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

Abstract

In eight cats and their offspring the mode of transmission of Toxocara cati following natural and experimental infection was investigated in three experiments. In experiments 1 and 2 the kittens of four cats with a chronic natural infection and of four cats with an acute experimental infection, respectively, were examined. In experiment 3 two queens of experiment 2 were mated again to examine whether in the adult cat "dormant" larvae exist in the tissue, that can be reactivated during pregnancy or lactation to infect the offspring. Additionally, the muscle tissue and organs of two adult cats, one with chronic one with acute infection, were examined for hypobiotic larvae. Pre-natal infections with T. cati did not occur in experiments 1 or 2. In none of the kittens that were examined directly after birth were larvae found. In the offspring of experiment 1 one single larva of T. cati was found 28 days post-partum. Whereas in the kittens of experiment 2 up to 333 larvae were found in one animal. Lactogenic transmission of larvae occurs after acute infection of the queen during late pregnancy but not during chronic natural infection. There is no evidence for the existence of arrested somatic larvae in the adult cat as an important host-finding strategy in the life cycle of T. cati. Following milk-borne infections, the majority of larvae seem to undergo direct development in the intestine without tracheal migration. Only a small number of larvae was found in other organs.

Full text

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
Ó Springer-Verlag 2003
Abstract In eight cats and their offspring the mode of
transmission of Toxocara cati following natural and
experimental infection was investigated in three experi-
ments. In experiments 1 and 2 the kittens of four cats wi th
a chronic natural infection and of four cats with an acute
experimental infection, respectively, were examined. In
experiment 3 two queens of experiment 2 were mated
again to examine whether in the adult cat ‘‘dormant’’
larvae exist in the tissue, that can be reactivated during
pregnancy or lactation to infect the offspring. Addition-
ally, the muscle tissue and organs of two adult cats, one
with chronic one with acute infection, were examined for
hypobiotic larvae. Pre-natal infections with T. cati did not
occur in experiments 1 or 2. In none of the kittens that
were examined directly after birth were larvae found. In
the offspring of experiment 1 one single larva of T. cati
was found 28 days post-partum. Whereas in the kittens of
experiment 2 up to 333 larvae were found in one animal.
Lactogenic transmission of larvae occurs after acute
infection of the queen during late pregnancy but not
during chronic natural infection. There is no evidence for
the existence of arrested somatic larvae in the adult cat
as an important host-finding strategy in the life cycle of
T. cati. Following milk-borne infections, the majority of
larvae seem to undergo direct development in the intestine
without tracheal migration. Only a small number of larvae
was 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 a
zoonotic parasite that can caus e human toxocarosis
(Dubinsky 1999; Janitschke 1999). Surprisingly there is
only 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 Toxocara
canis in the dog during the last decades. In fact, there are
only two investigations on this topic—one was published
by 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 egg s containing infective third-
stage larvae (L3), these undergo a tracheal migration via
the liver and lungs until they finally reach the small
intestine. During this migration the larvae develop to the
adult stage, and patency starts 8 weeks post-infection.
Some of the larv ae reach the muscle tissue where they
become embedded an d remain infective.
Sprent (1956) and Swerczek et al. (1971) examined
the vertical transmission of T. cati in the cat. Sprent
(1956) infected one pregnant cat once a week during the
last 4 weeks of gestation with a total of 30,000 infective
eggs. Three kittens born on the day after the third
infection were examined when 3 and 4 days old. One
larva was found in the muscle tissue of one of the 3-day-
old kittens. Additionally, no larvae were found in the
tissues of 17 kittens of naturally infected queens, that
were examined for larvae directly after birth via Cae-
sarian section. Swerczek et al. (1971) did not detect any
larvae 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 kittens
of five queens experimentally infected with 2,000 eggs/
day for 1–10 days pre-partum after the kittens had
nursed for 5–20 days. Most of the larvae were found in
the gastrointestinal tract of the offspring and only very
few in liver and lung tissue. One hundred larvae were
recovered from milk samples that were taken daily from
the five queens mentioned above and 663 larvae were
Parasitol Res (2004) 92: 142–146
DOI 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, Germany
E-mail: Christian.Epe@tiho-hannover.de
Tel.: +49-511-9538797
Fax: +49-511-9538583

recovered from the mammary glands of these five queens
15–22 days post-partum (p.p.). Furthermore, 198 larvae
were 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 via
diaplacental tra nsmission of larvae and that infection of
the offspring with T. cati always takes place after birth.
