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.

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Transplacental Transmission of Leishmania infantum as a Means for Continued Disease Incidence in North America

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic “cutoff” value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.

/pdf/infection-parameters-in-the-sand-fly-vector-that-predict-1edsyjjqeb.pdf

Infection Parameters in the Sand Fly Vector That Predict Transmission of Leishmania major

Leishmaniasis is a vector-borne neglected tropical disease caused by protozoan parasites of the Leishmania genus and transmitted by the female Phlebotomus and Lutzomyia sand flies. The currently prescribed therapies still rely on pentavalent antimonials, pentamidine, paromomycin, liposomal amphotericin B, and miltefosine. However, their low efficacy, long-course treatment regimen, high toxicity, adverse side effects, induction of parasite resistance and high cost require the need for better drugs given that antileishmanial vaccines may not be available in the near future. Although most drugs are still derived from terrestrial sources, the interest in marine organisms as a potential source of promising novel bioactive natural agents has increased in recent years. About 28,000 compounds of marine origin have been isolated with hundreds of new chemical entities. Recent trends in drug research from natural resources indicated the high interest of aquatic eukaryotic photosynthetic organisms, marine algae in the search for new chemical entities given their broad spectrum and high bioactivities including antileishmanial potential. This current review describes prepared extracts and compounds from marine macroalgae along with their antileishmanial activity and provides prospective insights for antileishmanial drug discovery.

Marine Algae as Source of Novel Antileishmanial Drugs: A Review

Predicting how Leishmania will respond to control efforts requires an understanding of their transmission strategy. Using real-time quantitative PCR to quantify infectious metacyclic and non-metacyclic forms in mouse skin from single sandfly bites we show that most transmissions were highly enriched for infectious parasites. However, a quarter of sandflies were capable of transmitting high doses containing more non-infectious promastigotes from the vector's midgut. Mouse infections replicating "high" to "low" quality, low-dose transmissions confirmed clear differences in the pathology of the infection and their onward transmissibility back to sandflies. Borrowing methods originally developed to account for exposure heterogeneity among hosts, we show how these high-dose, low-quality transmitters act as super-spreading vectors, capable of inflating Leishmania transmission potential by as much as six-fold. These results highlight the hidden potential of transmission of mixed Leishmania promastigote stages on disease prevalence and the role of dose heterogeneity as an underlying strategy for efficient transmission.

/pdf/quantifying-leishmania-metacyclic-promastigotes-from-52ltdx24u1.pdf

Quantifying Leishmania Metacyclic Promastigotes from Individual Sandfly Bites Reveals the Efficiency of Vector Transmission

Molecular targets and pathways for the treatment of visceral leishmaniasis.

The aim of this study is to improve the cultivation of Leishmania promastigotes without the use of common, semisolid culture media such as Evans' modified Tobie's medium (EMTM), liquid RPMI 1640, and Peptone-yeast extract medium (P-Y). Although EMTM medium permits the growth of a high number of parasites, it is technically difficult to prepare as it requires the use of fresh rabbit blood from animals bred on farms, while RPMI 1640 and P-Y show lower growth rates than the EMTM. There is, therefore, a need to develop new blood-free and time-saving culture systems. The aim of this paper is to propose new modified microbiological media, named RPMI-PY and Tobie-PY, to isolate Leishmania and cultivate parasites for research and diagnostic purposes. This study compares classic culture media to the new media, RPMI-PY and Tobie-PY, and demonstrates that the new media have superior performance in terms of time and parasitic load. The growth rate of the parasite was significantly higher at 24, 48, and 72 ...

https://bioone.org/journals/journal-of-parasitology/volume-100/issue-2/13-253.1/Evaluation-of-Two-Modified-Culture-Media-for-Leishmania-infantum-Cultivation/10.1645/13-253.1.pdf

Evaluation of two modified culture media for Leishmania infantum cultivation versus different culture media.

