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Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from symptomatic and asymptomatic naturally infected bitches
Veterinary Parasitology 165 (2009) 327–331
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from symptomatic and asymptomatic naturally infected bitches Kristel Kegler Pangrazio a, Erica A. Costa b, Shyrley P. Amarilla a, Ada G. Cino a, Teane M.A. Silva b, Tatiane A. Paixa˜o b, Luciana F. Costa b, Enrique G. Dengues a, Andres Avalos Ruiz Diaz a, Renato L. Santos b,* a
Departamento de Ciencias Patolo´gicas, Facultad de Ciencias Veterinarias, Universidad Nacional de Asuncio´n, Ruta Mcal. Estigarribia km 10, San Lorenzo, Paraguay Departamento de Clinica e Cirurgia Veterina´rias, Escola de Veterina´ria, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 March 2009 Received in revised form 3 July 2009 Accepted 8 July 2009
Visceral leishmaniasis (VL) is primarily transmitted by an invertebrate vector, but transmission in the absence of the vector has been reported. Vertical transmission of VL has been described in man and dogs. The aim of this study was to evaluate the distribution of Leishmania amastigotes in fetal organs and histopathologic changes associated with parasitism and to determinate the frequency of transplacental transmission and potential of vertical transmission by symptomatic and asymptomatic pregnant bitches. Symptomatic (n = 4) and asymptomatic (n = 4) pregnant bitches, serologically and parasitologically positive for Leishmania sp., carrying a total of 53 fetuses (26 from symptomatic and 27 from asymptomatic bitches) were selected at the Veterinary Hospital of the National University of Asuncion, Paraguay. Samples of placenta and fetal organs such as liver, spleen, lymph nodes, bone marrow, kidney and heart were histologically evaluated and processed for immunodetection of amastigotes and PCR. There were no lesions compatible with VL in fetal tissues in spite of the presence of amastigotes, particularly in lymphoreticular tissues. However, fetal hepatocytes had marked degenerative changes that were independent of the presence of amastigotes in liver. Twenty-six out of 53 placentas (13 symptomatic and 13 asymptomatic) and a total of 17 fetuses out of 53 (nine symptomatic and eight asymptomatic) were PCR positive. Together these findings indicate a high frequency of transplacental transmission and no differences in the potential of transmission when symptomatic were compared to asymptomatic pregnant bitches. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Visceral leishmaniasis Leishmania Vertical transmission Transplacental transmission
1. Introduction Visceral leishmaniasis (VL) is a zoonotic disease of major public health and veterinary importance that is caused by a protozoa belonging to the genus Leishmania, grouped into the donovani complex, including L. donovani
* Corresponding author. Tel.: +55 31 3409 2239; fax: +55 31 3409 2230. E-mail address: [email protected] (R.L. Santos). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.07.013
and L. infantum in the Old World and L. chagasi in New World (Shaw, 1994). VL is considered endemic in Mediterranean countries and has a wide distribution throughout Latin America extending from Mexico to Argentina (Lainson and Rangel, 2005). Although several wildlife species can potentially be infected (Luppi et al., 2008), the domestic dog is the major reservoir of VL for humans, particularly within urban areas (Diniz et al., 2008). In Asuncion (capital of Paraguay), where VL is endemic in periurban and metropolitan areas, canine
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seroprevalence ranged from 11.8% to 37% between 1999 and 2005 (Canese, 2000; Avalos et al., 2005 unpublished data). Importantly, our group has demonstrated that L. chagasi has a tropism for some genital organs in male dogs, namely the epididymis, glans penis and prepuce (Diniz et al., 2005). Although there is no apparent tropism to the canine non-pregnant female genital system (Silva et al., 2008), there are reports of abortion with necrotizing placentitis with large amounts of intralesional Leishmania amastigotes in a woman (Eltoum et al., 1992), and in an asymptomatic pregnant bitch (Dubey et al., 2005). Indeed, placenta with delayed maturation, chronic infarction and ischemic changes in placental villi with absence of Leishmania amastigotes has also been described in a woman with VL (Caldas et al., 2003). Transmission of VL among dogs and from dogs to humans usually occurs through the bite of infected sand flies belonging to the genus Phlebotomus in the Old World and Lutzomyia in the New World, which are the vectors of Leishmania (Killick-Kendrick, 1990). However, transmission in the absence of the vector has been reported, including cases due to blood transfusion in humans and dogs (Harris, 1994; Owens et al., 2001; Giger et al., 2002). Interestingly, it has been reported a prevalence of 41% in a foxhound kennel in the absence of a known suitable biological vector supporting the hypothesis of direct or vertical transmission (Gaskin et al., 2002). Genital lesions in male dogs are associated with shedding of Leishmania in the semen (Diniz et al., 2005), and we have recently demonstrated venereal transmission from naturally infected males to susceptible bitches (Silva et al., 2009). Congenital transmission of VL has been recognized in humans long ago (Low and Cooke, 1926). Indeed, more than 10 cases of