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ARTICLE ORIGINAL
Passive transmission of humoral and cellular
immunity in canine visceral leishmaniasis
° H.M. ANDRADE*, °° V. DE P.C.P. TOLEDO, °°° W. MAYRINK and °°° O. GENARO
° Department of Parasitology and Microbiology, Universidade Federal do Piauí, Teresina, PI, Brazil.
°° Department of Clinical and Toxicological Analyses, College of Pharmacy, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
°°° Department of Parasitology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
* Corresponding author : Hélida Monteiro de Andrade Universidade Federal do Piauí, Campus Ministro Petronio Portela - SG-16
Departamento de Parasitologia e Microbiologia, Teresina - Piauí, 64.049-550, Teresina - Piauí, Brazil - Telephone-Fax : (86) 237-1010
E-mail : [email protected]
This study received financial support from FAPEMIG and CAPES.
SUMMARY
RÉSUMÉ
Visceral leishmaniasis is becoming more frequent in many parts of the
world. Where it is endemic, dogs are considered the primary reservoir.
Serologic survey of canine populations is one of the measures used to identify positive animals to be sacrificed as a method of disease control.
However, since information on the passive transfer of immunity in canine
visceral leishmaniasis (CVL) is lacking, serologically positive offspring
may be being sacrificed when they are merely exhibiting passively-acquired
antibodies. To evaluate passive transmission of humoral immunity in CVL,
we used 10 serologically and parasitologically positive female dogs and
their 48 puppies, in which IgG and IgM levels were evaluated using indirect
immunofluorescence reaction over a period of four months. Passive transmission of cellular immunity was analyzed using the delayed test of hypersensitivity (DTH) in another group of 5 female dogs naturally infected and
their 25 offspring that were between 8 and 75 days old, as well as a control
dog and her 9 puppies 8 days after birth. IgG-class antibodies were identified in all the litters starting from the day of birth and lasting on average
32.1 ±15.3 days (14-70 days). IgM was not detected in the offspring but was
present in 7 of the 10 adult dogs. Among the adults with positive DTH, 72%
of their puppies also had positive DTH reactions. Passive transmission of
humoral and cellular immunity occur in CVL and therefore it is recommended that seropositive offspring less than 70 days of age should not be sacrificed based on serologic surveys for CVL.
Transmission passive de l’immunité humorale et cellulaire dans la
leishmaniose viscérale canine. Par H.M. ANDRADE, V. de P.C.P.
TOLEDO, W. MAYRINK et O. GENARO.
KEY-WORDS : passive immunity - canine visceral leishmaniasis - Leishmania chagasi.
MOTS-CLÉS : immunité passive - leishmaniose viscéral
canin - Leishmania chagasi.
Introduction
East Asia and northern Africa where the etiologic agent is
Leishmania infantum, as well as Central and South American
countries where the disease is caused by Leishmania chagasi
[5].
Visceral leishmaniasis is becoming more frequent in many
areas of the world due to epidemiological changes, like urbanization in Brazil and mass migrations on the Indian subcontinent, which lead to the introduction of non-immunized individuals into the disease cycle [27].
In many regions where the zoonotic visceral leishmaniasis
(ZVL) is endemic, dogs are considered the principal reservoir. This occurs in countries in southern Europe, the MiddleRevue Méd. Vét., 2001, 152, 4, 317-324
La Leishmaniose viscérale est en expansion dans diverses parties du
monde. En beaucoup d’endroits où la Leishmaniose viscérale est endémique
les chiens sont considérés comme son principal réservoir. La recherche
sérologique chez la population canine est l’une des mesures adoptées pour
identifier des animaux positifs pour qu’ils soient éliminés afin de maintenir
le contrôle de la maladie. Toutefois, comme il n’y a pas d’informations sur
le transfert passif de l’immunité dans la Leishmaniose viscérale canine
(LVC), des chiots séropositifs pourraient être en train d’être sacrifiés, alors
qu’ils seraient seulement en train d’exhiber des anticorps passivement
acquis. Pour évaluer la transmission passive de l’immunité humorale dans
la LVC, nous avons utilisés 10 chiennes séropositives et parasitées pour LV
et leurs 48 chiots, chez qui a été fait des mesures des IgG et des IgM par la
RIFI, pendant 4 mois. La transmission passive de l’immunité cellulaire a été
évaluée par le Test d’Hypersensibilité Retardée (THR) chez un autre groupe de 5 chiennes positives avec leurs 25 chiots âgés de 8 à 75 jours. Des
anticorps de classe IgG ont été rencontrés dans toutes les nichées de la
recherche à partir du jour de la naissance et ont duré pendant 32,1 ± 15,3
jours (14 - 70 jours). Les IgM n’ont pas été détectées chez les chiots mais
étaient présentes chez 7 des 10 mères. Des 25 chiots des chiennes THR
positives, 18 (72 %) ont présenté des résultats THR aussi positifs. La transmission passive de l’immunité humorale et cellulaire a lieu dans la LVC et
nous recommandons que les nouveau-nés séropositifs de moins de 70 jours
ne soient pas sacrifiés sur les seuls résultats des tests sérologiques pour la
LVC.