Due to the detec tion of numerous larvae in milk sam-
ples, mammary glands and the tissue of kittens, Swer-
czek et al. (1971) concluded that transmammary
infection of kittens plays an important role in the life
cycle of T. cati. They presumed that lactogenic trans-
mission can be achieved by the migration of infective
larvae to the mammary gland directly after infection, or
by reactivation of larvae localized in the tissue due to a
change in the hormonal balance and an increased blood
supply of the gland during lactation. As the majority of
larvae had been recovered from the small intestine of the
kittens, Swerczek et al. (1971) suggested that larvae
probably do not migrate through liver and lungs since
they already have migrated and matured in the preced-
ing host so that they can directly develop into adults in
the intestine. The few larvae that were found in other
organs presumably had been ingested by the kittens as
infective eggs from the contaminated environment as a
consequence of the da ily dosing of the queens.
Due to differing experimental designs, different ani-
mal material, an undefined history of the queens and a
missing description of worm burdens, and chronic or
fresh infections, the work presented in this paper should
summarise, clarify and supplement the findings of the
two existing investigations. In the course of this study
emphasis was put on the possibility of pre-natal and/or
lactogenic transmission of T. cati following natural and
experimental infection and the existence and reactiva-
tion of arrested somatic larvae in the queen. Addition-
ally, the further action of transmitted larvae in the kitten
was explored, especially with respect to in which organs
development would take place and if tracheal migration
through the liver, the vascular systems and the lungs
occurs.
Materials and methods
Experimental design
Three hypotheses were investigated in three experiments during the
course of the study.
Hypothesis 1
If chronic infection of the adult queen with T. cati leads to arrested
somatic larvae and vertical transmission, the offspring must be
infected.
Experiment 1
Group 1 consisted of four queens with a naturally acquired chronic
infection. The course of patency was monitored coproscopically
once a week. These cats were mated twice and the two consecutive
litters were examined for larvae. Half of each litter of the first
mating was examined directly after birth via Caesarian section
(group 1a) prior to the first milk-suckling in order to avoid any
potential larval uptake with the colostrum. The remaining kittens
were reared by the queens (group 1b). After the second mating of
the 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 arrested
somatic larvae the mammary glands of three queens were examined
after weaning and their gastrointestinal tracts were searched for
adult 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 infected
during 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 with
2,000 infective eggs of T. cati. All kittens were reared by their
mothers for up to 22 days and subsequently examined. The queens
were given an anthelmintic treatment after weaning.
Hypothesis 3
If high exposure of the adult queen to infective third-stage larvae of
T. cati leads to hypobiotic somatic larvae and their reactivation
during a consecutive pregnancy with vertical transmission, the
offspring must be infected. If this is an important transmission
strategy of the parasite, a reservoir of somatic larvae should be
present in the queens.
Experiment 3
Two cats of group 2 were mated a second time after anthelmintic
treatment without re-infection (group 3). Weekly coproscopical
examinations documented the absence of egg-shedding. The room
in which the cats and their litters were housed had been thoroughly
cleaned and disinfected. All fittings, like litter boxes or feeding
dishes, were either brand new or had been autoclaved before con-
tact with the experimental animals. Access to the room was possible
when wearing disposable protective clothing only.
