Isobolographic analysis of co-administration of two plant-derived antiplasmodial drug candidates, cryptolepine and xylopic acid, in Plasmodium berghei.

BACKGROUND: Increasing resistance to current anti-malarial therapies requires a renewed effort in searching for alternative therapies to combat this challenge, and combination therapy is the preferred approach to address this. The present study confirms the anti-plasmodial effects of two compounds, cryptolepine and xylopic acid and the relationship that exists in their combined administration determined. METHODS: Anti-plasmodial effect of cryptolepine (CYP) (3, 10, 30 mg kg-1) and xylopic acid (XA) (3, 10, 30 mg kg-1) was evaluated in Plasmodium berghei-infected male mice after a 6-day drug treatment. The respective doses which produced 50% chemosuppression (ED50) was determined by iterative fitting of the log-dose responses of both drugs. CYP and XA were then co-administered in a fixed dose combination of their ED50s (1:1) as well as different fractions of these combinations (1/2, 1/4, 1/8, 1/16 and 1/32) to find the experimental ED50 (Zexp). The nature of interaction between cryptolepine and xylopic acid was determined by constructing an isobologram to compare the Zexp with the theoretical ED50 (Zadd). Additionally, the effect of cryptolepine/xylopic acid co-administration on vital organs associated with malarial parasiticidal action was assessed. RESULTS: The Zadd and Zexp were determined to be 12.75 ± 0.33 and 2.60 ± 0.41, respectively, with an interaction index of 0.2041. The Zexp was significantly (P < 0.001) below the additive isobole indicating that co-administration of cryptolepine and xylopic acid yielded a synergistic anti-plasmodial effect. This observed synergistic antiplasmodial effect did not have any significant deleterious effect on the kidney, liver and spleen. However, the testis were affected at high doses. CONCLUSION: The co-administration of cryptolepine and xylopic acid produces synergistic anti-malarial effect with minimal toxicity.

Comparative Susceptibility of Different Mouse Strains to Liver-Stage Infection With Plasmodium berghei Sporozoites Assessed Using In Vivo Imaging.

BACKGROUND: The liver stages of Plasmodium parasites are important targets for the discovery and development of prophylactic drugs. METHODS: A real-time in vivo imaging system was used to determine the level of luminescence measured from firefly luciferase expression by sporozoites developing in hepatocytes in different strains of mice. RESULTS: The luminescence values (photon counts/sec) measured from the anatomical liver location in the untreated mice infected with 10,000 Plasmodium berghei sporozoites were 8.15 × 105 for C57BL/6 Albino, 2.12 × 105 for C3H/HeNCrL, 0.91 × 105 for C57BL/6 WT, 0.28 × 105 for BALB/c, and 0.16 × 105 for ICR/CD-1 mice. This data suggests that the C57BL/6 Albino strain is most susceptible to luminescent photon, mainly because the less light scattering and absorption from deeper tissues and the skin in the strain of mouse. The photon count observed in black C57BL/6 wild type mice was shown to be 88.83% lower compared to C57BL/6 Albino mice. Although the highest growth rate of sporozoites in hepatocytes was found for C57BL/6 wild type mice in this study, the black skin of this mouse significantly reduced parasite-associated bioluminescence. CONCLUSIONS: The minimal light scattering and absorption and also enhanced susceptibility to liver infection of C57BL/6 Albino mice makes this strain preferable sensitivity for discovery and development of prophylactic antimalarial drugs.

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice.

The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis.

Molecular and phylogenetic characterizations of an Eimeria krijgsmanni Yakimoff & Gouseff, 1938 (Apicomplexa: Eimeriidae) mouse intestinal protozoan parasite by partial 18S ribosomal RNA gene sequence analysis.

Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata.

Impact of treatment with praziquantel, silymarin and/or beta-glucan on pathophysiological markers of liver damage and fibrosis in mice infected with Mesocestoides vogae (Cestoda) tetrathyridia.