newborns, born to either symptomatic or asymptomatic mothers, who developed clinical symptoms of VL during their first year of life in absence of the biological vector have been documented (reviewed by Meinecke et al., 1999; Figueiro-Filho et al., 2004). Similarly, vertical route was demonstrated to occur in naturally infected dogs, resulting in positive newborn puppies at the time of birth (Masucci et al., 2003). Transplacental transmission of Leishmania sp. was also confirmed in experimentally infected BALB/mice, and in one Beagle whose fetuses were removed from the uterus by c-section thus preventing the possibility of transvaginal transmission (Rosypal et al., 2005; Rosypal and Lindsay, 2005). Considering these clear indications of vertical transmission of VL in dogs and the lack of information about frequency of transmission through this route and possible differences in the transmission potential of symptomatic and asymptomatic bitches, the aim of this study was to assess the distribution of Leishmania amastigotes in fetal organs and placenta as well as to evaluate histopathological changes associated with parasitism. 2. Materials and methods 2.1. Animals and experimental design Eight mixbreed bitches naturally infected with L. chagasi with 50–60 days of pregnancy were selected for this study
after clinical and ultrasound examination at the Veterinary Hospital ‘‘Prof. Dr. Vicente Nunez’’ (National University of Asuncion – UNA, Paraguay). These bitches were identified by letters, and they were all serologically positive for Leishmania. Serum samples were analyzed using a commercial kit containing immunochromatographic strips with a recombinant Leishmania antigen k39 and a dominant amastigote antigen of L. chagasi (rK39), which is highly sensitive and specific for Leishmania donovani complex (Sundar et al., 2002). Bone marrow, spleen and cervical lymph node aspirates were also obtained for parasitological diagnosis. Cytological smears were fixed with methanol, air-dried and stained with 10% Giemsa. Leishmania amastigotes were detected in bone marrow and spleen of all infected pregnant bitches, and in lymph nodes of all bitches except bitch H. These eight pregnant bitches were clinically classified into two groups, one (i) symptomatic (n = 4; bitches A, C, D, E) that exhibited classical sings of VL such as alopecia, dry exfoliative dermatitis and/or ulcers, lymphandenopathy, onychogryphosis, weight lose and anemia; and (ii) asymptomatic (n = 4; bitches B, F, G, H) without any clinical sign of VL. Five pregnant bitches (A, B, D, E, G) were euthanatized with an overdose of thiopental and intravenous injection of potassium chloride, and the necropsy was preformed immediately after euthanasia. Three bitches (C, F, H) underwent ovariohysterectomy. Fetal crown-rump length measurement was employed to confirm the age of fetuses (Noden and Lahunta, 1985). 2.2. Histopathology and immunohistochemistry Fragments of the placenta, mesenteric lymph nodes, spleen, kidney, liver, heart, and femoral bone marrow were collected for histopathology and immunohistochemistry. Tissue samples were fixed in 10% buffered formalin for 24– 48 h, dehydrated, cleared, embedded in paraffin, cut into 5mm-thick sections, and stained with hematoxylin and eosin (HE) or further processed for immunohistochemistry as previously described (Tafuri et al., 2004). Briefly, 4-mmthick sections from paraffin embedded tissue samples were mounted onto silane-coated slides, hydrated, incubated with 4% hydrogen peroxide for 30 min, washed in PBS, incubated in 26 mg/ml of skim milk as blocking solution for 30 min, incubated with the primary antibody at 4 8C in a humid chamber for 18–24 h, washed in PBS, incubated with biotinylated secondary antibody for 30 min at room temperature and incubated with streptavidin–peroxidase complex (LSAB+ Kit, DAKO Corporation, Carpinteria, CA) for 30 min. Finally, reactions were revealed by diaminobenzidine (DAB) and the slides counter-stained with hematoxylin. 2.3. PCR Samples of placenta, lymph node, spleen, kidney, liver, heart and femoral bone marrow were collected individually and stored at 20 8C until processing for DNA extraction using the guanidine isothiocyanate protocol (Pitcher et al., 1989). PCR was preformed as previously described by Lachaud et al. (2002). Briefly, 1 mg of template DNA was added to 15.6 ml of a solution containing 5.1 ml of 10 PCR
K.K. Pangrazio et al. / Veterinary Parasitology 165 (2009) 327–331
329
Table 1 PCR detection of Leishmania kDNA in fetal tissues and placentas from symptomatic and asymptomatic naturally infected bitches. Bitch
Symptomatic bitches
Asymptomatic bitches
A
C
D
E
Total
B
F
G
H
Total
Fetuses (n) Positive placentas (n) Positive fetuses (n)
8 7 0
6 0 1
3 3 2
9 3 6
26 (100.0%) 13 (50.0%) 9 (34.6%)
6 5 5
8 3 2
6 3 0
7 2 1
27 (100.0%) 13 (48.1%) 8 (29.6%)
buffer, 5.1 ml of a 200 mM dNTP solution, 0.2 mM primer A (50 -CTTTTCTGGTCCCGCGGGTAGG-30 ), 0.2 mM primer B (50 -CCACCTGGCCTATTTTACACCA-30 ), 3 mM MgCl2, and 1.5 U of Taq polymerase. Cycling parameters were denaturation at 94 8C for 4 min; 49 cycles of denaturation (94 8C for 30 s), annealing (59 8C for 30 s), and extension (72 8C for 30 s), and a final extension at 72 8C for 10 min. PCR products (145 bp) were resolved by 2% agarose gel electrophoresis. 2.4. Statistical analysis The frequency of positivity was compared among groups by Fisher’s exact test with Graphpad Instat software (version 3.05, Graphpad Software, Inc., San Diego, CA).