Additionally, two adult cats aged 5 years were infected with
625 embryonated T. cati eggs p.o. Both cats originated from the
institute’s cat colony. One cat was parasite naive, the other had a
history of former T. cati infections. Weekly coproscopical exam-
inations documented patency in both cats and necropsy took place
4 months after infection.
Experimental animals
Ten female cats (domestic short haired) aged between 1 and 5 years
were housed in groups with access to an outdoor area. Prior to
parturition, pregnant animals were housed individually in tiled
boxes. Boxes and accessories of all animals were cleaned daily with
60–80°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 to
water ad libitum. The room in which the cats and their litters of
group 3 were housed had been cleaned with a steam jet at 120°C
and 1,000 kPa pressure and disinfected with 5% sodium hydroxide.
143

Experimental infections
Infective T. cati eggs originated from a field isolate identified in
northern Germany in 1995. Eggs were collected from the uteri of
female T. cati worms and incubated at 25–26°C for 42 days in a
thin water layer. Until application, eggs were stored in Petri dishes
at 4–5°C. Only eggs with viable third-stage larvae not older than 12
months 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 were
infected daily with 2,000 infective eggs of T. cati. Larvae were
suspended in 1 ml water and administered with a blunt cannula per
os.
The two additional cats of experiment 3 were infected once with
625 infective eggs of T. cati p.o..
Anthelmintic treatment
Cats were treated orally with a fixed combination of 57.5 mg
pyrantel embonate and 5 mg praziquantel/kg body weight (Dron-
tal, Bayer Vital, Leverkusen).
Parasitological examinations
Fecal samples from queens and kittens were examined according to
a combined sedimentation-flotation method (Bauer 1988).
After necropsy the contents and the gently stripped mucosa of
the gastrointestinal tract of queens and kittens were macroscopically
examined for intestinal stages. Mucosa of the kittens was digested as
described below. Additionally, in each case the complete organs of
the 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 for
the presence of somatic larvae according to Herlich (1956). Each
organ was separately minced and digested for 2 h in pepsin/HCl on
a magnetic stirrer (pH 1–2, 500 IU pepsin/g tissue, 37°C) and cen-
trifuged (2,000 g, room temperature) for 30 min. After discarding
the supernatant, larvae in the remaining sediment were counted
microscopically (40-fold magnification). Tissue that was digested
incompletely was removed from the suspension and put into a
Baermann apparatus (Baermann 1917) to collect the remaining
larvae 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 under
rinsing water and examined for adult stages of T. cati. The sex of
the collected roundworms was determined under a binocular
microscope.
Clinical examinations
During the whole animal phase the queens and their litters were
examined daily by clinical observations. These general health
observations included physical appearance and behaviour, abnor-
malities of food and water consumption and appearance of urine
and feces if present.
All animal experiments complied with the German National
Code for Animal Protection (Tierschutzgesetz), issued May 1998
(version of April 2001).
Results
All animals were clinically health y throughout the study.
The gestation period of 63-67 days was within physio-
logical limits (Linde-Forsberg and Eneroth 199 8).
Experiment 1
All four queens of group 1 showed patent infections
until the end of the experiment in week 38. In eight
kittens that had been examined directly after birth by a
Cesarian 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 the
wall of the small intestine. In none of the remaining
kittens of group 1b and 1c (second mating) were stages
of T. cati detected after suckling periods that lasted for
up to 28 days. In the mammary glands of three queens
no larvae could be detected. From five to nine adult T.
cati were found in the contents of the gastr ointestinal
tract.
Experiment 2
All queens of group 2 shed eggs after experimental
infection 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 infection
and remained patent until the end of experiment 2
after week 23 (Table 1). A total of 952 T. cati larvae
were found in the tissue samples of 12 kittens. Nine
hundred and thirty-six larvae were located in the
gastrointestinal tract (98%), 13 larvae (1.4%) were
found in the liver tissue of four kittens, two larvae
(0.2%) in the lung tissue of one kitten and one single
larva (0.1%) was detected in the kidney sample of one
kitten (Tab. 3). All queens were given an anthelmintic
treatment 7–8 days p.p., and in the total feces col-
lected after 3 days six to ten adult stages of T. cati
were collected.
