Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Effect of Clindamycin on Intestinal Microbiome and Miltefosine Pharmacology in Hamsters Infected with Leishmania infantum.

Visceral leishmaniasis (VL), a vector-borne parasitic disease caused by Leishmania donovani and L. infantum (Kinetoplastida), affects humans and dogs, being fatal unless treated. Miltefosine (MIL) is the only oral medication for VL and is considered a first choice drug when resistance to antimonials is present. Comorbidity and comedication are common in many affected patients but the relationship between microbiome composition, drugs administered and their pharmacology is still unknown. To explore the effect of clindamycin on the intestinal microbiome and the availability and distribution of MIL in target organs, Syrian hamsters (120-140 g) were inoculated with L. infantum (108 promastigotes/animal). Infection was maintained for 16 weeks, and the animals were treated with MIL (7 days, 5 mg/kg/day), clindamycin (1 mg/kg, single dose) + MIL (7 days, 5 mg/kg/day) or kept untreated. Infection was monitored by ELISA and fecal samples (16 wpi, 18 wpi, end point) were analyzed to determine the 16S metagenomic composition (OTUs) of the microbiome. MIL levels were determined by LC-MS/MS in plasma (24 h after the last treatment; end point) and target organs (spleen, liver) (end point). MIL did not significantly affect the composition of intestinal microbiome, but clindamycin provoked a transient albeit significant modification of the relative abundance of 45% of the genera, including Ruminococcaceae UCG-014, Ruminococcus 2; Bacteroides and (Eubacterium) ruminantium group, besides its effect on less abundant phyla and families. Intestinal dysbiosis in the antibiotic-treated animals was associated with significantly lower levels of MIL in plasma, though not in target organs at the end of the experiment. No clear relationship between microbiome composition (OTUs) and pharmacological parameters was found.

Antileishmanial and Antitrypanosomes Drugs for the Current Century.

Human infections by trypanosomatids are widely distributed and prevalent in the tropical and subtropical regions. Diseases caused by Trypanosoma and Leishmania have variable clinical outcomes, ranging from self-healing to fatality, and are considered Neglected Tropical Diseases (NTD). In addition, animal trypanosomiases have a significant impact on animal health and production, apart from their potential role as reservoirs in zoonotic species. Control of these infections is progressing and, in some cases (such as human African trypanomiasis (HAT)), significant reductions have been achieved. In the absence of effective vaccination, chemotherapy is the most used control method. Unfortunately, the therapeutic arsenal is scarce, old, and of variable efficacy, and reports of resistance to most antiparasitic agents have been published. New drugs, formulations, or combinations are needed to successfully limit the spread and severity of these diseases within a One Health framework. In this Special Issue, contributions regarding the identification and validation of drug targets, underlying mechanisms of action and resistance, and potential new molecules are presented. These research contributions are complemented by an update revision of the current chemotherapy against African Trypanosoma species, and a critical review of the shortcomings of the prevailing model of drug discovery and development.

Leishmania infantum infection does not affect the main composition of the intestinal microbiome of the Syrian hamster.

BACKGROUND: Visceral leishmaniasis (VL) is the most severe form of all leishmanial infections and is caused by infection with protozoa of Leishmania donovani and Leishmania infantum. This parasitic disease occurs in over 80 countries and its geographic distribution is on the rise. Although the interaction between the intestinal microbiome and the immune response has been established in several pathologies, it has not been widely studied in leishmaniasis. The Syrian hamster is the most advanced laboratory model for developing vaccines and new drugs against VL. In the study reported here, we explored the relationship between the intestinal microbiome and infection with L. infantum in this surrogate host. METHODS: Male Syrian hamsters (120-140 g) were inoculated with 108 promastigotes of a canine-derived L. infantum strain or left as uninfected control animals. Infection was maintained for 19 weeks (endpoint) and monitored by an immunoglobulin G (IgG) enyzme-linked immunosorbent assay throughout the experiment. Individual faecal samples, obtained at weeks 16, 18 and 19 post-inoculation, were analysed to determine the 16S metagenomic composition (the operational taxonomic units [OTUs] of the intestinal microbiome and the comparison between groups were FDR (false discovery rate)-adjusted). RESULTS: Leishmania infantum infection elicited moderate clinical signs and lesions and a steady increase in specific anti-Leishmania serum IgG. The predominant phyla (Firmicutes + Bacteriodetes: > 90%), families (Muribaculaceae + Lachnospiraceae + Ruminococcaceae: 70-80%) and genera found in the uninfected hamsters showed no significant variations throughout the experiment. Leishmania infantum infection provoked a slightly higher-albeit non-significant-value for the Firmicutes/Bacteriodetes ratio but no notable differences were found in the relative abundance or diversity of phyla and families. The microbiome of the infected hamsters was enriched in CAG-352, whereas Lachnospiraceae UCG-004, the [Eubacterium] ventriosum group and Allobaculum were less abundant. CONCLUSIONS: The lack of extensive significant differences between hamsters infected and uninfected with L. infantum in the higher taxa (phyla, families) and the scarce variation found, which was restricted to genera with a low relative abundance, suggest that there is no clear VL infection-intestinal microbiome axis in hamsters. Further studies are needed (chronic infections, co-abundance analyses, intestinal sampling, functional analysis) to confirm these findings and to determine more precisely the possible relationship between microbiome composition and VL infection.