3. Results Congenital transmission of VL was demonstrated by PCR. Overall 32% (n = 53) of fetuses from naturally infected bitches were positive, with no significant difference in the frequency of positive fetuses when symptomatic bitches were compared to asymptomatic (P > 0.05; Table 1). Interestingly, none of the bitches had all fetuses infected. The rate of infection in fetuses from individual gestations ranged from 0 to 83.3% (5/6; bitch B). Leishmania kDNA was amplified in 26 of 53 placentas (49%) with no significant differences in the frequency of positivity between symptomatic and asymptomatic bitches. None of the 53 fetuses and their placentas had any gross lesion. Microscopically, no lesions were observed in any placenta, while placental maturation was compatible with their gestational age. Despite the high frequency of positive placentas by PCR, no amastigotes were observed in any of the samples either in HE-stained sections or by immunohistochemistry. Histologically, a few amastigotes mostly associated with macrophages were observed in the liver (Fig. 1), spleen, lymph node, and bone marrow of six, four, three, and two
fetuses, respectively, belonging to both groups (symptomatic and asymptomatic). No significant histological fetal changes were observed in association with Leishmania amastigotes. Interestingly, 55-day-old fetuses had moderate diffuse fatty degeneration of hepatocytes (Fig. 1), which was more severe in all 60-day-old fetuses. This degenerative hepatopathy was not been related to the degree of parasitism or with the clinical status of the bitch. No amastigotes were observed in the kidneys and hearts of all fetuses. As expected, a much higher sensitivity for detection of amastigotes was obtained with IHC when compared with HE-stained sections in fetal tissues. Amastigotes were labeled mostly intracellularly within macrophages in the spleen, bone marrow, lymph node and liver, from ten, nine, eight, and eight fetuses, respectively. There was an absence of immunolabeled amastigotes in the placenta, kidney and heart. Distribution of amastigotes in the placenta and fetal organs by PCR, IHC and HE techniques is summarized in Table 2.
4. Discussion Although, our previous work has demonstrated that VL is not associated with genital lesions and parasitism in female reproductive tract (Silva et al., 2008), vertical transmission has already been reported in humans, mice and dogs (Meinecke et al., 1999; Rosypal et al., 2005; Masucci et al., 2003). Here we demonstrated for the first time that frequency of transplacental transmission does not differ between symptomatic and asymptomatic pregnant bitches, indicating that the clinical status of the bitch (either asymptomatic or symptomatic) is not predictive of its potential for transplacental transmission of VL. Frequency of transplacental transmission was very low in pregnant BALB/c mice experimentally infected with L. infantum, corresponding to 4 of 88 pups and 3 of 16 placentas from inoculated mice (Rosypal et al., 2005). In
Table 2 Distribution of Leishmania sp. in fetal tissues and placenta. Comparison between polymerase chain reaction, immunohistochemistry and hematoxylin and eosin stained histological sections. Organ (n = 53)
PCRa
IHCa
HEa
Liver Spleen Bone marrow Lymph nodes Kidney Heart Placenta
15 15 15 11 9 9 26
8 10 9 8 0 0 0
6 4 2 3 0 0 0
a
(28.3%) (28.3%) (28.3%) (20.7%) (17.0%) (17.0%) (49.0%)
(15.1%) (18.9%) (17.0%) (15.1%)
(11.3%) (7.5%) (3.8%) (5.6%)
Abbreviations—PCR: polymerase chain reaction; IHC: immunohistochemistry; HE: hematoxylin and eosin.
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Fig. 1. Fetal liver. (A) Marked diffuse vacuolization of hepatocytes with intracellular amastigotes (inset). H.E. Bar = 50 mm. (B) Immunolabeling of intracellular Leishmania sp. amastigotes in a Kupffer cell. Streptavidin– peroxidase complex. Bar = 10 mm.
