiment 2 up to 333 larvae were found in one animal.Lactogenic transmission of larvae occurs after acuteinfection of the queen during late pregnancy but notduring chronic natural infection. There is no evidence forthe existence of arrested somatic larvae in the adult catas an important host-finding strategy in the life cycle ofT. cati. Following milk-borne infections, the majority oflarvae seem to undergo direct development in the intestinewithout trachealmigration.Only a small number of larvaewas found in other organs.

Introduction

Toxocara cati is the most common gastrointestinal hel-minth of the cat world-wide. It is important not only

because it infects young kittens but also because it is azoonotic parasite that can cause human toxocarosis(Dubinsky 1999; Janitschke 1999). Surprisingly there isonly very little knowledge about the biology of T. cati,especially as far as vertical infection is concerned,whereas a lot of research has been done on Toxocaracanis in the dog during the last decades. In fact, there areonly two investigations on this topic—one was publishedby Sprent (1956) and the other by Swerczek et al.(1971)—which is the reason why the present study fo-cuses on their results.

Sprent (1956) described the life cycle of T. cati: fol-lowing the oral uptake of eggs containing infective third-stage larvae (L3), these undergo a tracheal migration viathe liver and lungs until they finally reach the smallintestine. During this migration the larvae develop to theadult stage, and patency starts 8 weeks post-infection.Some of the larvae reach the muscle tissue where theybecome embedded and remain infective.

Sprent (1956) and Swerczek et al. (1971) examinedthe vertical transmission of T. cati in the cat. Sprent(1956) infected one pregnant cat once a week during thelast 4 weeks of gestation with a total of 30,000 infectiveeggs. Three kittens born on the day after the thirdinfection were examined when 3 and 4 days old. Onelarva was found in the muscle tissue of one of the 3-day-old kittens. Additionally, no larvae were found in thetissues of 17 kittens of naturally infected queens, thatwere examined for larvae directly after birth via Cae-sarian section. Swerczek et al. (1971) did not detect anylarvae in 92 kittens of seven naturally and 20 experi-mentally infected queens, that had been fed 300–2,000 eggs/day for 2–56 days pre-partum. However,7,959 larvae were isolated from the tissues of 17 kittensof five queens experimentally infected with 2,000 eggs/day for 1–10 days pre-partum after the kittens hadnursed for 5–20 days. Most of the larvae were found inthe gastrointestinal tract of the offspring and only veryfew in liver and lung tissue. One hundred larvae wererecovered from milk samples that were taken daily fromthe five queens mentioned above and 663 larvae were

Parasitol Res (2004) 92: 142–146DOI 10.1007/s00436-003-1019-y

N. Coati Æ T. Schnieder Æ C. Epe (&)Institute of Parasitology, Hannover School of Veterinary Medicine,Buenteweg 17, 30559 Hannover, GermanyE-mail: Christian.Epe@tiho-hannover.deTel.: +49-511-9538797Fax: +49-511-9538583

recovered from the mammary glands of these five queens15–22 days post-partum (p.p.). Furthermore, 198 larvaewere recovered from the mammary glands of six natu-rally infected queens. Sprent (1956) and Swerczek et al.(1971) agree that there is no infection of the offspring viadiaplacental transmission of larvae and that infection ofthe offspring with T. cati always takes place after birth.Due to the detection of numerous larvae in milk sam-ples, mammary glands and the tissue of kittens, Swer-czek et al. (1971) concluded that transmammaryinfection of kittens plays an important role in the lifecycle of T. cati. They presumed that lactogenic trans-mission can be achieved by the migration of infectivelarvae to the mammary gland directly after infection, orby reactivation of larvae localized in the tissue due to achange in the hormonal balance and an increased bloodsupply of the gland during lactation. As the majority oflarvae had been recovered from the small intestine of thekittens, Swerczek et al. (1971) suggested that larvaeprobably do not migrate through liver and lungs sincethey already have migrated and matured in the preced-ing host so that they can directly develop into adults inthe intestine. The few larvae that were found in otherorgans presumably had been ingested by the kittens asinfective eggs from the contaminated environment as aconsequence of the daily dosing of the queens.