First description of Lotmaria passim and Crithidia mellificae haptomonad stages in the honeybee hindgut.

The remodelling of flagella into attachment structures is a common and important event in the trypanosomatid life cycle. Lotmaria passim and Crithidia mellificae can parasitize Apis mellifera, and as a result they might have a significant impact on honeybee health. However, there are details of their life cycle and the mechanisms underlying their pathogenicity in this host that remain unclear. Here we show that both L. passim promastigotes and C. mellificae choanomastigotes differentiate into haptomonad stages covering the ileum and rectum of honeybees. These haptomonad cells remain attached to the host surface via zonular hemidesmosome-like structures, as revealed by transmission electron microscopy. This work describes for the first known time the haptomonad morphotype of these species and their hemidesmosome-like attachments in A. mellifera, a key trait used by other trypanosomatid species to proliferate in the insect host hindgut.

Experimental evidence of harmful effects of Crithidia mellificae and Lotmaria passim on honey bees.

The trypanosomatids Crithidia mellificae and Lotmaria passim are very prevalent in honey bee colonies and potentially contribute to colony losses that currently represent a serious threat to honey bees. However, potential pathogenicity of these trypanosomatids remains unclear and since studies of infection are scarce, there is little information about the virulence of their different morphotypes. Hence, we first cultured C. mellificae and L. passim (ATCC reference strains) in six different culture media to analyse their growth rates and to obtain potentially infective morphotypes. Both C. mellificae and L. passim grew in five of the media tested, with the exception of M199. These trypanosomatids multiplied fastest in BHI medium, in which they reached a stationary phase after around 96 h of growth. Honey bees inoculated with either Crithidia or Lotmaria died faster than control bees and their mortality was highest when they were inoculated with 96 h cultured L. passim. Histological and Electron Microscopy analyses revealed flagellated morphotypes of Crithidia and Lotmaria in the lumen of the ileum, and adherent non-flagellated L. passim morphotypes covering the epithelium, although no lesions were evident. These data indicate that parasitic forms of these trypanosomatids obtained from the early stationary growth phase infect honey bees. Therefore, efficient infection can be achieved to study their intra-host development and to assess the potential pathogenicity of these trypanosomatids.

Molecular characterisation of five Sarcocystis species in domestic sheep (Ovis aries) from Spain.

The major aim of the present study was to determine by molecular methods whether the wide and narrow types of macroscopic sarcocysts in Spanish sheep belonged to different species, that is, Sarcocystis gigantea and Sarcocystis medusiformis, respectively. Additionally, we wanted to identify and characterize molecularly the species forming microscopic sarcocysts and determine the phylogenetic placement of all species found. Portions of the oesophagus, diaphragm and hind legs containing macroscopic sarcocysts were collected from slaughtered culled ewes at an abattoir in the Province of Madrid, Central Spain, but both macroscopic and microscopic sarcocysts were isolated for molecular examination. Genomic DNA from 63 sarcocysts (21 macroscopic, 42 microscopic) were examined at the cytochrome c oxidase subunit I gene (cox1), while selected isolates of each species found were further examined at the 18S and 28S ribosomal RNA (rRNA) genes. The 63 sarcocysts comprised five cox1 sequence types, each corresponding to a particular sarcocyst type, and thus represented five Sarcocystis spp. The slender fusiform and thick macrocysts belonged to S. medusiformis and S. gigantea, respectively. The microscopic sarcocysts belonged to Sarcocystis arieticanis, Sarcocystis tenella and a Sarcocystis mihoensis-like species with slanting thorn-like cyst wall protrusions, which was characterised molecularly for the first time. Based on its phylogenetic position, the S. mihoensis-like species probably uses corvids as definitive hosts.

Discovery and Pharmacological Studies of 4-Hydroxyphenyl-Derived Phosphonium Salts Active in a Mouse Model of Visceral Leishmaniasis.

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.

Immunization with recombinant rHc23 partially protects lambs against trickle infections by Haemonchus contortus.