contrast, Masucci et al. (2003) reported a high frequency of positive newborn puppies born to naturally infected bitches, which contrasted to the mouse model (Rosypal et al., 2005), suggesting that possibly transvaginal infection could increase the number of infected newborn puppies. In this study, 32% of fetuses and 49% of the placentas were PCR positive. This finding support the notion that Leishmania is transmitted to the progeny mostly though the placenta in dogs, and that the transvaginal route seems to play a secondary role, which is supported by our previous study indicating absence of amastigotes in the vaginal mucosa of naturally infected bitches (Silva et al., 2008). Despite the absence of lesions and immune-detection of amastigotes in the placenta, Leishmania kDNA was detected in 49% of the placentas from both symptomatic and asymptomatic bitches, indicating that although in extremely low loads the organism is often present in the placenta. The placenta has an extremely high blood flow, which could carried amastigotes, resulting in DNA amplification, although the absence of amastigotes in HE- and IHC-stained sections indicate that the placenta is not likely a target tissue for Leishmania infection, whereas it clearly serves as a transmission route to the fetuses. It is noteworthy that the PCR protocol employed in this study is
extremely sensitive, detecting up to 0.0001 amastigotes (parasite equivalent) per reaction (Lachaud et al., 2002). This study demonstrated that the distribution of Leishmania amastigotes in fetal organs is similar to that observed in post-natal or adults dogs, with amastigotes distributed mostly in lymphoreticular organs such as the spleen, bone marrow, lymph nodes and liver, in which amastigotes were observed in HE- or IHC-stained tissue sections. Although amastigotes were not observed in fetal kidneys and hearts, in some cases these organs were PCR positive indicating infection with a very low parasite load. Apparently the clinical course of the disease seems to be identical in congenital transmitted and otherwise acquired VL in humans (Meinecke et al., 1999), but there is no information available about the clinical features of vertically transmitted VL in dogs, which may be similar to the disease transmitted through the biological vector since the tissue distribution of the parasite in the fetus is similar to naturally infected adult dogs as demonstrated in this study. Importantly, the organism in the fetus is not associated with the classical lesions of VL in adult dogs, which may be due to the immature state of the fetal immune system in spite of the fact that the fetus may be responsive in utero to a range of antigens during the final stages of gestation in dogs (Krakowka, 1998). It is noteworthy that during pregnancy the immune response is modulated to avoid deleterious effect on the developing fetus biasing adaptive immunity towards a Th2 response. It is thought that the down-regulation of adaptive immune responses make pregnant females more susceptible to a wide variety of pathogens and thus increasing the risk of transplacental transmission (Wegmann et al., 1993). There was a moderate to severe fatty degeneration of hepatocytes in all fetal livers, particularly at the end of gestation (60-day-old fetuses), which was independent of the presence of amastigotes. In canine VL, hepatic involvement has been described in symptomatic and asymptomatic adult dogs. Histopathologic changes includes swelling of hepatocytes associated with changes in fat content in response to inflammation, mainly related to the presence of amastigotes within Kupffer cells (Keenan et al., 1984; Melo et al., 2008). The mechanism of this degenerative hepatic changes is not clear, although it has been demonstrated that pathogen components may deplete glutathione (GSH), a potent antioxidant, and increase the expression of iNOS and thus NO production in fetal liver promoting degeneration by lipid peroxidation (Xu et al., 2005). Moreover, in late gestation, GSH is apparently physiological down-regulated to promote regression of hematopoietic cells exposing hepatocytes to some oxidative stress (Sato et al., 2003). However, our results do not support any cause and effect association between this hepatic degenerative changes and parasite loads either in the fetus or in the pregnant bitch. In conclusion, distribution of amastigotes is similar in canine fetal and post-natal adult organs. Therefore, puppies born to infected bitches should be considered as potential reservoirs and source of infection of VL. Hence, the use of infected bitches for reproduction should definitely be avoided to prevent vertical transmission, which according to this study is an important route of transmission and therefore spreading of canine VL.
