Molecular evidence of early vertical transmission of Leishmania ( Leishmania ) infantum in a dog

Evidência molecular da transmissão vertical precoce de Leishmania (Leishmania) infantum em um cão

Vinícius Vasconcelos Gomes de Oliveira Rafael Antonio Nascimento Ramos Carlos Alberto do Nascimento Ramos Neurisvan Ramos Guerra Frederico Celso Lyra Maia Leucio Câmara Alves Valdemiro Amaro da Silva JuniorAbout the authors

ABSTRACT:

Visceral leishmaniasis (VL) is caused by the protozoon Leishmania infantum . Transmission of this parasite to hosts occurs mainly through the bite of infected sand flies. However, alternative infection routes have been hypothesized, especially in areas where the biological vector is absent. The exact time of infection and whether in utero transmission occurs have still not been fully elucidated. This report demonstrates molecular evidence of vertical transmission ofL. infantum from a pregnant dog to the embryo. Samples (e.g. vulva, vagina, cervix, uterine body, uterine horn and ovaries) from a female naturally infected byL. infantum and from her embryo were molecularly analyzed by means of qPCR and cPCR followed by DNA sequencing. The gestational age was estimated to be 23±1 day. Through qPCR, the presence ofL. infantum DNA was detected in all the samples analyzed (n=7), including the embryo, conversely through cPCR, only four samples (vagina, cervix, uterine body and embryo) were positive. This study demonstrated that transmission ofL. infantum from a pregnant dog to the embryo might occur in the early days of pregnancy. In conclusion, this is the first report showingL. infantum infecting a canine embryo.

Key words:
leishmaniasis; dog; vertical infection; embryo

RESUMO:

Leishmaniose visceral (LV) é causada pelo protozoárioLeishmania infantum . A transmissão deste parasito para hospedeiros é principalmente vetorial. No entanto, rotas alternativas de infecção têm sido investigadas, especialmente em áreas onde o vetor biológico está ausente. O período exato de infecção e se a transmissão in utero ocorre ainda não está totalmente elucidada. Este estudo demonstra uma evidência molecular da transmissão vertical deL. infantum de uma cadela prenhe para o embrião. As amostras (vulva, vagina, cérvix, corpo do útero, corno do útero e ovários) de uma fêmea naturalmente infectada por L. infantum e de seu embrião foram analisados molecularmente por meio de qPCR e cPCR, seguido de sequenciamento de DNA. A estimativa da idade gestacional foi de 23±1 dia. Na qPCR, a presença de DNA deL. infantum foi detectado em todas as amostras (n=7) analisadas, incluindo o embrião. No entanto, na PCR convencional, apenas quatro amostras (vagina, cérvix, corpo do útero e embrião) foram positivas. Este estudo demonstrou que a transmissão deL. infantum de uma cadela prenhe para o embrião pode ocorrer nos primeiros dias de prenhez. Em conclusão, de acordo com o conhecimento dos autores, este é o primeiro relatório evidenciando infecção de embrião canino por L. infantum.

Palavras-chave:
leishmaniose; cão; infecção vertical; embrião

Visceral Leishmaniasis is an important parasitic disease that affect mammals and is primarily transmitted by insect vectors belonging to the sub-family Phlebotominae . Nonetheless, alternative transmission routes among dogs have been hypothesized in VL (OLIVEIRA et al., 2015OLIVEIRA, V.V.G. et al. Transmission routes of visceral leishmaniasis in mammals. Ciência Rural, v.45, p.1622-1628, 2015. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782015000901622>. Accessed: Ago. 12, 2016. doi: 10.1590/0103-8478cr20141368.
http://www.scielo.br/scielo.php?script=s...
). For instance, involvement of ticks in the transmission of Leishmania infantum , especially Rhipicephalus sanguineus sensu lato, was first demonstrated in France in the 1930s, through observation of the ability of these ticks to mechanically transmit the protozoon to rodents (BLANC & CAMINOPETROS, 1930BLANC, G.; CAMINOPTEROS, J. La transmission du Kala-Azar méditerranéen par une tique: Rhipicephalus sanguineus. Comptes Rendus de l'Académie des Sciences, v.191, p.1162-1164, 1930.). Similarly, the potential role of fleas has already been assessed (FERREIRA et al., 2009FERREIRA, M.G.P.A.et al. Potential role for dog fleas inthe cycle of Leishmania spp. Veterinary Parasitology , v.165, p.150-154, 2009. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19595512>. Accessed: Aug. 11, 2016. doi: 10.1016/j.vetpar.2009.06.026.
http://www.ncbi.nlm.nih.gov/pubmed/19595...
), but the real role of these ectoparasites in the epidemiology of L. infantum remains a matter of controversy.

