Transplacental transmission of Leishmania infantum as a means for continued disease incidence in North America

Abstract

Background: Dogs are the predominant domestic reservoir for human L. infantum infection. Zoonotic visceral leishmaniasis (ZVL) is an emerging problem in some U.S. dog breeds, with an annual quantitative PCR prevalence of greater than 20% within an at-risk Foxhound population. Although classically Leishmania is transmitted by infected sand flies and phlebotomine sand flies exist in the United States, means of ongoing L. infantum transmission in U.S. dogs is currently unknown. Possibilities include vertical (transplacental/transmammary) and horizontal/venereal transmission. Several reports have indicated that endemic ZVL may be transmitted vertically.

Aims: Our aims for this present study were to establish whether vertical/transplacental transmission was occurring in this population of Leishmania-infected US dogs and determine the effect that this means of transmission has on immune recognition of Leishmania.

Methodology: A pregnant L. infantum-infected dam donated to Iowa State University gave birth in-house to 12 pups. Eight pups humanely euthanized at the time of birth and four pups and the dam humanely euthanized three months post-partum were studied via L. infantum-kinetoplast specific quantitative PCR (kqPCR), gross and histopathological assessment and CD4+ T cell proliferation assay.

Key results: This novel report describes disseminated L. infantum parasites as identified by kqPCR in 8 day old pups born to a naturally-infected, seropositive U.S. dog with no travel history. This is the first report of vertical transmission of L. infantum in naturally-infected dogs in North America, emphasizing that this novel means of transmission could possibly sustain infection within populations.

Major conclusions: Evidence that vertical transmission of ZVL may be a driving force for ongoing disease in an otherwise non-endemic region has significant implications on current control strategies for ZVL, as at present parasite elimination efforts in endemic areas are largely focused on vector-borne transmission between canines and people. Determining frequency of vertical transmission and incorporating canine sterilization with vector control may have a more significant impact on ZVL transmission to people in endemic areas than current control efforts.

Figure 1. Disseminated L. infantum infection in pups born to an infected female.

Tissue samples from target organs, as indicated, were collected at necropsy. DNA was isolated from 1 g of tissue using the QIAGEN DNA isolation kit. DNA was then analyzed via quantitative RT-PCR for the presence of L. infantum kinetoplast DNA. Data indicates detection of parasites in each organ and the number of L. infantum kinetoplast genome copies for (A) tissues from neonatal pups, 1–8, euthanized within 24 hours of birth, and (B) pups A–D euthanized at 3 months of age and the dam.

Figure 2. L. infantum-specific CD4⁺ T cell proliferative responses in vertically-infected pups.

Peripheral blood mononuclear cells (PBMC) were isolated from blood samples from neonatal pups (4–7), 3-month-old pups (A–D) and the dam. PBMC were stained with CFSE and cultured in the presence of L. infantum freeze-thawed antigen. PMBC were harvested at day 7 and the CD4⁺ T cell proliferation response was assessed via flow cytometry. Shown are the percentages of proliferating CD4⁺ T cells in response to L. infantum antigen.