Experiment 3
Two queens of group 2 were mated a seco nd time. No
adult worms were found in the total feces produced
during 3 consecutive days after anthelmintic treatment.
Both cats remained coproscopically negative through-
out the gestation period and lactation which was
documented by daily examination of fecal samples.
From day 21 p.p. until day 57 p.p. fecal samples of
the eight kittens were examined. On days 44, 53 and
55 p.p. all three kittens of litter 1 became patent. The
shedding of eggs continued until the end of the
experiment on day 57 p.p. All five kittens of litter 2
remained coproscopically negative between days 21
and 57 p.p.
The two adult cats with a chronic infection reached
patency after 56 days and were necropsied 63 days
after reaching patency. The tissue digestion revealed
few larvae (L3) in the muscle tissue and lung: animal
1 showed four L3 in muscle tissue and two L3 in
lung tissue, animal 2 showed one single L3 in muscle
tissue.
144

Discussion
In these investigations the mode of transmission fol-
lowing natural and experimental infection with T. cati
was examined. The fact that there are only two publi-
cations on the vertical transmission of T. cati in the cat
and no research has been done for the last 30 years made
this study necessary. Additionally, the differing methods
and experimental designs used in these two investiga-
tions needed to be improved upon and supplemented
using a sufficient but minimum number of animals.
The pepsin digestion of tissue was first described by
Herlich (1956) who isolated nematodes from the
abomasal mucosa of ruminants. Due to a number of
factors (e.g. immunoreactivity of the host, pathogenicity
of the parasite) it is impossible to determine the sensi-
tivity and specificity of this method since experimental
infection with a certain number of infective stages does
not necessarily lead to the same amount of tissue larvae.
Therefore, no gold standard for method evaluation
could be established and the quality of results was highly
dependent on the experience and accuracy of the labo-
ratory staff. Methodological details like reagents and
temperature used for the tissue digestion in the present
investigation are identical to those described by Sprent
(1956) and Swerczek et al. (1971), with the exception of
the incubation period. It is possible that the incubation
period used in these studies, i.e. 18 h (Sprent 1956) and
12–18 h (Swerczek et al. 1971), might have influenced
the number of larvae detected.
To exclude post-natal lactogenic infection via the
colostrum, a Caesarian section was necessary in the four
queens after the first mating and the offspring were
examined before any ingestion of colostrum. No stages
of T. cati were detected in any of the kittens. This result
is identical to that of Sprent (1956) who examined the
tissues of 17 neonatal kittens from naturally infected
queens without finding any larvae. Similarly, Swerczek
et al. (1971) did not detect any larvae in 78 kittens of
naturally infected queens and 14 kittens of experimen-
tally infected queens, respectively, prior to their first
milk feed. Obviously, pre-natal infection with T. cati
does not occur in the cat which is in contrast to T. canis
in the dog, where pre-n atal transmission is the major
route of infection for the puppy. In 24 kittens examined
after a lactation period of up to 28 days only one larva
was found in the small intestine. An explanation of this
finding cannot be really given since either a methodo-
logical error (i.e. a false-positive finding due to materials
which had not been cleaned) or a misdiagnosis should be
excluded first. Nevertheless, this larva does not represent
a population in terms of a survival strategy of the par-
asite, so one explanation may be the pure chance of
finding this single larva after an unusual migration. No
stages 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 the
former isolated 198 larvae of the mammary glands of six
queens with a natural T. cani infection. However, there
is only very little information given about these cats, for
example whether infections were acute or chronic. De-
tails about the number of adult T. cati located in the
gastrointestinal tract of the queens, distribution of lar-
vae among the different cats and time of examination
p.p. are missing. The statement of Swerczek et al. (1971)
concerning lactogenic transmission being the most
important way of infection for the kitten is based on the
examination of kittens from experimentally infected
queens and mammary glands of six naturally infected
queens. Unfortunately, the offspring of these six natu-
rally infected queens are not mentioned at all. Further-
more, the artificial situation achieved by regularly
infecting pregnant queens with high numb ers of infective
eggs of T. cati is unlikely to represent reality and cannot
be co mpared to the situation of the common domestic
cat.
