Self-emulsifying drug delivery systems (SEDDS) containing Cymbopogon citratus essential oil: Enhancing the stability and acaricide efficacy against Rhipicephalus (Boophilus) microplus.

The objectives of this study were to develop a self-emulsifying drug delivery system (SEDDS) to enhance the stability and efficacy of Cymbopogon citratus essential oil or lemongrass oil (LEO) against cattle tick larvae and engorged females. The system with the highest oil loading in SEDDS was composed of LEO (23.33%w/w), Tween 80: SGKH 4000 in a 2:1 ratio as surfactant (66.67%w/w), and propylene glycol as co-surfactant (10%w/w). The selected SEDDS-LEO has a particle size of 18.78 nm with a narrow size distribution (polydispersity index of 0.27). Notably, the stability of SEDDS was superior to that of the original oil, both during long-term storage and under accelerated conditions. SEDDS-LEO at oil concentrations ranging from 1.458% to 5.833% w/v showed a significantly higher percentage of egg-laying reduction against adult ticks compared with the original oil at the same concentrations (p < 0.05). Furthermore, SEDDS-LEO demonstrated greater larvicidal efficacy than the original oil, with lower LC50 and LC90 values of 0.91 mg/mL and 1.20 mg/mL, respectively, whereas the original oil's LC50 and LC90 values were 1.17 mg/mL and 1.74 mg/mL, respectively. Our findings indicate that SEDDS-LEO is a promising candidate for use as an acaricide in the control of tick populations in dairy cattle.

Inhibitory effects on bovine babesial infection by iron chelator, 1-(N-acetyl-6-aminohexyl)- 3-hydroxy-2-methylpyridin-4-one (CM1), and antimalarial drugs.

BACKGROUND: Babesiosis is an infectious disease caused by protozoa of the apicomplexan phylum, genus Babesia. It is a malaria-like parasitic disease that can be transmitted via tick bites. The apicomplexan phylum of eukaryotic microbial parasites has had detrimental impacts on human and veterinary medicine. There are only a few drugs currently available to treat this disease; however, parasitic strains that are resistant to these commercial drugs are increasing in numbers. Plasmodium and Babesia are closely related as they share similar biological features including mechanisms for host cell invasion and metabolism. Therefore, antimalarial drugs may be useful in the treatment of Babesia infections. In addition to antimalarials, iron chelators also inhibit parasite growth. In this study, we aimed to evaluate the in vitro inhibitory efficacy of iron chelator and different antimalarials in the treatment of Babesia bovis. METHODS: Cytotoxicity of antimalarial drugs; pyrimethamine, artefenomel, chloroquine, primaquine, dihydroarthemisinine, and the iron chelator, 1-(N-acetyl-6-aminohexyl)- 3-hydroxy-2 methylpyridin-4-one (CM1), were evaluated against Madin Darby Bovine Kidney (MDBK) cells and compared to diminazene aceturate, which is the currently available drug for animal babesiosis using an MTT solution. Afterwards, an evaluation of the in vitro growth-inhibitory effects of antimalarial drug concentrations was performed and monitored using a flow cytometer. Half maximal inhibitory concentrations (IC50) of each antimalarial and iron chelator were determined and compared to the antibabesial drug, diminazine aceturate, by interpolation using a curve-fitting technique. Subsequently, the effect of the drug combination was assessed by constructing an isobologram. Values of the sum of fractional inhibitions at 50% inhibition were then estimated. RESULTS: Results indicate that all drugs tested could safely inhibit babesia parasite growth, as high as 2500 µM were non-toxic to mammalian cells. Although no drugs inhibited B. bovis more effectively than diminazine aceturate in this experiment, in vitro growth inhibition results with IC50 values of pyrimethamine 6.25 ± 2.59 µM, artefenomel 2.56 ± 0.67 µM, chloroquine 2.14 ± 0.76 µM, primaquine 22.61 ± 6.72 µM, dihydroarthemisinine 4.65 ± 0.22 µM, 1-(N-acetyl-6-aminohexyl)- 3-hydroxy-2 methylpyridin-4-one (CM1) 9.73 ± 1.90 µM, and diminazine aceturate 0.42 ± 0.01 µM, confirm that all drugs could inhibit B. bovis and could be used as alternative treatments for bovine babesial infection. Furthermore, the efficacy of a combination of the iron chelator, CM1, in combination with artefenomel dihydroarthemisinin or chloroquine, and artefenomel in combination with the iron chelator, CM1, dihydroarthemisinin or chloroquine, exhibited synergism against B. bovis in vitro. CONCLUSION: Our evaluation of the inhibitory efficacy of the iron chelator CM1, antimalarial drugs, and a combination of these drugs against B. bovis could be potentially useful in the development and discovery of a novel drug for the treatment of B. bovis in the future.

Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response.

Both strong innate and adaptive immune responses are an important component of protection against intraerythrocytic protozoan parasites. Resistance to bovine babesiosis is associated with interferon (IFN)-γ mediated responses. CD4+ T cells and macrophages have been identified as major effector cells mediating the clearance of pathogens. Previously, the apical membrane antigen 1 (AMA-1) was found to significantly induce the immune response inhibiting B. bovis merozoite growth and invasion. However, a detailed characterization of both humoral and cellular immune responses against the structure of B. bovis AMA-1 (BbAMA-1) has not yet been established. Herein, the present study aimed to express the recombinant BbAMA-1 domain I+II protein [rBbAMA-1(I/II)], which is the most predominant immune response region, and to characterize its immune response. As a result, cattle vaccinated with BbAMA-1(I/II) significantly developed high titters of total immunoglobulin (Ig) G antibodies and a high ratio of IgG2/IgG1 when compared to control groups. Interestingly, the BbAMA-1(I/II)-based formulations produced in our study could elicit CD4+ T cells and CD8+ T cells producing IFN-γ and tumor necrosis factor (TNF)-α. Collectively, the results indicate that immunization of cattle with BbAMA-1(I/II) could induce strong Th1 cell responses. In support of this, we observed the up-regulation of Th1 cytokine mRNA transcripts, including IFN-γ, TNF-α, Interleukin (IL)-2 and IL-12, in contrast to down regulation of IL-4, IL-6 and IL-10, which would be indicative of a Th2 cytokine response. Moreover, the up-regulation of inducible nitric oxide synthase (iNOS) was observed. In conclusion, this is the first report on the in-depth immunological characterization of the response to BbAMA-1. According to our results, BbAMA-1 is recognized as a potential candidate vaccine against B. bovis infection. As evidenced by the Th1 cell response, it could potentially provide protective immunity. However, further challenge-exposure with virulent B. bovis strain in immunized cattle would be needed to determine its protective efficacy.

Liver- and Spleen-Specific Immune Responses in Experimental Leishmania martiniquensis Infection in BALB/c Mice.

Leishmania martiniquensis is a neglected cause of an emerging leishmaniasis in many countries, including France, Germany, Switzerland, the United States of America, Myanmar, and Thailand, with different clinical manifestations ranging from asymptomatic, cutaneous (CL), visceral (VL), and atypically disseminated CL and VL. The persistence of parasites and the recurrence of the disease after treatment are challenges in controlling the disease. To explore efficient prophylaxis and therapy, this study aimed to investigate infection outcome and organ-specific immune responses after inoculation with L. martiniquensis (MHOM/TH/2011/PG; 5 x 106 promastigotes) in BALB/c mice via intravenous and intraperitoneal routes. A quantitative PCR technique, targeting L. martiniquensis ITS1, was primarily established to estimate the parasite burden. We found that the infection in the liver resolved; however, persistent infection was observed in the spleen. Histopathology with Leishmania-specific immunostaining revealed efficient hepatic granuloma formation, while splenic disorganization with parasitized macrophages at different locations was demonstrated. The mRNA expression of Th1 cytokines (IFN-γ, TNF-α, IL-12p40) and iNOS in the liver and spleen was upregulated. In addition, high expression of IL-10 was observed in the spleen in the chronic phase, revealing a significant moderate correlation with the parasite persistence [r(12) = 0.72, P = 0.009]. Further clarification of the mechanisms of persistent infection and experimental infection in immunosuppressed murine models are warranted.

Biliary cystadenoma associated with Opisthorchis viverrini infection in a domestic cat (Felis catus).

A 12-year-old, female, domestic cat (Felis catus) presented with dehydration, emaciation, anorexia, and lethargy. The cat was unresponsive to medical treatment and euthanized; the carcass was submitted for pathological diagnosis. Necropsy revealed icteric mucous membranes. The liver was enlarged, with multinodular, cystic, white masses, 0.5-4.0 cm in diameter, scattered throughout. Microscopically, the biliary epithelium presented with a proliferation of multifocal cystic masses, occasionally with periodic acid-Schiff-positive fluid within the cysts. Simple cuboidal epithelial cells showed small, round to oval, vesicular nuclei and rare mitotic figures. There were also multifocal trematode-like parasites situated within the biliary tracts. Immunohistochemistry of the cystic masses was positive for pan-cytokeratin and proliferating cell nuclear antigen, while negative for vimentin. Molecular analysis and gene sequencing of the parasite indicated that it was Opisthorchis viverrini. Based on the pathological findings and molecular analysis, the cat was diagnosed with biliary cystadenoma related to O. viverrini infection. This report described an unusual case of O. viverrini infection associated with biliary tumor in a cat, and raises the possibility of domestic cats as a reservoir host of the human liver fluke.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

BACKGROUND: Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE: The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS: We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS: Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Taeniasis among Refugees Living on Thailand-Myanmar Border, 2012.