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Acknowledgements R.L.S. is a recipient of a fellowship from the ‘‘Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico’’ (CNPq, Brası´lia, Brazil). E.A.C. is supported by the ‘‘Fundac¸a˜o de Amparo a Pesquisa do Estado de Minas Gerais’’ (FAPEMIG, Belo Horizonte, Brazil). Work in RLS lab is supported by grants from CNPq and FAPEMIG. References Caldas, J.M., Costa, M.L., Gama, E.A., Ramos, A.G., Barral, A., 2003. Visceral Leishmaniasis in pregnancy: a case report. Acta Trop. 88, 39–43. Canese, A., 2000. Leishmaniasis visceral canina en el a´rea metropolitana de la ‘‘Gran Asuncio´n’’, Paraguay 2000. Medicina (Buenos Aires) 60 (Suppl. III), 65. Diniz, S.A., Melo, M.S., Borges, A.M., Bueno, R., Reis, B.P., Tafuri, W.L., Nascimento, E.F., Santos, R.L., 2005. Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs. Vet. Pathol. 42, 650–658. Diniz, S.A., Silva, F.L., Carvalho Neta, A.V., Bueno, R., Guerra, R.M.S.N.C., Abreu-Silva, A.L., Santos, R.L., 2008. Animal reservoirs for visceral leishmaniasis in densely populated urban areas. J. Infect. Develop. Countries 2, 24–33. Dubey, J.P., Rosypal, A.C., Pierce, V., Scheinberg, S.N., Lindsay, D.S., 2005. Placentitis associated with leishmaniasis in a dog. J. Am. Vet. Med. Assoc. 227, 1266–1269. Eltoum, I.A., Ziljstra, E.E., Ali, M.S., Ghalib, H.W., Satti, M.M.H., Eltoum, B., El-Hassan, A.M., 1992. Congenital kala-azar and leishmaniasis in the placenta. Am. J. Trop. Med. Hyg. 46, 57–62. Figueiro-Filho, A.E., Duarte, G., El-Beitune, P., Quintana, S.M., Lemos Maia, T., 2004. Visceral leishmaniasis (kala-azar) and pregnancy. Infect. Dis. Obstet. Gynecol. 12, 31–40. Gaskin, A.A., Schantz, P., Jackson, J., Birkenheuer, A., Tomlinson, L., Gramiccia, M., Levy, M., Steurer, F., Kollmar, E., Hegarty, B.C., Ahn, A., Breitschwerdt, E.B., 2002. Visceral leishmaniasis in a New York foxhound kennel. J. Vet. Intern. Med. 16, 34–44. Giger, U., Oakley, D.A., Owens, S.D., Schantz, P., 2002. Leishmania donovani transmission by packed RBC transfusion to anemic dogs in the United States. Transfusion 42, 381–383. Harris, M.P., 1994. Suspected transmission of leishmaniasis. Vet. Rec. 135, 339. Keenan, C.M., Hendricks, L.D., Lightner, L., Johnson, A.J., 1984. Visceral leishmaniasis in the German shepherd dog. II. Pathology. Vet. Pathol. 21, 80–86. Killick-Kendrick, R., 1990. Phlebotomine vectors of the leishmaniases: a review. Med. Vet. Entomol. 4, 1–24. Krakowka, S., 1998. Immunology of the dog: ontogeny of the immune system. In: Pastoret, P., Bazin, H., Briebel, P., Govaerts, A. (Eds.), Handbook of Vertebrate Immunology. Academic Press, San Diego, p. 271. Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J.P., Bastien, P., 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40, 210–215. Lainson, R., Rangel, E.F., 2005. Lutzomyia longipalpis and eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil—a review. Mem. Inst. Oswaldo Cruz 100, 811–827.
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Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from symptomatic and asymptomatic naturally infected bitches Kristel Kegler Pangrazio a, Erica A. Costa b, Shyrley P. Amarilla a, Ada G. Cino a, Teane M.A. Silva b, Tatiane A. Paixa˜o b, Luciana F. Costa b, Enrique G. Dengues a, Andres Avalos Ruiz Diaz a, Renato L. Santos b,* a
Departamento de Ciencias Patolo´gicas, Facultad de Ciencias Veterinarias, Universidad Nacional de Asuncio´n, Ruta Mcal. Estigarribia km 10, San Lorenzo, Paraguay Departamento de Clinica e Cirurgia Veterina´rias, Escola de Veterina´ria, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 March 2009 Received in revised form 3 July 2009 Accepted 8 July 2009
Visceral leishmaniasis (VL) is primarily transmitted by an invertebrate vector, but transmission in the absence of the vector has been reported. Vertical transmission of VL has been described in man and dogs. The aim of this study was to evaluate the distribution of Leishmania amastigotes in fetal organs and histopathologic changes associated with parasitism and to determinate the frequency of transplacental transmission and potential of vertical transmission by symptomatic and asymptomatic pregnant bitches. Symptomatic (n = 4) and asymptomatic (n = 4) pregnant bitches, serologically and parasitologically positive for Leishmania sp., carrying a total of 53 fetuses (26 from symptomatic and 27 from asymptomatic bitches) were selected at the Veterinary Hospital of the National University of Asuncion, Paraguay. Samples of placenta and fetal organs such as liver, spleen, lymph nodes, bone marrow, kidney and heart were histologically evaluated and processed for immunodetection of amastigotes and PCR. There were no lesions compatible with VL in fetal tissues in spite of the presence of amastigotes, particularly in lymphoreticular tissues. However, fetal hepatocytes had marked degenerative changes that were independent of the presence of amastigotes in liver. Twenty-six out of 53 placentas (13 symptomatic and 13 asymptomatic) and a total of 17 fetuses out of 53 (nine symptomatic and eight asymptomatic) were PCR positive. Together these findings indicate a high frequency of transplacental transmission and no differences in the potential of transmission when symptomatic were compared to asymptomatic pregnant bitches. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Visceral leishmaniasis Leishmania Vertical transmission Transplacental transmission
1. Introduction Visceral leishmaniasis (VL) is a zoonotic disease of major public health and veterinary importance that is caused by a protozoa belonging to the genus Leishmania, grouped into the donovani complex, including L. donovani
* Corresponding author. Tel.: +55 31 3409 2239; fax: +55 31 3409 2230. E-mail address: [email protected] (R.L. Santos). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.07.013
and L. infantum in the Old World and L. chagasi in New World (Shaw, 1994). VL is considered endemic in Mediterranean countries and has a wide distribution throughout Latin America extending from Mexico to Argentina (Lainson and Rangel, 2005). Although several wildlife species can potentially be infected (Luppi et al., 2008), the domestic dog is the major reservoir of VL for humans, particularly within urban areas (Diniz et al., 2008). In Asuncion (capital of Paraguay), where VL is endemic in periurban and metropolitan areas, canine