In addition, over the past few years, venereal and vertical transmissions of this protozoon have been demonstrated as possible infection routes, especially in areas where the biological vectors are absent (OLIVEIRA et al., 2015OLIVEIRA, V.V.G. et al. Transmission routes of visceral leishmaniasis in mammals. Ciência Rural, v.45, p.1622-1628, 2015. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782015000901622>. Accessed: Ago. 12, 2016. doi: 10.1590/0103-8478cr20141368.
http://www.scielo.br/scielo.php?script=s...
). This hypothesis is supported by the retrieval of viable forms of Leishmania spp. in semen of infected dogs (SILVA et al., 2009aSILVA, F.L. et al. Venereal transmission of canine visceral leishmaniasis. Veterinary Parasitology , v.160, p.55-59, 2009a. Available from: http://www.sciencedirect.com/science/article/pii/S0304401708006031>. Accessed: Aug. 10, 2016. doi: 10.1016/j.vetpar.2008.10.079.
http://www.sciencedirect.com/science/art...
). Indeed, it has been demonstrated that this type of infection reported above can occur between an infected male and a non-infected female (SILVA et al., 2009bSILVA, S.M. et al. First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil. Veterinary Parasitology , v.166, p.159-162, 2009b. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19733439>. Accessed: Aug. 10, 2014. doi: 10.1016/j.vetpar.2008.10.079
http://www.ncbi.nlm.nih.gov/pubmed/19733...
).

Conversely, although vertical transmission in humans has been known for a long time (LOW & COOKE, 1926LOW, G.C.; COOKE, W.E. A congenital infection of kala azar. Lancet, v.2, p.1209-1211, 1926.), this event has not been completely elucidated in dogs. In fact, so far, all cases of vertical transmission reported in the scientific literature have described transmission between the female and fetus or newborn puppies (NAUCKE & LORENTZ, 2012NAUCKE, T.J.; LORENTZ, S. First report of venereal and vertical transmission of canine leishmaniosis from naturally infected dogs in Germany. Parasites & Vectors, v.5, p.67, 2012. Available from: http://www.parasitesandvectors.com/content/5/1/67>. Accessed: Aug. 12, 2016. doi: 10.1186/1756-3305-5-67.
http://www.parasitesandvectors.com/conte...
). The exact time of infection and whether in utero transmission occurs remain unknown. Therefore, the aim of this report was to provide molecular evidence of vertical transmission of L. infantum from a pregnant dog to the embryo in early stages of pregnancy.

A female mixed-breed dog naturally infected by L. infantum diagnosed by immunofluorescence antibody test (IFAT≥40) and parasitological examination of the bone marrow (RAMOS et al., 2013RAMOS, R.A.N. et al. Quantification of Leishmania infantum DNA in the bone marrow, lymph node and spleen of dogs. Brazilian Journal ofVeterinary Parasitology , v.22, p.346-350, 2013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24142164>. Accessed: Aug. 12, 2016. doi: 10.1590/S1984-29612013000300005.
http://www.ncbi.nlm.nih.gov/pubmed/24142...
) was used in this study. This animal came from the Zoonotic Disease Control Center of the city of Caruaru (8°16'59" S and 35°58'33" W), in the state of Pernambuco, Brazil.

During the necropsy, it was possible to observe that the animal was in an early stage of pregnancy. Therefore, the whole reproductive tract was carefully removed, put into a sterile plastic vial and sent to the laboratory. Subsequently, the uterus was opened and five embryos were found. Fragments from the vulva, vagina, cervix, uterine body, uterine horn and ovaries and from one embryo were collected and stored in sterile plastic tubes for molecular processing.

Genomic DNA (samples from vulva, vagina, cervix, uterine body, uterine horn, ovaries, and embryo) was extracted using the QIAamp DNA Blood and Tissue(r) kit (Qiagen(r), Hilden, Germany), following the manufacturer's instructions. All the procedures were performed using micropipette barrier tips to prevent contamination.

The qPCR reactions were performed using the primers LEISH-1 (5'-AACTTTTCTGGTCCTCCGGGTAG-3') and LEISH-2 (5'-ACCCCCAGTTTCCCGCC-3') and the FAM probe (5'-AAAAATGGGTGCAGAAAT-3'-NFQ-MGB) that amplify the kinetoplast minicircle DNA of Leishmania spp. (FRANCINO et al., 2006FRANCINO, O. et al. Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Veterinary Parasitology , v.137, p.214-221, 2006. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16473467>. Accessed: Jul. 13, 2016. doi: 10.1016/j.vetpar.2006.01.011.
http://www.ncbi.nlm.nih.gov/pubmed/16473...
). The reaction mixture (12.5μL) contained 6.25μL of Taqman Universal PCR Master Mix, each primer at a concentration of 900nM, the probe at a concentration of 200nM and 100ηg of template DNA. The amplification protocol consisted of initial denaturation at 95°C for 3 minutes, followed by 42 cycles of denaturation (at 95°C for 10 seconds) and annealing-extension (at 60°C for 30 seconds). All assays were carried out in triplicate, with each battery containing a negative control (bone marrow DNA from a dog that was negative in microscopic, molecular and serological examinations) and a positive control (DNA from an in vitro culture of L. infantum ).