Our results indicate that, unlike in the dog, reacti-
vation of arrested somatic larvae and their vertical
transmission via the milk in chronically infected queens
and their offspring does not play a strategic role in the
life cycle of T. cati in the sense of a survival strategy.
Of 952 larvae of T. cati isolated from kitten organ
tissue samples, 98% were located in the gastrointestinal
tract. Larvae were found only sporadically in the liver,
Table 1 Recovery of Toxocara
cati larvae from kittens of
experimentally infected queens.
p.p. Post-partum
Cat no. Kitten no. Days p.p. Liver Lung Kidney Muscle Small
intestine
P
11 1 00000 0
12 1 00000 0
33 3 00000 0
14 8 00001818
15 9 00001 1
1 6 11 0 0 0 0 82 82
2 7 17 0 0 0 0 232 232
4 8 21 1 0 0 0 76 77
4 9 21 2 0 0 0 63 65
410 21 02004042
4 11 21 9 0 1 0 92 102
1 12 22 1 0 0 0 332 333
P
– 13 2 1 0 936 952
145

lungs and kidneys. These data show that lactogenic
transmission of T. cati occurs after infection of the
queen during late pregnancy. This result is similar to
that of Swerczek et al. (1971) who also examined the
offspring of experimentally infected queens and detected
the majority of larvae in the gastrointestinal tract and
only very few in lungs and liver tissue. These results
confirm the assumption that, following milk-borne
infections, larvae undergo direct development in the
intestine without tracheal migration. Sprent (1956)
speculated a ripening progress of the larvae in the tissue
of the queens that makes migration in the offspring
superfluous and would explain the altered development.
No larvae were found in the kitten samples collected
during the first 3 days of lactation which indicates that
excretion of the majority of the larval population via the
milk starts only some days after parturition.
During experiment 2 the queens had been exposed to
high infective doses of T. cati during late pregnancy. If
somatic migration with following hypobiosis had taken
place in these queens, a reactivation with vertical
transmission during another pregnancy would be pos-
sible. Sprent (1956) detected 38 larvae in the muscle
tissue 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 implemented
hygienic measures. The two queens had been given an
anthelmintic treatment, pyrantel embonate, twice during
pregnancy and no eggs were shed during the whole
animal phase. Pyrantel embonate has no effect on extra-
intestinal stages of T. canis in the dog (Neu 1974) .
Unfortunately there are no corresponding investigations
for the cat, so these facts were assumed to be true for
T. cati as well. All kittens of cat 1 started shedding eggs
from day 44 p.p. onwards, where as all kittens of cat 4
remained coproscopically negative until the end of the
study. A contamination from the environment has to be
discussed although the hygienic measures were strictly
implemented and an environmental infection seems to
be very unlikely. Nevertheless, these data definitely do
not support hypobiosis and vertical transmission as a
major survival and transmission strategy of T. cati.This
is supported by the additional data of two chronically
infected animals with very few larvae found in the
completely digested tissue. These larvae represent bio-
logical variations of stages lost during tissue migration
rather than a parasite’s strategy of survival via hypobi-
osis.
The results of the present investigations correspond
with the literature as far as the absence of pre-natal
transuterine transmission of larvae is concerned. The
biology of T. cati leaves transmission of larvae via the
milk as the only possible means of vertical infection.
This route of infec tion is followed regularly after acute
infection of the queen during late pregnancy, but was
observed only in one case following chronic natural
infection where a single larva was found. Following
milk-borne infection, most of the larvae seem to undergo
direct development in the intestine without tracheal
migration since only a small number of larvae was found
in other organs.
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