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seroprevalence ranged from 11.8% to 37% between 1999 and 2005 (Canese, 2000; Avalos et al., 2005 unpublished data). Importantly, our group has demonstrated that L. chagasi has a tropism for some genital organs in male dogs, namely the epididymis, glans penis and prepuce (Diniz et al., 2005). Although there is no apparent tropism to the canine non-pregnant female genital system (Silva et al., 2008), there are reports of abortion with necrotizing placentitis with large amounts of intralesional Leishmania amastigotes in a woman (Eltoum et al., 1992), and in an asymptomatic pregnant bitch (Dubey et al., 2005). Indeed, placenta with delayed maturation, chronic infarction and ischemic changes in placental villi with absence of Leishmania amastigotes has also been described in a woman with VL (Caldas et al., 2003). Transmission of VL among dogs and from dogs to humans usually occurs through the bite of infected sand flies belonging to the genus Phlebotomus in the Old World and Lutzomyia in the New World, which are the vectors of Leishmania (Killick-Kendrick, 1990). However, transmission in the absence of the vector has been reported, including cases due to blood transfusion in humans and dogs (Harris, 1994; Owens et al., 2001; Giger et al., 2002). Interestingly, it has been reported a prevalence of 41% in a foxhound kennel in the absence of a known suitable biological vector supporting the hypothesis of direct or vertical transmission (Gaskin et al., 2002). Genital lesions in male dogs are associated with shedding of Leishmania in the semen (Diniz et al., 2005), and we have recently demonstrated venereal transmission from naturally infected males to susceptible bitches (Silva et al., 2009). Congenital transmission of VL has been recognized in humans long ago (Low and Cooke, 1926). Indeed, more than 10 cases of newborns, born to either symptomatic or asymptomatic mothers, who developed clinical symptoms of VL during their first year of life in absence of the biological vector have been documented (reviewed by Meinecke et al., 1999; Figueiro-Filho et al., 2004). Similarly, vertical route was demonstrated to occur in naturally infected dogs, resulting in positive newborn puppies at the time of birth (Masucci et al., 2003). Transplacental transmission of Leishmania sp. was also confirmed in experimentally infected BALB/mice, and in one Beagle whose fetuses were removed from the uterus by c-section thus preventing the possibility of transvaginal transmission (Rosypal et al., 2005; Rosypal and Lindsay, 2005). Considering these clear indications of vertical transmission of VL in dogs and the lack of information about frequency of transmission through this route and possible differences in the transmission potential of symptomatic and asymptomatic bitches, the aim of this study was to assess the distribution of Leishmania amastigotes in fetal organs and placenta as well as to evaluate histopathological changes associated with parasitism. 2. Materials and methods 2.1. Animals and experimental design Eight mixbreed bitches naturally infected with L. chagasi with 50–60 days of pregnancy were selected for this study
after clinical and ultrasound examination at the Veterinary Hospital ‘‘Prof. Dr. Vicente Nunez’’ (National University of Asuncion – UNA, Paraguay). These bitches were identified by letters, and they were all serologically positive for Leishmania. Serum samples were analyzed using a commercial kit containing immunochromatographic strips with a recombinant Leishmania antigen k39 and a dominant amastigote antigen of L. chagasi (rK39), which is highly sensitive and specific for Leishmania donovani complex (Sundar et al., 2002). Bone marrow, spleen and cervical lymph node aspirates were also obtained for parasitological diagnosis. Cytological smears were fixed with methanol, air-dried and stained with 10% Giemsa. Leishmania amastigotes were detected in bone marrow and spleen of all infected pregnant bitches, and in lymph nodes of all bitches except bitch H. These eight pregnant bitches were clinically classified into two groups, one (i) symptomatic (n = 4; bitches A, C, D, E) that exhibited classical sings of VL such as alopecia, dry exfoliative dermatitis and/or ulcers, lymphandenopathy, onychogryphosis, weight lose and anemia; and (ii) asymptomatic (n = 4; bitches B, F, G, H) without any clinical sign of VL. Five pregnant bitches (A, B, D, E, G) were euthanatized with an overdose of thiopental and intravenous injection of potassium chloride, and the necropsy was preformed immediately after euthanasia. Three bitches (C, F, H) underwent ovariohysterectomy. Fetal crown-rump length measurement was employed to confirm the age of fetuses (Noden and Lahunta, 1985). 2.2. Histopathology and immunohistochemistry Fragments of the placenta, mesenteric lymph nodes, spleen, kidney, liver, heart, and femoral bone marrow were collected for histopathology and immunohistochemistry. Tissue samples were fixed in 10% buffered formalin for 24– 48 h, dehydrated, cleared, embedded in paraffin, cut into 5mm-thick sections, and stained with hematoxylin and eosin (HE) or further processed for immunohistochemistry as previously described (Tafuri et al., 2004). Briefly, 4-mmthick sections from paraffin embedded tissue samples were mounted onto silane-coated slides, hydrated, incubated with 4% hydrogen peroxide for 30 min, washed in PBS, incubated in 26 mg/ml of skim milk as blocking solution for 30 min, incubated with the primary antibody at 4 8C in a humid chamber for 18–24 h, washed in PBS, incubated with biotinylated secondary antibody for 30 min at room temperature and incubated with streptavidin–peroxidase complex (LSAB+ Kit, DAKO Corporation, Carpinteria, CA) for 30 min. Finally, reactions were revealed by diaminobenzidine (DAB) and the slides counter-stained with hematoxylin. 