BACKGROUND: Haemonchosis is one of the most economically important parasitic diseases affecting small ruminants all over the world. Chemotherapeutic control has several shortcomings (limited anthelmintic arsenal, frequent resistance) and is hardly affordable by many farm economies. A recombinant antigen (rHc23) was shown to induce significant protection in vaccination trials with single dose challenges and different adjuvants. RESULTS: Lambs were vaccinated with 100 µg rHc23/dose + bacterial immunostimulant (BI) (LPS from Escherichia coli + Propionibacterium acnes extract) (days - 2, 0, 7 and 14) and subjected to a trickle infection with two dosages [6x, 1000 infective larvae (L3) or 6x, 2000 L3]. Vaccinated lambs showed a significant antibody response against rHc23 and Haemonchus contortus soluble extract as assessed by ELISA and Western blot (WB). Fecal egg counts (epg) along the experiment of vaccinated and BI treated lambs were significantly reduced. All vaccinated animals showed total egg output and abomasal helminth burdens (median, average) lower than those from unvaccinated or BI-treated animals lambs although differences were not statistically significant. CONCLUSIONS: Vaccination with 100 µg rHc23/dose + BI against H.contortus trickle infections apparently induced lower epg values and helminth burdens at the end of the experiment. Intragroup individual variations did not allow to obtain conclusive results and more research is needed including adjuvants and larger groups of animals to validate the potential value of rHc23 as candidate to develop a recombinant vaccine for lambs haemonchosis.

Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics.

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 µM for Leishmania infantum, 3.4 µM for L. donovani, 6.7 µM for L. major), Trypanosoma cruzi (EC50 7.5 µM) and T. brucei (EC50 0.8 µM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.

SAR Studies and Biological Characterization of a Chromen-4-one Derivative as an Anti-Trypanosoma brucei Agent.

Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (1), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure-activity relationship studies around hit compound 1. The pivaloyl derivative (13) showed significant anti-T. brucei activity (EC50 = 1.1 µM) together with a selectivity index higher than 92. The early in vitro ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and hERG inhibition) were determined for compound 1 and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform.

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

In-Vitro Activity of Silybin and Related Flavonolignans against Leishmania infantum and L. donovani.

Flavonolignans from the seeds of the milk thistle (Silybum marianum) have been extensively used in folk medicine for centuries. Confirmation of their properties as hepatoprotective, antioxidant and anticancer has been obtained using standardized extracts and purified flavonolignans. Information on their potential effect on Leishmania is very scarce. We have investigated the effect of silymarin, silybin and related flavonolignans on the multiplication of promastigotes in vitro and ex vivo on intracellular amastigotes of L. infantum (Li) and L. donovani (Ld), causative agents of human and canine visceral leishmaniasis (VL). In addition, the potential synergistic effect of the most active molecule and well-established antileishmanial drugs against promastigotes was explored. Dehydroisosilybin A elicited the highest inhibition against Ld and Li promastigotes with an approximate IC50 of 90.23 µM. This molecule showed a moderate synergism with amphotericin B (AmB) but not with SbIII or paromomycin, although it was ineffective against amastigotes. Antileishmanial activity on intracellular amastigotes of the two diastereoisomers of dehydrosilybin (10 µM) was comparable to that elicited by 0.1 µM AmB. Antiproliferative activity and safety of flavonolignans suggest the interest of exploring their potential value in combination therapy against VL.

Morphological and molecular characteristics of six Sarcocystis spp. from red deer (Cervus elaphus) in Spain, including Sarcocystis cervicanis and three new species.

Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500-800 × 80-180 µm. By LM, the sarcocysts either had densely packed, about 8-µm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5' end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.

Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds.

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 µM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-ß-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Sarcocystosis in Cervus elaphus: Comparison of diagnostic methods.

Red deer (Cervus elaphus) from a National Wildlife Reserve near Toledo in central Spain were surveyed for Sarcocystis infection. A total of 61 deer were examined. Tissue compression and histology were used to examine samples from diaphragm and heart from each animal included in the study, and results from the two techniques and the two tissues were compared to determine the tissue and technique that provide the most accurate measure of prevalence and intensity. Prevalence and intensity were then compared between calves, yearlings and adults. Sarcocystis was detected in 59 (97%) of the 61 deer. Comparison between tissues showed that (a) prevalence based on histology was similar for heart and diaphragm, (b) prevalence based on compression was significantly higher for heart than for diaphragm and (c) intensity was significantly higher for heart than for diaphragm, regardless of the technique used. Comparison between techniques showed that (a) both techniques rendered similar prevalences and intensities of Sarcocystis infection with heart samples and (b) both techniques were not comparable with diaphragm samples (compression rendered lower prevalence but higher intensity than histology). Together these data suggest that heart is the preferable tissue for estimating prevalence and intensity, regardless of the technique used. A preliminary species identification of isolated cysts from three animals showed two morph types, corresponding to Sarcocystis cervicanis (syn. S. cf. grueneri; S. wapiti) in the heart and diaphragm of three animals and S. hjorti, only in the diaphragm of two animals. Given the different location of those morph types, both heart and diaphragm should be sampled and preferably assessed using histology to most reliably detect infection. Based on histology of heart, prevalence and intensity of Sarcocystis were significantly lower in calves than in yearlings or adults.