Mesocestoides vogae tetrathyridia infection in mice causes hepatocyte injury, hepatic granulomatous inflammmation, liver fibrosis and chronic peritonitis manifested with portal hypertension. To reduce the detrimental effect of parasites on the host liver, the effect of the anthelmintic drug praziquantel (PZQ) in combination with natural products silymarin (an antioxidant) and beta-glucan (an immunomodulator) was investigated. The therapeutic effect of drugs was assessed by means of aminotransferase (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) activities, content of albumin, total proteins and hyaluronic acid (HA) in sera of ICR mice infected with M. vogae larvae. Animals were treated with PZQ suspended in oil emulsion (Group 1), PZQ combined with silymarin incorporated into lipid microspheres (LMS) (Group 2), PZQ combined with beta-glucan incorporated into liposomes (LG) (Group 3), PZQ co-administered with LMS and LG (Group 4). Untreated animals (Group 5) served as the control. Treatment of animals started at the early chronic phase of infection (day 14 p.i.) and lasted 10 days; serum samples were collected on days 0, 7, 14, 25, 28, 31, 35 and 45 p.i. ALT and AST activities were significantly (P < 0.05) decreased in Groups 2, 3 and 4. HA content was significantly (P < 0.05 and 0.01) lower in Groups 2 and 4. Albumin levels were decreased in Groups 2 and 4, total protein concentration decreased in Groups 1 and 3 (P < 0.05 and 0.01). These results showed that combined treatment of PZQ with silymarin and/or beta-glucan was able to ameliorate or suppress fibrogenesis in the liver, protect liver cells from oxidative damage and, possibly, stimulate regeneration of the parenchyma.

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis.

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.

Susceptibility of Peromyscus leucopus and Mus musculus to infection with Baylisascaris procyonis.

In this study, we compared the susceptibility of Peromyscus leucopus (white-footed mouse), a common natural intermediate host, and Mus musculus, a commonly used experimental model, to infection with larvae of the raccoon ascarid, Baylisascaris procyonis. Three groups of 10 mice of each species were given 50, 250, or 500 infective B. procyonis eggs by gavage. The mice were observed daily for clinical signs of central nervous system (CNS) disease and at necropsy the distribution of larvae in 10 body regions and organs was determined and compared. Clinical CNS disease developed in 57% of P. leucopus and 93% of M. musculus. The average clinical incubation period was significantly longer in P. leucopus (20.6 days postinfection [PI]) than in M. musculus (10.7 days PI), and clinical disease progressed slower in P. leucopus. Significantly fewer larvae were recovered from P. leucopus than from M. musculus. Most larvae were recovered from the anterior carcass and viscera of P. leucopus and from the carcass, head, and brain of M. musculus. CNS invasion was dose dependent in M. musculus but not in P. leucopus. Few or no grossly visible larval granulomas were present in P. leucopus but were abundant in M. musculus. We concluded that P. leucopus was less susceptible than M. musculus to B. procyonis infection, based on a decreased intensity of infection, longer clinical incubation period or lack of clinical disease, slower progression of disease, different larval distribution, and lower tissue reactivity to larvae.

A protocol for the production of Neospora caninum tissue cysts in mice.

Identification of a definitive host for Neospora caninum has been inhibited by lack of an efficient method for producing bradyzoites, needed for oral infectivity trials. An improved protocol for producing bradyzoite-containing tissue cysts in mouse brains is described. Six variables, including mouse strain (Balb/C, CBA/Ca, and ICR), sex, N. caninum isolate (NC-2 and NC-Liverpool), tachyzoite inoculum dose, immunosuppression with methylprednisolone acetate (MPA), and sulfadiazine treatment were tested. Tissue cyst numbers were estimated using an immunohistologic staining procedure specific for bradyzoites. Male ICR mice (> or = 30 g) that were immunosuppressed with 2 mg MPA 7 days prior to and 2.5 mg MPA at the time of subcutaneous inoculation with 400,000 N. caninum tachyzoites produced the highest numbers of tissue cysts. Significant numbers were produced by methods using the NC-2 strain of N. caninum; however, protocols using NC-Liverpool produced greater numbers of tissue cysts. Sulfadiazine treatment did not appear to contribute to tissue cyst production. The procedure described is superior to previously described methods with regard to numbers of tissue cysts produced, protocol reproducibility, and survival of mice until tissue cyst formation.