Positive qPCR samples were further tested by means of cPCR using the primers MC1 (5'GTTAGCCGATGGTGGTCTTG3') and MC2 (5'CACCCATTTTTCCGATTTTG3') (CORTES et al., 2004CORTES, S. et al. PCR as a rapid and sensitive tool in the diagnosis of human and canine leishmaniasis using Leishmania donovani s.l.-specific kinetoplastid primers. Transactions of the Royal Society of Tropical Medicine and Hygiene, v.98, p.12-17, 2004. Available from: http://trstmh.oxfordjournals.org/content/98/1/12.long>. Accessed: Aug. 16, 2016. doi: 10.1016/S0035-9203(03)00002-6.
http://trstmh.oxfordjournals.org/content...
). Amplicons (embryo and uterine body) were then purified using the Qiaex II kit (Qiagen(r), Hilden, Germany) and sequenced in both directions by means of Sanger's method (SANGER et al., 1977SANGER, F. et al. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America, v.74, p.5463-5467, 1977. ) in an automated sequencer (ABI-3130; Applied Biosystems). Identity of the DNA sequences was determined by making comparisons with sequences available in Genbank(r) using the BLASTn search tool (ALTSCHUL et al., 1990ALTSCHUL, S.F. et al. Basic local alignment search tool. Journal of Molecular Biology, v.21, p.403-410, 1990. Available from: http://www.sciencedirect.com/science/article/pii/S0022283605803602>. Accessed: Jul. 13, 2016. doi: 10.1016/S0022-2836(05)80360-2.
http://www.sciencedirect.com/science/art...
).

Based on the macroscopic characteristics of the reproductive system (e.g. uterine edema) and embryo morphology (embryo in C shape), the canine gestational age was estimated as 23±1 days. The embryo was 1cm in length and was particularly characterized by the presence of anterior and posterior limb buds.

Through qPCR, the presence of L. infantum DNA was detected in all the samples analyzed (n=7), including the embryo. Conversely, through cPCR, only four samples were reported to be positive (e.g. vagina, cervix, uterine body and embryo). The search for homology in the Genbank(r) database showed similarity of 98-99% (for both sequences) with DNA sequences of L. infantum . Sequences obtained in this study were deposited in Genbank(r) under accession numbers KT735370 and KT735371.