2.3. PCR Samples of placenta, lymph node, spleen, kidney, liver, heart and femoral bone marrow were collected individually and stored at 20 8C until processing for DNA extraction using the guanidine isothiocyanate protocol (Pitcher et al., 1989). PCR was preformed as previously described by Lachaud et al. (2002). Briefly, 1 mg of template DNA was added to 15.6 ml of a solution containing 5.1 ml of 10 PCR
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Table 1 PCR detection of Leishmania kDNA in fetal tissues and placentas from symptomatic and asymptomatic naturally infected bitches. Bitch
Symptomatic bitches
Asymptomatic bitches
A
C
D
E
Total
B
F
G
H
Total
Fetuses (n) Positive placentas (n) Positive fetuses (n)
8 7 0
6 0 1
3 3 2
9 3 6
26 (100.0%) 13 (50.0%) 9 (34.6%)
6 5 5
8 3 2
6 3 0
7 2 1
27 (100.0%) 13 (48.1%) 8 (29.6%)
buffer, 5.1 ml of a 200 mM dNTP solution, 0.2 mM primer A (50 -CTTTTCTGGTCCCGCGGGTAGG-30 ), 0.2 mM primer B (50 -CCACCTGGCCTATTTTACACCA-30 ), 3 mM MgCl2, and 1.5 U of Taq polymerase. Cycling parameters were denaturation at 94 8C for 4 min; 49 cycles of denaturation (94 8C for 30 s), annealing (59 8C for 30 s), and extension (72 8C for 30 s), and a final extension at 72 8C for 10 min. PCR products (145 bp) were resolved by 2% agarose gel electrophoresis. 2.4. Statistical analysis The frequency of positivity was compared among groups by Fisher’s exact test with Graphpad Instat software (version 3.05, Graphpad Software, Inc., San Diego, CA).
3. Results Congenital transmission of VL was demonstrated by PCR. Overall 32% (n = 53) of fetuses from naturally infected bitches were positive, with no significant difference in the frequency of positive fetuses when symptomatic bitches were compared to asymptomatic (P > 0.05; Table 1). Interestingly, none of the bitches had all fetuses infected. The rate of infection in fetuses from individual gestations ranged from 0 to 83.3% (5/6; bitch B). Leishmania kDNA was amplified in 26 of 53 placentas (49%) with no significant differences in the frequency of positivity between symptomatic and asymptomatic bitches. None of the 53 fetuses and their placentas had any gross lesion. Microscopically, no lesions were observed in any placenta, while placental maturation was compatible with their gestational age. Despite the high frequency of positive placentas by PCR, no amastigotes were observed in any of the samples either in HE-stained sections or by immunohistochemistry. Histologically, a few amastigotes mostly associated with macrophages were observed in the liver (Fig. 1), spleen, lymph node, and bone marrow of six, four, three, and two
fetuses, respectively, belonging to both groups (symptomatic and asymptomatic). No significant histological fetal changes were observed in association with Leishmania amastigotes. Interestingly, 55-day-old fetuses had moderate diffuse fatty degeneration of hepatocytes (Fig. 1), which was more severe in all 60-day-old fetuses. This degenerative hepatopathy was not been related to the degree of parasitism or with the clinical status of the bitch. No amastigotes were observed in the kidneys and hearts of all fetuses. As expected, a much higher sensitivity for detection of amastigotes was obtained with IHC when compared with HE-stained sections in fetal tissues. Amastigotes were labeled mostly intracellularly within macrophages in the spleen, bone marrow, lymph node and liver, from ten, nine, eight, and eight fetuses, respectively. There was an absence of immunolabeled amastigotes in the placenta, kidney and heart. Distribution of amastigotes in the placenta and fetal organs by PCR, IHC and HE techniques is summarized in Table 2.
4. Discussion Although, our previous work has demonstrated that VL is not associated with genital lesions and parasitism in female reproductive tract (Silva et al., 2008), vertical transmission has already been reported in humans, mice and dogs (Meinecke et al., 1999; Rosypal et al., 2005; Masucci et al., 2003). Here we demonstrated for the first time that frequency of transplacental transmission does not differ between symptomatic and asymptomatic pregnant bitches, indicating that the clinical status of the bitch (either asymptomatic or symptomatic) is not predictive of its potential for transplacental transmission of VL. Frequency of transplacental transmission was very low in pregnant BALB/c mice experimentally infected with L. infantum, corresponding to 4 of 88 pups and 3 of 16 placentas from inoculated mice (Rosypal et al., 2005). In
Table 2 Distribution of Leishmania sp. in fetal tissues and placenta. Comparison between polymerase chain reaction, immunohistochemistry and hematoxylin and eosin stained histological sections. Organ (n = 53)
PCRa
IHCa
HEa
Liver Spleen Bone marrow Lymph nodes Kidney Heart Placenta
15 15 15 11 9 9 26
8 10 9 8 0 0 0
6 4 2 3 0 0 0
a
(28.3%) (28.3%) (28.3%) (20.7%) (17.0%) (17.0%) (49.0%)
(15.1%) (18.9%) (17.0%) (15.1%)
(11.3%) (7.5%) (3.8%) (5.6%)
Abbreviations—PCR: polymerase chain reaction; IHC: immunohistochemistry; HE: hematoxylin and eosin.