Maintenance of the life cycle of Echinostoma trivolvis (Trematoda) in dexamethasone-treated ICR mice and laboratory-raised Helisoma trivolvis (Gastropoda).

Echinostoma trivolvis is a ubiquitous 37-collar-spined echinostome found in aquatic birds and mammals and in the planorbid snail Helisoma trivolvis. This echinostome has not been cycled continuously in the laboratory. The present report provides details on the continuous life cycle of E. trivolvis in dexamethasone-treated ICR mice and laboratory-raised H. trivolvis snails. Previous attempts to obtain patent adult of E. trivolvis in mice hosts failed because of worm injection within 2 weeks of infection. ICR mice infected with encysted metacercariae and injected with 2 mg/kg dexamethasone daily for 28 days yielded gravid worms that produced 250-500 eggs/worms at 21 and 28 days postinfection (p.i.). Miracidia derived from these eggs or eggs containing fully developed miracidia were capable of infecting 3- to 5-mm shell-diameter, laboratory-reared H. trivolvis snails. These snails released cercariae by 35 days p.i. Cercariae encysted in the kidney-pericardium of the snails. Encysted metacercariae could be excysted in vitro in an alkaline trypsin-bile salts medium or in vivo when fed to domestic chicks.

Schistosoma mansoni: relocation of parasites to lungs from hepatic portal system in rodents.

The prevalence and development of adult worms in the lungs of mice and gerbils infected with Schistosoma mansoni was investigated. All infected BALB/c mice harbored the schistosomes in their lungs at 10-12 weeks post-infection, showing the distinct relocation of adult worms to the lungs, from the hepatic portal system. The male and female flukes from lungs of BALB/c mice were significantly smaller than those from livers. The percentage of gravid females in lungs was considerably lower than that in the livers. The number of eggs recovered from lungs of BALB/c mice and gerbils having lung female worms, however, was higher than that from animals without lung females, indicating egg deposition of lung females. The number of eggs detected in the brains correlated well with the number of eggs from the lungs in BALB/c and ICR mice. Out of 119 infected gerbils at 8 weeks post-infection, only two animals had egg-emboli in the brain vessels, although many eggs embolized in the lungs of those animals. These data suggest that transfer of worms to the lungs from livers involves reduction of worm recovery from the portal circulation, and also pulmonary pathology of the disease.

In vitro excystation of Spironucleus muris.

In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia, were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.

Effect of irradiation on the viability of Angiostrongylus cantonensis and A. costaricensis infective larvae.

Infective larvae (third-stage larvae) of both Angiostrongylus cantonensis and A. costaricensis from their snail intermediate host were subjected to either X-ray or gamma-ray irradiation. The viability of the irradiated larvae was assayed by oral inoculation of the larvae into rodents (A. cantonensis in mice and rats, A. costaricensis in mice only). From the results of worm recovery, the minimal dose of irradiation that inhibited the infectivity of the third-stage larvae of A. cantonensis and A. costaricensis was 2 and 4 kGy, respectively.

Trichomonas vaginalis: strain differences in adhesion to plastic and virulence in vitro and in vivo.