ZVL control is limited and includes combating the vector,
eliminating reservoirs and treating the infected human population [8,17]. These measures can help limit ZVL transmission in endemic areas, as was shown by MAGALHÃES et al.
(1980) [18] in Vale do Rio Doce, Minas Gerais, Brazil.
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ANDRADE (H.M.) ET COLLABORATEURS
Canine visceral leishmaniasis (CVL) diagnosis can be
made based on serologic tests. Once the antibody response is
exceptionally strong [26], a serologic survey of the canine
population is one of the measures that has been adopted to
identify positive animals to be sacrificed as a form of disease
control. However, because information on the transfer of passive immunity in CVL is lacking, serologically positive offspring may be sacrificed when in fact they are merely exhibiting passively-acquired antibodies.
cally positive animals are sacrificed in ZVL control campaigns. Such information can help prevent the unnecessary
sacrificed of uninfected dogs that are seropositive due to the
passive transfer of antibodies.
The immune relationship between mother and neonate is
complex. Antibodies, parasitic antigens, immune cells, citokynes and probably other still-unknown factors may be transferred during the fetal phase across placenta and/or postnatally [6].
Sixteen adult female dogs with confirmed gestation were
studied. Fifteen dogs were diagnosed with visceral leishmaniasis through specific anti-Leishmania antibody detection
using indirect immunofluorescence test and parasitologic
examination of Giemsa-stained bone marrow smears. An
adult female that was negative on the same tests was used as
a control. The positive dogs were naturally infected and were
obtained from the kennel of the Zoonosis Control Center of
the City of Belo Horizonte, Minas Gerais. The control dog
came from our study kennel (Institute of Biological Sciences
of the Universidade Federal de Minas Gerais). A total of 82
offspring were part of the study starting from the day of birth.
The study animals were of the following breeds: Two adult
Dobermans with 16 offspring; 1 adult Rotweiller and 8 offspring; 1 adult Boxer with 8 offspring and 12 adults of mongrels with 50 offspring. The adults, together with their litters,
were maintained in separate cages. The animals were identified by number according to the order of entrance into the
study. Offspring received the same number as their mother,
followed by a letter in alphabetical order.
After weaning, puppies and adults were fed an age-appropriate, balanced diet (Purina®) ad libitum. All animals were
inspected daily and examined weekly. Periodic evaluation of
the environment was done and pyrethroid insecticide (KOthrine®) was applied quarterly to have a control the presence of sandflies.
In dogs, as in cats, the placenta is the endotheliocorial type,
in which the epithelium of the chorium is in contact with
maternal capillary endothelia. In these species, small quantities of immunoglobulin G (IgG) (5-10 %) may be transferred
from mother to fetus, but the majority is obtained through the
colostrum [25].
The period of intestinal permeability of proteins in offspring varies between species and between isotypes.
Generally it is higher immediately following birth and
declines after about 6 hours, possibly because intestinal cells
that absorb immunoglobulins are replaced by a population of
more mature cells. The length of the absorption period is not
well established [25].
Normally, non-nursing animals have extremely low serum
levels of IgG. Immunoglobulins, particularly IgG, is coming
to the serum through colostrum absorption. Due to the nature
of the absorption process, IgG peak serum levels usually
occur between 12 and 24 hours postpartum. These passivelyacquired antibodies immediately begin to decline through
normal catabolic processes once absorption ends. The rate of
decline differs depending on the Ig isotype, however the time
required to reach non-protective levels also depends on initial
concentration [25].
Passively-acquired maternal antibodies do not only inhibit
Ig synthesis, they also avoid successful vaccination of young
animals. This refractory period can last several months,
depending on the quantity of antibodies transferred to the
neonate and the half-life of the immunoglobulins [25].