Due to differing experimental designs, different ani-mal material, an undefined history of the queens and amissing description of worm burdens, and chronic orfresh infections, the work presented in this paper shouldsummarise, clarify and supplement the findings of thetwo existing investigations. In the course of this studyemphasis was put on the possibility of pre-natal and/orlactogenic transmission of T. cati following natural andexperimental infection and the existence and reactiva-tion of arrested somatic larvae in the queen. Addition-ally, the further action of transmitted larvae in the kittenwas explored, especially with respect to in which organsdevelopment would take place and if tracheal migrationthrough the liver, the vascular systems and the lungsoccurs.

Materials and methods

Experimental design

Three hypotheses were investigated in three experiments during thecourse of the study.

Hypothesis 1

If chronic infection of the adult queen with T. cati leads to arrestedsomatic larvae and vertical transmission, the offspring must beinfected.

Experiment 1

Group 1 consisted of four queens with a naturally acquired chronicinfection. The course of patency was monitored coproscopically

once a week. These cats were mated twice and the two consecutivelitters were examined for larvae. Half of each litter of the firstmating was examined directly after birth via Caesarian section(group 1a) prior to the first milk-suckling in order to avoid anypotential larval uptake with the colostrum. The remaining kittenswere reared by the queens (group 1b). After the second mating ofthe queens all newborn kittens remained with their mothers(group 1c). At different timepoints but within the first 28 days p.p.all kittens of group 1b and 1c were examined. To detect arrestedsomatic larvae the mammary glands of three queens were examinedafter weaning and their gastrointestinal tracts were searched foradult T. cati.

Hypothesis 2

If acute infection of the adult queen with T. cati during late preg-nancy leads to vertical transmission, the offspring must be infected.

Experiment 2

Four parasite-naive queens (group 2) were experimentally infectedduring late pregnancy. Weekly coproscopical examinations docu-mented the absence of egg-shedding. Ten days prior to the calcu-lated date of birth the queens were infected daily with2,000 infective eggs of T. cati. All kittens were reared by theirmothers for up to 22 days and subsequently examined. The queenswere given an anthelmintic treatment after weaning.

Hypothesis 3

If high exposure of the adult queen to infective third-stage larvae ofT. cati leads to hypobiotic somatic larvae and their reactivationduring a consecutive pregnancy with vertical transmission, theoffspring must be infected. If this is an important transmissionstrategy of the parasite, a reservoir of somatic larvae should bepresent in the queens.

Experiment 3

Two cats of group 2 were mated a second time after anthelmintictreatment without re-infection (group 3). Weekly coproscopicalexaminations documented the absence of egg-shedding. The roomin which the cats and their litters were housed had been thoroughlycleaned and disinfected. All fittings, like litter boxes or feedingdishes, were either brand new or had been autoclaved before con-tact with the experimental animals. Access to the room was possiblewhen wearing disposable protective clothing only.

Additionally, two adult cats aged 5 years were infected with625 embryonated T. cati eggs p.o. Both cats originated from theinstitute’s cat colony. One cat was parasite naive, the other had ahistory of former T. cati infections. Weekly coproscopical exam-inations documented patency in both cats and necropsy took place4 months after infection.

Experimental animals

Ten female cats (domestic short haired) aged between 1 and 5 yearswere housed in groups with access to an outdoor area. Prior toparturition, pregnant animals were housed individually in tiledboxes. Boxes and accessories of all animals were cleaned daily with60–80�C hot water and detergent (Sarox Allzweckreiniger, Deut-sche-Hahnerol, Sarstedt). The cats were fed a standard diet (Allco-Tapsy and Allco-Cat; Allco, Morsum-Wilmstorf) and had access towater ad libitum. The room in which the cats and their litters ofgroup 3 were housed had been cleaned with a steam jet at 120�Cand 1,000 kPa pressure and disinfected with 5% sodium hydroxide.