Trichomonas vaginalis isolated from clinical cases were grown axenically in TYI-S-33 medium. Various strains of this flagellate showed different adhesion characteristics in medium containing bovine serum or Cohn fractions thereof. These were not inversely related to the time in axenic culture. The adsorption of serum components to the parasites was assessed in relationship to adhesion but in many cases was probably not adhesion-related. All strains tested for virulence in vitro were almost equally cytotoxic for HeLa tissue cultures. However, in infectivity trials, one of these strains exhibited the highest adhesion capability and proved to be the most virulent for mice, and another (cloned) strain with the poorest adhesion capability failed to cause infection. Other strains exhibited lessened virulence following their extended axenic cultivation. It therefore appears that the in vivo pathogenic potential of the parasites growing in axenic culture is inherently strain-dependent. The findings suggest that although adhesion in whole serum-containing medium is sufficient to differentiate between various Trichomonas isolates, it is insufficiently sensitive to correlate adhesion with virulence. It apparently is important to identify the adhesion-mediating factor(s) in serum or in its Cohn fractions IV-1 and IV-4 and to use it (them) to elucidate the possible correlation between the parasite's capacity to adhere and its pathogenicity.

Gametocytocidal and sporontocidal activity of antimalarials against Plasmodium berghei ANKA in ICR Mice and Anopheles stephensi mosquitoes.

The gametocytocidal and sporontocidal activity of three 8-aminoquinolines (primaquine, WR-238605, and WR-242511), three dihydroacridine-diones (floxacrine, WR-250547, and WR-250548), a 1,4-naphthoquinone (menoctone), a synthetic aminoalcohol (halofantrine), and a guanide (WR-182393) was determined against a cloned line of Plasmodium berghei ANKA. Gametocytocidal activity was assessed by treating mice with a single intraperitoneal inoculation of a given compound (25 mg base drug/kg mouse body weight) four days after the mice were infected with P. berghei. Thin blood smears were made every other day, and the percent parasitemia and macrogametocyte and microgametocyte rates were determined. Floxacrine, menoctone, WR-242511, WR-250547, and WR-250548 effectively cleared sexual and asexual parasites from the peripheral circulation within six days of drug administration. Halofantrine, primaquine, WR-182393, and WR-238605 were ineffective at clearing P. berghei ANKA from circulating erythrocytes at the doses tested; however, mice survival time increased markedly with these compounds when compared with the controls. Significant numbers of macrogametocytes and microgametocytes were present throughout the duration of the infection in mice treated with halofantrine, primaquine, WR-182393, and WR-238605. Sporontocidal activity was evaluated by allowing Anopheles stephensi mosquitoes to feed on P. berghei-infected mice 90 min after treatment with a particular drug. Halofantrine and WR-182393 exhibited no sporontocidal activity, while floxacrine, menoctone, primaquine, WR-238605, WR-242511, WR-250547, and WR-250548 exhibited significant activity. Minimum effective doses (mg base drug/kg of mouse body weight) that prevented mosquitoes from developing sporozoite-infected salivary glands were 0.1563 mg/kg for WR-250547, 0.625 mg/kg for menoctone, 1.25 mg/kg for primaquine, 10 mg/kg for floxacrine, 10 mg/kg for WR-242511, 10 mg/kg for WR-250548, and 25 mg/kg for WR-238605.

The expulsion of Echinostoma trivolvis (Trematoda) from ICR mice: scanning electron microscopy of the worms.

Echinostoma trivolvis adults are rejected from ICR mice within 3 weeks postinfection (p.i.) but are retained in golden hamsters for greater than 15 weeks. The present study used scanning electron microscopy (SEM) to examine worm topography in ICR mice, particularly that of the collar spines, and to correlate worm loss with tegumentary changes. The topography of the worm in ICR mice was similar to that observed in previous studies on this echinostome in domestic chick embryos, chickens, and golden hamsters. Observations were made on the pattern of collar spines in 115 worms from ICR mice at 3-14 days p.i. All worms examined at 3 days exhibited extended spines, whereas about 70% of the worms examined at 14 days displayed retracted or missing spines. Eight worms from golden hamsters examined at 14 days p.i. showed extended collar spines. The retraction or loss of collar spines may play a role in the expulsion of E. trivolvis from ICR mice.

Infectivity, growth, and distribution of Echinostoma caproni (Trematoda) in the ICR mouse.