There is evidence that cell-mediated immunity is transferred to newborns through colostrum [25]. Parasite-specific
skin tests were positive in children of mothers infected with
Schistosoma mansoni [6] and cellular immunity, mediated by
antigen-specific T cells, was passed through maternal milk in
mice infected with Trichinella spiralis and that immunity
may have been protected the offspring against intestinal
pathogens [15].
The maternal-fetal exchange, mainly the passage of molecules from maternal to fetal circulation is well-established,
but the quantification and the effects of this exchange, particularly in CVL, are not documented. Information is necessary regarding levels of passive immunity in newborn dogs,
which is of significant importance considering that serologi-
2. Material and Methods
A) ANIMALS
B) PLASMA AND MILK COLLECTION
Blood and milk samples of 10 adult females and blood
samples of their 48 offspring were collected on the day of
birth and weekly for 120 days postpartum. Punctures were
made in the jugular vein of puppies and the radial vein of
adults to draw blood samples using disposable sterile
syringes containing heparin. The plasma was separated, aliquoted in individual vials, marked and stored at -20°C until
use.
C) OBTAINEMENT OF ANTIGENS FOR IMMUNOLOGIC TESTS
a) Antigen for indirect fluorescent antibody assay
(IFA).
L. mexicana (strain MHOM/BR/1960/BH6) promastigotes
were used, which had been maintained in logarithmic growth
in liver infusion triptose culture medium. After 48 hours of
cultivation, the parasites were fixed in 5 % formaldehyde for
thirty minutes followed by 500-g centrifugation for 10
minutes. The sediment was resuspended in PBS, pH 7.2 and
Revue Méd. Vét., 2001, 152, 4, 317-324
PASSIVE TRANSMISSION OF HUMORAL AND CELLULAR IMMUNITY IN CANINE VISCERAL LEISHMANIASIS
319
submitted to further centrifugation. This process was repeated three times and the sediment was then resuspended in
PBS. Ten microliter of the suspension were placed on slides
for immunofluorescence with a concentration of 10-20 promastigotes per field. After being air-dried, the slides were
wrapped in blotting paper then aluminum foil and stored at
-20°C until use.
b) Antigens for delayed hypersensitivity skin tests.
Antigens used for the skin test were made separately from
extracts of two Leishmania strains : L. (L) chagasi (strain
MHOM/BR/1972/BH 46) and L. (V.) braziliensis (strain
MCAN/BR/1972/C348) according to the method described
by MELO et al. (1977) [22].
D) IMMUNOLOGIC TESTS
a) Humoral immunity.
Was evaluated by IFA according to the method described
by CAMARGO (1964) [4] in the plasma and milk of adults
and in the plasma of offspring using anti-IgG
(Biomanguinhos, Fiocruz, Rio de Janeiro) and anti-IgM
(Bethyl-Laboratories, Inc., USA). The results were expressed
in antibody titers, which were obtained by serially diluting
the plasma by a factor of 2 until a negative result was reached. IgG and IgM were analyzed in all samples collected
from puppies starting on the day of birth until 120 days postpartum.
b) Cellular immunity.
Was analyzed by skin test to evaluate the delayed hypersensitivity type of response. The test was performed on the
control animal and her litter (n=9) at 9 days postpartum and
in 5 adult females and their respective litters that were aged
8, 15, 36, 39, 57 and 75 days.
Each animal received 200 µl of antigen containing 200 µg
of protein via the intradermal route on the inside of the thigh
according to the technique described in GENARO et al.
(1988) [9]. The L. (L.) chagasi antigen was injected into the
right thigh and the L. (V.) braziliensis antigen into the left,
both solutions were prepared as described above. All adults
were given the test on the same day as their litter. Animal 13
had a litter of 10 offspring, half of which received the skin
test at 8 days of age and the other half at 75 days postpartum.
The diameter of the injection site was measured 72 hours
after the test was given. Reactions measuring 5mm or more
were considered positive [9].
Results
A) IgG TITER
IgG-class anti-Leishmania antibodies were detected in all
litters. These antibodies were present in the plasma of offspring starting on the day of birth and in the blood, colostrum
and milk of seropositive adults.