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Experimental infections

Infective T. cati eggs originated from a field isolate identified innorthern Germany in 1995. Eggs were collected from the uteri offemale T. cati worms and incubated at 25–26�C for 42 days in athin water layer. Until application, eggs were stored in Petri dishesat 4–5�C. Only eggs with viable third-stage larvae not older than 12months were used for the experimental infections.

Fifty-five days after mating [from day )10 prior to the calcu-lated date of parturition (day 0)] all pregnant cats of group 2 wereinfected daily with 2,000 infective eggs of T. cati. Larvae weresuspended in 1 ml water and administered with a blunt cannula peros.

The two additional cats of experiment 3 were infected once with625 infective eggs of T. cati p.o..

Anthelmintic treatment

Cats were treated orally with a fixed combination of 57.5 mgpyrantel embonate and 5 mg praziquantel/kg body weight (Dron-tal, Bayer Vital, Leverkusen).

Parasitological examinations

Fecal samples from queens and kittens were examined according toa combined sedimentation-flotation method (Bauer 1988).

After necropsy the contents and the gently stripped mucosa ofthe gastrointestinal tract of queens and kittens were macroscopicallyexamined for intestinal stages. Mucosa of the kittens was digested asdescribed below. Additionally, in each case the complete organs ofthe kittens (lungs, liver, gastrointestinal tract plus contents, kidneys,total prepared muscle tissue) and queens (mammary gland, lungs,liver and kidneys, 50-g samples of muscle tissue) were examined forthe presence of somatic larvae according to Herlich (1956). Eachorgan was separately minced and digested for 2 h in pepsin/HCl ona magnetic stirrer (pH 1–2, 500 IU pepsin/g tissue, 37�C) and cen-trifuged (2,000 g, room temperature) for 30 min. After discardingthe supernatant, larvae in the remaining sediment were countedmicroscopically (40-fold magnification). Tissue that was digestedincompletely was removed from the suspension and put into aBaermann apparatus (Baermann 1917) to collect the remaininglarvae after an overnight migration period. To facilitate microscopy,a detergent containing potassium hydroxide was added to the sed-iment (Neodisher; Weigert, Hamburg).

After anthelmintic treatment of the queens the total feces pro-duced during 3 days were collected, passed through a sieve underrinsing water and examined for adult stages of T. cati. The sex ofthe collected roundworms was determined under a binocularmicroscope.

Clinical examinations

During the whole animal phase the queens and their litters wereexamined daily by clinical observations. These general healthobservations included physical appearance and behaviour, abnor-malities of food and water consumption and appearance of urineand feces if present.

All animal experiments complied with the German NationalCode for Animal Protection (Tierschutzgesetz), issued May 1998(version of April 2001).

Results

All animals were clinically healthy throughout the study.The gestation period of 63-67 days was within physio-logical limits (Linde-Forsberg and Eneroth 1998).

Experiment 1

All four queens of group 1 showed patent infectionsuntil the end of the experiment in week 38. In eightkittens that had been examined directly after birth by aCesarian section no larvae were found in the tissues(group 1a). In one kitten of group 1b which was exam-ined 28 days p.p., a single larva (L3) was found in thewall of the small intestine. In none of the remainingkittens of group 1b and 1c (second mating) were stagesof T. cati detected after suckling periods that lasted forup to 28 days. In the mammary glands of three queensno larvae could be detected. From five to nine adult T.cati were found in the contents of the gastrointestinaltract.

Experiment 2

All queens of group 2 shed eggs after experimentalinfection with 14,000–24,000 infective eggs of T. cati—depending on the length of gestation—with a pre-patency period of 6 or 7 weeks after the first infectionand remained patent until the end of experiment 2after week 23 (Table 1). A total of 952 T. cati larvaewere found in the tissue samples of 12 kittens. Ninehundred and thirty-six larvae were located in thegastrointestinal tract (98%), 13 larvae (1.4%) werefound in the liver tissue of four kittens, two larvae(0.2%) in the lung tissue of one kitten and one singlelarva (0.1%) was detected in the kidney sample of onekitten (Tab. 3). All queens were given an anthelmintictreatment 7–8 days p.p., and in the total feces col-lected after 3 days six to ten adult stages of T. catiwere collected.