This report provides molecular evidence of vertical in utero transmission of L. infantum from a pregnant dog to the embryo in early days of pregnancy. Over the last years, the vectorial role of invertebrates in the transmission of L. infantum among mammals has been studied; however other potential routes of infection have been speculated. In dogs, it has been suggested that this transmission may occur through blood transfusion (OWENS et al., 2001OWENS, S.D. et al. Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs. Journal of the American Veterinary Medical Association , v.219, p.1076-1083, 2001. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11700704>. Accessed: Aug. 12, 2016. doi: 10.1016/S0887-7963(02)80076-1.
http://www.ncbi.nlm.nih.gov/pubmed/11700...
), and through venereal and vertical routes (OLIVEIRA et al., 2015OLIVEIRA, V.V.G. et al. Transmission routes of visceral leishmaniasis in mammals. Ciência Rural, v.45, p.1622-1628, 2015. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782015000901622>. Accessed: Ago. 12, 2016. doi: 10.1590/0103-8478cr20141368.
http://www.scielo.br/scielo.php?script=s...
). In fact, vertical transmission in these domestic carnivores was first demonstrated in Italy, where L. infantum parasites were isolated from the liver, spleen and lymph nodes of newborn puppies (MANCIANTI & SOZZI, 1995MANCIANTI, F.; SOZZI, S. Isolation of Leishmania from a newborn puppy. Transactions of the Royal Society of Tropical Medicine and Hygiene , v.89, p.402, 1995. Available from: http://www.ncbi.nlm.nih.gov/pubmed/7570879>. Accessed: Aug. 12, 2016. doi: 10.1016/0035-9203(95)90028-4.
http://www.ncbi.nlm.nih.gov/pubmed/75708...
). However, this infection route remained controversial for a long time and several authors stated that this was an unusual transmission route. For instance, a previous study conducted in a Brazilian endemic area, in which 63 puppies from 18 naturally infected dogs were analyzed, concluded that L. infantum was not transmitted vertically, since at the end of the study, all the puppies were reported to be negative through molecular examination (ANDRADE et al., 2002ANDRADE, H.M. et al. Leishmania (Leishmania) chagasi is not vertically transmitted in dogs. Veterinary Parasitology, v.103, p.71-81, 2002. Available from: http://www.sciencedirect.com/science/article/pii/S0304401701005520>. Accessed: Aug. 16, 2016. doi: 10.1016/S0304-4017(01)00552-0.
http://www.sciencedirect.com/science/art...
). Conversely, more recently, this transmission route was demonstrated in Brazil (SILVA et al., 2009aSILVA, F.L. et al. Venereal transmission of canine visceral leishmaniasis. Veterinary Parasitology , v.160, p.55-59, 2009a. Available from: http://www.sciencedirect.com/science/article/pii/S0304401708006031>. Accessed: Aug. 10, 2016. doi: 10.1016/j.vetpar.2008.10.079.
http://www.sciencedirect.com/science/art...
), in the USA (BOGGIATTO et al., 2011BOGGIATTO, P.M. et al. Transplacental transmission of Leishmania infantum as a means for continued disease incidence in North America. PLoS Neglected Tropical Diseases, v.5, p.1019, 2011. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075227/>. Accessed: Aug. 16, 2016. doi: 10.1371/journal.pntd.0001019.
http://www.ncbi.nlm.nih.gov/pmc/articles...
) and in Germany (NAUCKE & LORENTZ, 2012NAUCKE, T.J.; LORENTZ, S. First report of venereal and vertical transmission of canine leishmaniosis from naturally infected dogs in Germany. Parasites & Vectors, v.5, p.67, 2012. Available from: http://www.parasitesandvectors.com/content/5/1/67>. Accessed: Aug. 12, 2016. doi: 10.1186/1756-3305-5-67.
http://www.parasitesandvectors.com/conte...
). However, all cases of vertical transmission reported in the scientific literature so far have described transmission between a female and fetus or newborn puppies, but never in an embryo.

This is the first report of detection of L. infantum DNA in an embryo of estimated age 23±1 days. In this study, sampling of the embryo directly from the uterus prevented contact with the vaginal canal, thereby reducing the possibility of contamination. Indeed, L. infantum DNA has already been detected in the uterus of a pregnant female (ROSYPAL et al., 2005ROSYPAL, A.C. et al. Transplacental transmission of a North American isolate of Leishmania infantum in a experimentally infected beagle. Journal of Parasitolology, v.91, p.970-972, 2005. Available from: http://www.journalofparasitology.org/doi/abs/10.1645/GE-483R.1?journalCode=para>. Accessed: Aug. 09, 2016. doi: 10.1645/GE-483R.1.
http://www.journalofparasitology.org/doi...
), and amastigote forms have been observed in the placental trophoblast (DUBEY et al., 2005DUBEY, J.P. et al. Placentitis associated with leishmaniasis in a dog. Journal of the American Veterinary Medical Association, v.227, p.1266-1269, 2005. Available from: http://www.researchgate.net/publication/7502033_Placentitis_associated_with_leishmaniasis_in_a_dog>. Accessed: Aug. 11, 2016. doi: 10.2460/javma.2005.227.1266.
http://www.researchgate.net/publication/...
). It has been demonstrated that circulation of parasites in the blood from the female to the placenta and annexes allows passage of L. infantum to the fetus (SILVA et al., 2009aSILVA, F.L. et al. Venereal transmission of canine visceral leishmaniasis. Veterinary Parasitology , v.160, p.55-59, 2009a. Available from: http://www.sciencedirect.com/science/article/pii/S0304401708006031>. Accessed: Aug. 10, 2016. doi: 10.1016/j.vetpar.2008.10.079.
http://www.sciencedirect.com/science/art...
).

The exact time of infection within vertical transmission is still unknown. Based on the findings of this report, the passage of L. infantum from a female to the embryo may occur in the early days of pregnancy. Therefore, this transmission route needs to be taken into consideration with regard to spreading of this infection among dogs. Finally, this is the earliest detection of L. infantum infecting a canine embryo.

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

The present study was approved by the Ethics Committee for Animal Experimentation of the Universidade Federal Rural de Pernambuco (UFRPE) (protocol ECAE: 23082.015186/2012).

ACKNOWLEDGEMENTS

The authors thank the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for its financial support.

REFERENCES

  • 1
    CR-2016-0553.R1

Publication Dates

  • Publication in this collection
    21 Nov 2016
  • Date of issue
    2017

History

  • Received
    06 June 2016
  • Accepted
    11 Aug 2016
  • Reviewed
    28 Oct 2016
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