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Fig. 1. Fetal liver. (A) Marked diffuse vacuolization of hepatocytes with intracellular amastigotes (inset). H.E. Bar = 50 mm. (B) Immunolabeling of intracellular Leishmania sp. amastigotes in a Kupffer cell. Streptavidin– peroxidase complex. Bar = 10 mm.
contrast, Masucci et al. (2003) reported a high frequency of positive newborn puppies born to naturally infected bitches, which contrasted to the mouse model (Rosypal et al., 2005), suggesting that possibly transvaginal infection could increase the number of infected newborn puppies. In this study, 32% of fetuses and 49% of the placentas were PCR positive. This finding support the notion that Leishmania is transmitted to the progeny mostly though the placenta in dogs, and that the transvaginal route seems to play a secondary role, which is supported by our previous study indicating absence of amastigotes in the vaginal mucosa of naturally infected bitches (Silva et al., 2008). Despite the absence of lesions and immune-detection of amastigotes in the placenta, Leishmania kDNA was detected in 49% of the placentas from both symptomatic and asymptomatic bitches, indicating that although in extremely low loads the organism is often present in the placenta. The placenta has an extremely high blood flow, which could carried amastigotes, resulting in DNA amplification, although the absence of amastigotes in HE- and IHC-stained sections indicate that the placenta is not likely a target tissue for Leishmania infection, whereas it clearly serves as a transmission route to the fetuses. It is noteworthy that the PCR protocol employed in this study is
extremely sensitive, detecting up to 0.0001 amastigotes (parasite equivalent) per reaction (Lachaud et al., 2002). This study demonstrated that the distribution of Leishmania amastigotes in fetal organs is similar to that observed in post-natal or adults dogs, with amastigotes distributed mostly in lymphoreticular organs such as the spleen, bone marrow, lymph nodes and liver, in which amastigotes were observed in HE- or IHC-stained tissue sections. Although amastigotes were not observed in fetal kidneys and hearts, in some cases these organs were PCR positive indicating infection with a very low parasite load. Apparently the clinical course of the disease seems to be identical in congenital transmitted and otherwise acquired VL in humans (Meinecke et al., 1999), but there is no information available about the clinical features of vertically transmitted VL in dogs, which may be similar to the disease transmitted through the biological vector since the tissue distribution of the parasite in the fetus is similar to naturally infected adult dogs as demonstrated in this study. Importantly, the organism in the fetus is not associated with the classical lesions of VL in adult dogs, which may be due to the immature state of the fetal immune system in spite of the fact that the fetus may be responsive in utero to a range of antigens during the final stages of gestation in dogs (Krakowka, 1998). It is noteworthy that during pregnancy the immune response is modulated to avoid deleterious effect on the developing fetus biasing adaptive immunity towards a Th2 response. It is thought that the down-regulation of adaptive immune responses make pregnant females more susceptible to a wide variety of pathogens and thus increasing the risk of transplacental transmission (Wegmann et al., 1993). There was a moderate to severe fatty degeneration of hepatocytes in all fetal livers, particularly at the end of gestation (60-day-old fetuses), which was independent of the presence of amastigotes. In canine VL, hepatic involvement has been described in symptomatic and asymptomatic adult dogs. Histopathologic changes includes swelling of hepatocytes associated with changes in fat content in response to inflammation, mainly related to the presence of amastigotes within Kupffer cells (Keenan et al., 1984; Melo et al., 2008). The mechanism of this degenerative hepatic changes is not clear, although it has been demonstrated that pathogen components may deplete glutathione (GSH), a potent antioxidant, and increase the expression of iNOS and thus NO production in fetal liver promoting degeneration by lipid peroxidation (Xu et al., 2005). Moreover, in late gestation, GSH is apparently physiological down-regulated to promote regression of hematopoietic cells exposing hepatocytes to some oxidative stress (Sato et al., 2003). However, our results do not support any cause and effect association between this hepatic degenerative changes and parasite loads either in the fetus or in the pregnant bitch. In conclusion, distribution of amastigotes is similar in canine fetal and post-natal adult organs. Therefore, puppies born to infected bitches should be considered as potential reservoirs and source of infection of VL. Hence, the use of infected bitches for reproduction should definitely be avoided to prevent vertical transmission, which according to this study is an important route of transmission and therefore spreading of canine VL.
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