Experiment 3

Two queens of group 2 were mated a second time. Noadult worms were found in the total feces producedduring 3 consecutive days after anthelmintic treatment.Both cats remained coproscopically negative through-out the gestation period and lactation which wasdocumented by daily examination of fecal samples.From day 21 p.p. until day 57 p.p. fecal samples ofthe eight kittens were examined. On days 44, 53 and55 p.p. all three kittens of litter 1 became patent. Theshedding of eggs continued until the end of theexperiment on day 57 p.p. All five kittens of litter 2remained coproscopically negative between days 21and 57 p.p.

The two adult cats with a chronic infection reachedpatency after 56 days and were necropsied 63 daysafter reaching patency. The tissue digestion revealedfew larvae (L3) in the muscle tissue and lung: animal1 showed four L3 in muscle tissue and two L3 inlung tissue, animal 2 showed one single L3 in muscletissue.

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Discussion

In these investigations the mode of transmission fol-lowing natural and experimental infection with T. catiwas examined. The fact that there are only two publi-cations on the vertical transmission of T. cati in the catand no research has been done for the last 30 years madethis study necessary. Additionally, the differing methodsand experimental designs used in these two investiga-tions needed to be improved upon and supplementedusing a sufficient but minimum number of animals.

The pepsin digestion of tissue was first described byHerlich (1956) who isolated nematodes from theabomasal mucosa of ruminants. Due to a number offactors (e.g. immunoreactivity of the host, pathogenicityof the parasite) it is impossible to determine the sensi-tivity and specificity of this method since experimentalinfection with a certain number of infective stages doesnot necessarily lead to the same amount of tissue larvae.Therefore, no gold standard for method evaluationcould be established and the quality of results was highlydependent on the experience and accuracy of the labo-ratory staff. Methodological details like reagents andtemperature used for the tissue digestion in the presentinvestigation are identical to those described by Sprent(1956) and Swerczek et al. (1971), with the exception ofthe incubation period. It is possible that the incubationperiod used in these studies, i.e. 18 h (Sprent 1956) and12–18 h (Swerczek et al. 1971), might have influencedthe number of larvae detected.

To exclude post-natal lactogenic infection via thecolostrum, a Caesarian section was necessary in the fourqueens after the first mating and the offspring wereexamined before any ingestion of colostrum. No stagesof T. cati were detected in any of the kittens. This resultis identical to that of Sprent (1956) who examined thetissues of 17 neonatal kittens from naturally infectedqueens without finding any larvae. Similarly, Swerczeket al. (1971) did not detect any larvae in 78 kittens ofnaturally infected queens and 14 kittens of experimen-tally infected queens, respectively, prior to their firstmilk feed. Obviously, pre-natal infection with T. cati

does not occur in the cat which is in contrast to T. canisin the dog, where pre-natal transmission is the majorroute of infection for the puppy. In 24 kittens examinedafter a lactation period of up to 28 days only one larvawas found in the small intestine. An explanation of thisfinding cannot be really given since either a methodo-logical error (i.e. a false-positive finding due to materialswhich had not been cleaned) or a misdiagnosis should beexcluded first. Nevertheless, this larva does not representa population in terms of a survival strategy of the par-asite, so one explanation may be the pure chance offinding this single larva after an unusual migration. Nostages of T. cati were detected in the mammary glands.Neither Swerczek et al. (1971) nor Sprent (1956) exam-ined the offspring of naturally infected queens, but theformer isolated 198 larvae of the mammary glands of sixqueens with a natural T. cani infection. However, thereis only very little information given about these cats, forexample whether infections were acute or chronic. De-tails about the number of adult T. cati located in thegastrointestinal tract of the queens, distribution of lar-vae among the different cats and time of examinationp.p. are missing. The statement of Swerczek et al. (1971)concerning lactogenic transmission being the mostimportant way of infection for the kitten is based on theexamination of kittens from experimentally infectedqueens and mammary glands of six naturally infectedqueens. Unfortunately, the offspring of these six natu-rally infected queens are not mentioned at all. Further-more, the artificial situation achieved by regularlyinfecting pregnant queens with high numbers of infectiveeggs of T. cati is unlikely to represent reality and cannotbe compared to the situation of the common domesticcat.