We tested refugee camp residents on the Thailand-Myanmar border for Taenia solium infection. Taeniasis prevalence was consistent with that for other disease-endemic regions, but seropositivity indicating T. solium taeniasis was rare. Seropositivity indicating cysticercosis was 5.5% in humans, and 3.2% in pigs. Corralling pigs and providing latrines may control transmission of these tapeworms within this camp.

Comparison of PCR methods for detection of Leishmania siamensis infection.

BACKGROUND: Leishmania siamensis, a newly identified species, has been reported as a causative agent of leishmaniasis in Thailand. This organism has been identified and genetically characterized using PCR techniques based on several target genes. However, the sensitivities and specificities of these methods for the diagnosis of L. siamensis infection have never been evaluated. METHODS: To evaluate the sensitivities and specificities of PCR methods to detect L. siamensis infection, PCR for different genetic markers, i.e., the small subunit ribosomal RNA region (SSU-rRNA), the internal transcribed spacer 1 region (ITS1), cysteine protease B (cpb), cytochrome b (cyt b), heat shock protein 70 (hsp70), the spliced leader mini-exon, and the triose-phosphate isomerase (tim) genes were compared. RESULTS: Both the ITS1-PCR and the SSU rRNA-PCR could detect promastigote of L. siamensis at concentrations as low as 0.05 parasites/µl or the DNA concentration at 2.3 pg/µl. Though the ITS1-PCR method only recognized 8 samples as positive, all of these could be assessed as true positive according to microscopic diagnosis and/or amplifying the results of the PCR and their sequencing, while other methods also produced false positive results. Compared with the ITS1-PCR method, the PCR amplified SSU-rRNA and cpb gene showed 100% sensitivity for the detection of L. siamensis in clinical specimens. The PCR amplified mini-exon and hsp70 gene also gave a high sensitivity of 87.5%. In contrast, the PCR methods for cyt b and tim gene showed low sensitivity. The PCR methods for cyt b, mini-exon and tim gene showed 100% specificity compared with the ITS1-PCR. CONCLUSION: As a result, the ITS1-PCR method is a suitable target for PCR-based detection of L. siamensis infection in clinical specimens due to its high sensitivity and specificity. The results of this study can be used for molecular diagnosis as well as in epidemiological studies of L. siamensis in affected areas.

Involvement of CD4⁺ Foxp3⁺ regulatory T cells in persistence of Leishmania donovani in the liver of alymphoplastic aly/aly mice.

Visceral leishmaniasis (VL) is a chronic and fatal disease in humans and dogs caused by the intracellular protozoan parasites, Leishmania donovani and L. infantum (L. chagasi). Relapse of disease is frequent in immunocompromised patients, in which the number of VL cases has been increasing recently. The present study is aimed to improve the understanding of mechanisms of L. donovani persistence in immunocompromised conditions using alymphoplastic aly/aly mice. Hepatic parasite burden, granuloma formation and induction of regulatory T cells were determined for up to 7 months after the intravenous inoculation with L. donovani promastigotes. While control aly/+ mice showed a peak of hepatic parasite growth at 4 weeks post infection (WPI) and resolved the infection by 8 WPI, aly/aly mice showed a similar peak in hepatic parasite burden but maintained persistent in the chronic phase of infection, which was associated with delayed and impaired granuloma maturation. Although hepatic CD4(+)Foxp3(+) but not CD8(+)Foxp3(+) T cells were first detected at 4 WPI in both strains of mice, the number of CD4(+)Foxp3(+) T cells was significantly increased in aly/aly mice from 8 WPI. Immunohistochemical analysis demonstrated the presence of Foxp3(+) T cells in L. donovani-induced hepatic granulomas and perivascular neo-lymphoid aggregates. Quantitative real-time PCR analysis of mature granulomas collected by laser microdissection revealed the correlation of Foxp3 and IL-10 mRNA level. Furthermore, treatment of infected aly/aly mice with anti-CD25 or anti-FR4 mAb resulted in significant reductions in both hepatic Foxp3(+) cells and parasite burden. Thus, we provide the first evidence that CD4(+)Foxp3(+) Tregs mediate L. donovani persistence in the liver during VL in immunodeficient murine model, a result that will help to establish new strategies of immunotherapy against this intracellular protozoan pathogen.