Host-parasite interactions of the intestinal trematode Echinostoma caproni were studied in ICR laboratory mice. All of 40 mice, each fed 25 metacercarial cysts of Echinostoma caproni, were infected 1-20 wk postinfection (PI) with a mean of 17.2 worms/host. At 24 and 29 wk PI only 2 of 6 mice (33%) were infected, with a mean of 4.2 worms/host. Mean body area of worms increased rapidly to about 5 mm2 by week 2, increased less rapidly to 8.8 mm2 by week 12, plateaued until week 24, and then declined. Mean dry weight of worms increased rapidly to about 0.5 mg by week 2, less rapidly to 1.4 mg by week 12, and then plateaued until week 24 PI. From 1 to 8 wk PI most worms localized in the jejunum and ileum; later most worms were in the jejunum and duodenum. Considerable differences were seen in the growth and distribution of E. caproni in the ICR mouse compared with previous studies on this echinostome species in the NMRI mouse.

[Isolation and identification of Cryptosporidium from various animals in Korea. II. Identification of Cryptosporidium muris from mice].

Each of SPF mice(Scl: ICR strain, 3-week-old males) was inoculated with 5 x 10(4) oocysts of Cryptosporidium by stomach tube. The oocysts were large type one which was previously isolated from Korean mice, and passaged in 3-week-old SPF mice. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of developmental stages the stomach of the mice was examined by transmission electron microscopy (TEM) at 4 weeks post-inoculation. The prepatent period for 6 mice was 5.6 days post-inoculation on the average, and the patent period was 63.2 days. The number of oocysts discharged per day from the mice reached peak on day 36.6 post-inoculation on the average. A large number of oocysts were found in fecal samples obtained from inoculated mice on days 30-50 post-inoculation. C. muris was larger than C. parvum at almost every developmental stages, the size difference being 1.4 times in oocysts, 2.4 times in sporozoites, 1.6 times in merozoites, and 1.5 times in microgametes. The ultrastructural features of the attachment site of C. muris to the mucus cells were remarkably different from those of C. parvum and its closely related species. The anterior projection of the protozoa (C. muris), the outer aspect of which was surrounded by a thick filamentous process of the host cell, has not been reported at any developmental stages of C. parvum or its closely related species. The size of the oocysts of strain RN 66 was larger than that of Korean mice origin. The above results reveal that the large type Cryptosporidium of Korean mice origin is identified as Cryptosporidium muris and this type was named as C. muris (strain MCR).

Infectivity of Trichinella pseudospiralis isolated from carrion.

The reproductive capacity index for Trichinella pseudospiralis infective larvae was similar for worms isolated from mouse carcasses on the day upon which mice were killed (day 0: 104.4 +/- 18.6 [mean +/- SD]) and on day 5 following mouse death (106.1 +/- 23.6), but was reduced for worms recovered from carcasses on day 10 postkill (PK: 22.7 +/- 5.7). Larvae isolated from mouse carcasses held at 24 C after day 10 PK were not infective. The percentage of viable worms (tightly coiled or moving) isolated from carrion was similar on days 0 and 5 PK but had declined to 40.4% by day 10 PK and showed a further reduction to 11.8% for worms isolated from carrion on day 15 PK. Viable worms were not recovered from carcasses after day 15 PK.

Single- and five-worm infections of Echinostoma caproni (Trematoda) in the ICR mouse.

Twenty-nine (64%) of 44 ICR mice fed a single metacercarial cyst of Echinostoma caproni and all of 23 mice each fed five cysts were infected with ovigerous worms at necropsy 2-4 weeks post-infection. Each host fed five cysts had two to five worms at necropsy, and all worms were either paired or clustered. Distribution of worms in the small intestine was similar in single- and five-worm infections and all worms were located 17-20 cm anterior to the ileo-cecal valve. Both single and multiple worms produced eggs with fully-developed miracidia. The number of eggs per uterus in 2-week-old multiple worms was almost twice that of single worms. The body area of 3- and 4-week-old multiple worms was significantly greater than that of single worms of the same age.