Our results indicate that, unlike in the dog, reacti-vation of arrested somatic larvae and their verticaltransmission via the milk in chronically infected queensand their offspring does not play a strategic role in thelife cycle of T. cati in the sense of a survival strategy.

Of 952 larvae of T. cati isolated from kitten organtissue samples, 98% were located in the gastrointestinaltract. Larvae were found only sporadically in the liver,

Table 1 Recovery of Toxocaracati larvae from kittens ofexperimentally infected queens.p.p. Post-partum

Cat no. Kitten no. Days p.p. Liver Lung Kidney Muscle Smallintestine

P

1 1 1 0 0 0 0 0 01 2 1 0 0 0 0 0 03 3 3 0 0 0 0 0 01 4 8 0 0 0 0 18 181 5 9 0 0 0 0 1 11 6 11 0 0 0 0 82 822 7 17 0 0 0 0 232 2324 8 21 1 0 0 0 76 774 9 21 2 0 0 0 63 654 10 21 0 2 0 0 40 424 11 21 9 0 1 0 92 1021 12 22 1 0 0 0 332 333P

– 13 2 1 0 936 952

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lungs and kidneys. These data show that lactogenictransmission of T. cati occurs after infection of thequeen during late pregnancy. This result is similar tothat of Swerczek et al. (1971) who also examined theoffspring of experimentally infected queens and detectedthe majority of larvae in the gastrointestinal tract andonly very few in lungs and liver tissue. These resultsconfirm the assumption that, following milk-borneinfections, larvae undergo direct development in theintestine without tracheal migration. Sprent (1956)speculated a ripening progress of the larvae in the tissueof the queens that makes migration in the offspringsuperfluous and would explain the altered development.No larvae were found in the kitten samples collectedduring the first 3 days of lactation which indicates thatexcretion of the majority of the larval population via themilk starts only some days after parturition.

During experiment 2 the queens had been exposed tohigh infective doses of T. cati during late pregnancy. Ifsomatic migration with following hypobiosis had takenplace in these queens, a reactivation with verticaltransmission during another pregnancy would be pos-sible. Sprent (1956) detected 38 larvae in the muscletissue of ten cats, 10–42 days after experimental infec-tion.

Patency of the litters was to be the evidence for lac-togenic infection, since infection through the environ-ment should be excluded by thoroughly implementedhygienic measures. The two queens had been given ananthelmintic treatment, pyrantel embonate, twice duringpregnancy and no eggs were shed during the wholeanimal phase. Pyrantel embonate has no effect on extra-intestinal stages of T. canis in the dog (Neu 1974).Unfortunately there are no corresponding investigationsfor the cat, so these facts were assumed to be true forT. cati as well. All kittens of cat 1 started shedding eggsfrom day 44 p.p. onwards, whereas all kittens of cat 4remained coproscopically negative until the end of thestudy. A contamination from the environment has to bediscussed although the hygienic measures were strictlyimplemented and an environmental infection seems tobe very unlikely. Nevertheless, these data definitely donot support hypobiosis and vertical transmission as a

major survival and transmission strategy of T. cati. Thisis supported by the additional data of two chronicallyinfected animals with very few larvae found in thecompletely digested tissue. These larvae represent bio-logical variations of stages lost during tissue migrationrather than a parasite’s strategy of survival via hypobi-osis.

The results of the present investigations correspondwith the literature as far as the absence of pre-nataltransuterine transmission of larvae is concerned. Thebiology of T. cati leaves transmission of larvae via themilk as the only possible means of vertical infection.This route of infection is followed regularly after acuteinfection of the queen during late pregnancy, but wasobserved only in one case following chronic naturalinfection where a single larva was found. Followingmilk-borne infection, most of the larvae seem to undergodirect development in the intestine without trachealmigration since only a small number of larvae was foundin other organs.

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