It was possible to collect blood from the offspring of adult
number 4 (Figure 1) before they had contact with maternal
Revue Méd. Vét., 2001, 152, 4, 317-324
FIGURE 1. — Levels of IgG-class anti-Leishmania antibodies detected by
IFA in adult N° 4, in her litter and in her milk on the respective days postpartum.
colostrum. These samples did not have anti-Leishmania antibodies but the next samples (day 7) had an average titer of
1:220, which decreased until day 28, the last collection that
contained antibodies.
Adults’ plasma IgG levels were constant in adults numbered 1, 4, 5, and 10. The remainder had slight fluctuations
during the postpartum period. That was not the case with IgG
in the milk of all adults, which declined in general between
days 7 and 14.
Table I lists the plasma and milk antibody titers of adult
dogs on the day they gave birth, the average IgG titer during
the nursing phase and the duration of IgG in the milk and in
the plasma of offspring. The duration of passive antiLeishmania antibodies in the offspring varied between 14
and 70 days with an average of 32.1 ±15.3 days.
B) IgM TITER
Table I shows the results of IFA analysis of IgM. The adult
females’ IgM titer on the day of delivery varied between 1:20
and 1:80, with 3 of the 10 tested adults presenting negative
IFA for this immunoglobulin. None of the offspring had positive IgM on any of the tests, which were performed on
samples collected on the day of birth and weekly until 120
days postpartum.
C) DELAYED TEST OF HYPERSENSITIVITY (DTH)
All leishmaniasis seropositive adults used in this study also
had positive reactions on the DTH (skin test) for one or both
of the Leishmania antigens studied here.
The results for each animal can be seen in Table II along
with the animal’s age and the increase in size in millimeters
at the application site of each antigen. Of the 25 offspring of
positive mothers used in this experiment, 18 (72 %) had positive results. Of the seven with negative results, two were
from the same litter and were 39 days old while the other 5
also came from one litter and were 75 days old. However,
when only offspring less than 60 days old were considered,
18/20 (90 %) were positive, and in tests done after this age,
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ANDRADE (H.M.) ET COLLABORATEURS
ND = test not done, Neg- negative test
TABLE I. — Antibody levels in plasma and milk of adult female dogs with visceral leishmaniasis on day of birth
and during nursing, and the duration of IgG in maternal milk and offspring plasma.
none of 5 that were originally positive were reactive. The
control animal and her litter (n=9) were not reactive to any of
the antigens.
The litter of animal 13 was tested at two different ages.
Half the litter was tested at 8 days old, with 100 % positive
and the other half at 75 days old, with 100 % negative (Table
II).
Figure 2 illustrates the number of offspring tested and the
number of positive reactions on the DTH according to age on
the day of the test.
In dogs, passive antibody transmission is better described
for viral diseases like canine distemper, parvoviruses and
infectious canine hepatitis. An in-utero presence of canine
distemper-neutralizing antibodies in gnotobiotic litters deprived of colostrum and in offspring free of pathogens has been
observed. These maternally-derived antibodies protected the
offspring from fatal infection with the virulent canine distemper virus. This protection was associated with lymphope-
Discussion
VL diagnosis can easily be made based on serologic tests.
Exceptionally strong reactions to VL antibodies indicate the
presence of a large number of parasites in the bone marrow or
the spleen, organs that play a fundamental role in the production and subsequent maturation of antibodies [26].
In Brazil, serologic survey of canine populations is one of
the measures that has been adopted to identify positive animals to be eliminated as a means of controlling the disease.
However, as there is not adequate data related to the passive
transfer of immunity in CVL, serologically positive offspring
may be being destroyed when they are merely exhibiting passively-acquired antibodies.
FIGURE 2. — Delayed test of hypersensitivity. Number of offspring tested
(■) and number of offspring with positive DHT ( ) according to animals’ age.
Revue Méd. Vét., 2001, 152, 4, 317-324
MISE AU POINT ET ÉVALUATION D’UNE MÉTHODE PAR PCR
*Results from test done when offspring were 8 days old.
TABLE II. — Results of delayed test of hypersensitivity using Leishmania
antigens in adult female dogs with visceral leishmaniasis. Adults are
represented by number and their respective offspring are identified by
that number followed by a letter.
nia, viremia and the temporary suppression of the lymphatic
response to PHA-P [14]. In the presence of maternal antibodies, the ability of vaccines to elicit a serologic response is
dose related (antibody titer) [3]. Newborn hamsters of
mothers infected with L. donovani responded better to immunization with amastigotes than offspring of uninfected
mothers. This suggests specific sensitivity, which could have
been provoked by the passage of soluble parasite antigens or
from the sensitized cells of the mother to fetus during gestation and/or nursing [12].