Pentadecane is an organic compound that is made up of primarily carbon and hydrogen atoms. Pentadecane is a floral volatile found in many different plants and essential oils and also in crude extracts of some plants, and it shows antimicrobial activity. This study investigated in vitro effects of pentadecane in Leishmania infantum parasites and found that it decreases growth by 86 ± 2% both in promastigotes (half maximal inhibitory concentration [IC50] = 65.3 μM) and in amastigotes (IC50 = 60.5 μM), resulting in a reduction of macrophage infection; growth inhibition was 77% at 300 μM. Analysis of propidium iodide incorporation in L. infantum, treated with pentadecane at 48 hr, suggested that cells were arresting in the sub-G0/G1 and G1 phases of the cell cycle, whereas cytotoxicity assay of pentadecane in immortalized cells lines DH82 and U937 and in primary epithelial cells of Cercopiteco showed that it caused negligible cytotoxic effect. This study shows that pentadecane has antimicrobial act...

/pdf/cytotoxic-screening-and-in-vitro-evaluation-of-pentadecane-4li9kmpf1a.pdf

Cytotoxic Screening and In Vitro Evaluation of Pentadecane Against Leishmania infantum Promastigotes and Amastigotes

Abstract Leishmaniasis is a disease with a worldwide distribution affecting both humans and animals. There is a need to identify and develop new drugs for the treatment of leishmaniasis. This study showed that crude ethanolic extracts of the red alga Asparagopsis taxiformis have a powerful effect against L. infantum, the prevalent species of the genus Leishmania in the Mediterranean basin. L. infantum demonstrated decreased vitality with increasing concentration of the algal extracts. At a concentration of 40 μg/mL, the extracts achieved 100% mortality of the parasite and the LD50 value was 25 μg/mL for promastigotes and 9 μg/mL for amastigotes. Algal extracts caused morphological alterations and apoptosis in Leishmania cells. The potential cytotoxic action of crude extracts was investigated by a MTT viability assay on DH82 and Vero cell lines but there was no cytotoxic effect. The potential of red alga A. taxiformis metabolites as anti-leishmanial agents merits further pharmacological investigation.

Effectiveness of red alga Asparagopsis taxiformis extracts against Leishmania infantum

The geographical diffusion of leishmaniasis can depend on the factors limiting the distribution of sandfly vectors. In Sicily, as in all Mediterranean areas, sandflies are present almost all year round because the climate permits perpetuation of this vector's life cycle. Transmission can occurs in rural and domestic habitats through the bite of infected female phlebotomine sandflies. In Italy, the visceral form of leishmaniasis is commonly found, which is due exclusively to L. infantum. Two species of sandfly are considered the main vectors: Phlebotomus perniciosus and P. perfiliewi. Fluorescence resonance energy transfer technology was used to determine the parasite load in phlebotomine vectors, and the test was targeted on a 117-bp fragment of kinetoplast DNA minicircles. The assay was evaluated, focusing on analytical sensitivity, discriminatory power, and reliability of quantification. During 2005, a total of 1686 sandflies were trapped in various Sicilian provinces and in farms randomly selected using black light traps. We found 20, 30, and 16 sandflies positive for Leishmania for each kind of analyzed phlebotomine sandfly, respectively, corresponding to 6.5% for the gravid, 2.7% for the fed, and 6.3% for other groups. Previously the insects were identified on the basis of morphology and the most prevalent sandfly species were P. Sergentomyia minuta, P. perfiliewi, and P. perniciosus.

Maria Piazza

Papers

Evaluation of two modified culture media for Leishmania infantum cultivation versus different culture media.

Cytotoxic Screening and In Vitro Evaluation of Pentadecane Against Leishmania infantum Promastigotes and Amastigotes

Effectiveness of red alga Asparagopsis taxiformis extracts against Leishmania infantum

Fluorescence-based detection of Leishmania infantum DNA in phlebotomus vectors.