Intranasal immunization of lambs with serine/threonine phosphatase 2A against gastrointestinal nematodes.

Seven 3-month-old, female, helminth-free lambs were immunized intranasally with three doses (1 mg total) of a recombinant part of the catalytic region of the serine/threonine phosphatase 2A (PP2Ar) (group 1 [G1]). In addition, four lambs were used as an adjuvant control group (G2), four as unimmunized, infected controls (G3), and four as unimmunized, uninfected controls (G4). Fifteen days after the last immunization, lambs from G1, G2, and G3 were challenged with 10,000 larval stage 3 (L3) organisms in a plurispecific nematode infection composed of ca. 40% Trichostrongylus colubriformis, 40% Haemonchus contortus, and 20% Teladorsagia circumcincta. All the lambs were clinically monitored throughout the experiment. Parasitological (fecal egg output and immunological response), biopathological (packed-cell volume and leukocyte and eosinophil counts), and zootechnical (live-weight gain) analyses were conducted. On day 105 of the experiment, all the animals were slaughtered and the adult worm population in their abomasa examined. Intranasal administration of PP2Ar with bacterial walls as an adjuvant elicited a strong immune response in the immunized lambs, as evidenced by their humoral immune response. Immunized animals and animals receiving the adjuvant shed significantly (P < 0.001) fewer numbers of parasites' eggs in their feces. The immunization significantly reduced the helminth burden in the abomasa by the end of the experiment (>68%), protection being provided against both Haemonchus and Teladorsagia. Live-weight gain in the immunized lambs was similar to that in the uninfected controls versus the infected or adjuvanted animal groups. Our results suggest that heterologous immunization of ruminants by intranasal administration may be efficacious in the struggle to control gastrointestinal helminths in these livestock.

Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue.

The value of resazurin-based Alamar Blue redox indicator to determine multiplication of Leishmania promastigotes in 96-well microtiter plates was examined. In addition, assay was validated with amphotericin B (AmB) and allicin. The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variable air-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed that the gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating cell densities of 2.5 × 105 promastigotes/well, up to 72 h incubation and 5% CO2 atmosphere or reduced air availability (sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results could be obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results. Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration (IC50) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditions this one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferation rates of Leishmania isolates or strains in a 96-well format.

Effect of allicin on promastigotes and intracellular amastigotes of Leishmania donovani and L. infantum.

Anti-leishmanial activity of allicin (=diallyl thiosulphinate) has been tested in vitro against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania infantum. Macrophage infections have been carried out in vitro in the murine cell line J774 and ex vivo with peritoneal macrophages from BALB/c mice with a modified method to isolate metacyclic promastigotes. The compound has shown a significant in vitro effect on the multiplication of promastigotes of L. donovani and L. infantum in a time- and dose-dependent manner. It has been shown for the first time the inhibition of multiplication of intracellular amastigotes of Leishmania by allicin. Inhibitory concentrations of the compound were in the micromolar range (10-30 µM) for both Leishmania species. Antileishmanial effect of allicin apparently was not related to products of degradation of the molecule as assessed by mass spectrometry analysis. Inhibitory activity of allicin against promastigotes and intracellular amastigotes increased when the compound was added to the cultures every 24 h. Two administrations of 5 µM allicin inhibited by ca. 50% the proliferation of Leishmania amastigotes. Low toxicity for mammalian cells of this compound suggests the interest of exploring the value of allicin in combined therapy against leishmanial infections.

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani.

The toxicity and antileishmanial activity of 20 betulin derivatives were studied. The toxicity of betulin and synthesized compounds was determined using a bacterial test (Microtox) and two mammalian cell lines (CHO-K1 and J774). The antileishmanial activity of compounds (50 µM) was examined in both the promastigote and intracellular amastigote stages of Leishmania infantum and L. donovani. No correlation was found among the toxicity tests. All the compounds showed significant antipromastigote activity. The antiproliferative capacity of derivatives was dependent on the parasite stage studied, and no substantial differences were found between Leishmania species. Betulin, 3,28-di-O-acetylbetulin and L-aspartyl amide of betulonic acid showed moderate activity against amastigotes. The highest inhibition of intracellular amastigote multiplication was achieved with a low micromolar concentration (IC(50) ca 9 µM) of heterocyclic betulin derivative 3,28-di-O-acetyllup-13(18)-ene with N-ethyltriazolo moiety 16, without significant toxicity for mammalian cells. These results point to the interest of this lead compound for further in vitro and in vivo tests.