The leishmaniasis are chronic afflictions in which, as is
expected, there is an important production of IgM in the iniRevue Méd. Vét., 2001, 152, 4, 317-324
321
tial stages of the disease and subsequently the development
of a predominantly IgG response [16]. The present study has
proven that IgG-class anti-Leishmania antibodies are transferred from positive mothers to their offspring, since this type
of immunoglobulin was detected in all the litters in concentrations that were inversely proportional to the age of the offspring. Even though IgM was not detected in 0-120-day-old
puppies, the diagnosis of acute infection in offspring through
the detection of specific IgM cannot be recommended due to
the fact that this immunoglobulin is not always be detected in
the acute phase of CVL [7, 11, 19].
The present study showed that passive transfer of IgG-class
antibodies occurs through colostrum since these antibodies
were not detected in offspring that did not ingest the colostrum of their infected mothers, yet were identified shortly
after the first nursing session. Passively-acquired antibodies
remained between 14 and 70 days with average duration of
32.1 ±15.3 days. In dogs and cats, postpartum transmission
of immunoglobulins is not limited to a few hours after birth
but can continue for a long period, often extending over
almost the entire course of lactation [2].
In all the litters of infected mothers studied here, antibody
levels in the offspring declined quicker than in the milk,
which can be explained by both the decrease in immunoglobulin permeability in the intestine [25], as well as by the
young animals’ rapid growth since this growth increases the
amount of blood and consequently dilutes the concentration
of maternal antibodies [2].
These results are important in determining the best age to
vaccinate animals against leishmaniasis, when such vaccine
is available. According to TIZARD (1996) [25], maternal
antibodies do not only inhibit immunoglobulin synthesis but
they also avoid successful vaccination in young animals and
the duration of these antibodies, called the refractory period,
depends on the quantity of transferred antibodies and the
half-life of the involved immunoglobulins.
On the other hand, it is important to determine the age at
which serologically positive animals can be sacrificed
without the risk of them being positive due to passive immunity. As seen here, IgG-class anti-Leishmania antibodies
were detected in puppies up to 70 days postpartum, which
indicates that canine offspring exhibiting positive serology
for leishmaniasis up to that age should not be sacrificed.
A positive serologic test in dogs less than 90 days old could
raise the suspicion of premature infection via phlebotomines
shortly after birth. However, GENARO et al. (1988) [9] and
MELO et al. (1998) [21] showed that in dogs, IgG and IgM
anti-Leishmania antibodies were only detected between 3
and 5 months after experimental infection with Leishmania
(l.) chagasi.
The evaluation of cellular immunologic response to leishmaniasis in dogs using DTH has been performed by several
authors who used antigens of different Leishmania species
[1, 9, 10, 13, 20, 23, 24]. Analyzing cellular response in offspring using the DTH and L. chagasi and L. braziliensis antigens, we found that L. chagasi antigens had good results for
this type of test and that 90% of offspring under 60 days of
age were reactive to L. chagasi and/or L. braziliensis anti-
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ANDRADE (H.M.) ET COLLABORATEURS
gens, whereas not one of the puppies older than 60 days had
this type of reaction.
These data regarding passive transmission of cellular
immunity seem to indicate that mothers with positive DTH
for Leishmania antigens have offspring with the same response, while this reaction is absent in animals over 75 days
old. These results agree with those of TIZARD (1996) [25],
which hold that there is evidence that cell-mediated immunity can be transferred to newborns via colostrum.
Our data indicate that offspring of VL-positive mothers
passively acquired cellular and humoral immunity and that
immunity was transitory. Further experiments evaluating IgG
subclasses and the phenotype of mononuclear cells could
provide new insight to better understand the maternal-fetal
relationship in CVL.
Based on these findings, we suggest that seropositive offspring, under 70 days of age, of infected mothers should not
be sacrificed as part of ZVL control programs. Rather, these
animals should be monitored in order to confirm diagnosis.
This is an important measure considering the high psychological and/or commercial value of the animals, as well as the
need to maintain genetic characteristics of certain races.
Acknowledgements
We are grateful to the Zoonosis Control Center of the City
of Belo Horizonte for donating naturally-infected animals
that were to be sacrifyed part of the ZVL control program.
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