Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major Induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Detection of Asymptomatic Carriers of Plasmodium vivax among Treated Patients by Nested PCR Method in Minab,Rudan and Bashagard,Iran

Background

Plasmodium vivax is the most widespread species of Plasmodium in humans and causing about 80 million clinical cases annually. This study was undertaken to detect P. vivax in asymptomatic treated vivax malaria patients to trace latent/sub-patent malaria infection.

Method

The venous blood of all detected cases with P. vivax in Bashagard, Minab and Roodan Districts in Hormozgan Province from 2009 to 2010 was examined by microscopic and nested PCR methods for presence of the parasite.

Results

In microscopic examination of peripheral blood smears, all samples were negative for the presence of the parasites. But, we detected two P. vivax related bands in the electrophoresis of the nested PCR products (120 bp).

Conclusion

Following up the malaria cases after treatment by a combination of methods, or new diagnostics such as RDTs can be included in the priorities of malaria elimination program in Iran.     

Absence of Asymptomatic Malaria Infection in Endemic Area of Bashagard District,Hormozgan Province,Iran

Background

A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran.

Methods

Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques.

Results

Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1₁₉-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1₁₉, using ELISA.

Conclusion

This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.     

Detection and Identification of Toxoplasma gondii Type One Infection in Sheep Aborted Fetuses in Qazvin Province of Iran

Background

The aim of this study was to apply the nested-PCR and bioassay methods in detection and genotyping of Toxoplasma gondii infection in provided sheep aborted fetus samples from Qazvin Province of Iran.

Methods

Eighteen sheep aborted fetal samples were studied by nested-PCR-RFLP, histopathological observation and microbiological assay. Bioassay in mice was carried out by inoculating the brain samples intraperitoneally.

Results

The results demonstrated the frequency of 66% infected sheep aborted fetal samples with T. gondii type one. Although we could not isolate any parasite from inoculated mice even after three passages, but it was confirmed histopathologically formation of cyst like bodies in prepared mice brain sections.

Conclusion

The results of the performed nested-PCR and formation of brain cyst in inoculated mice exhibited that T. gondii type one infection might be considered as one of the major causative agents for abortion in ewes.     

Aspicularis tetraptera Induced Hematological Parameters in Infected and Vaccinated Mice

Background

The present study deals with the effect of helminthic infection as Nematode parasite like Aspiculuris tetraptera on the haematological parameters of infected and vaccinated mice.

Methods

Totally 15 mice were used. Five mice were used for positive control, 5 mice used for negative control and 5 mice used for experiment. The hematological parameters were studied viz. RBC, Hb, and serum protein values.

Results

The mice carrying heavy infection showed decrease in the Hb, RBC, and serum protein but in the vaccinated mice, all studied parameters were become on normal range. The level of immune response was assessed based on above studied hematological parameters in infected and vaccinated mice with Aspiculuris tetraptera.

Conclusion

The increased value of RBC, Hb and Serum protein in infected and vaccinated mice compared to infected and non vaccinated suggested the involvement of blood parameters in immune response. This study also proves that somatic antigen of A. tetraptera was effective in imparting immunity in mice     

Immunogenicity of MHC Class I Peptides Derived from Leishmania mexicana Gp63 in HLA-A2.1 Transgenic (HHDII) and BALB/C Mouse Models

Background

Leishmania is an intracellular parasite infecting humans and many wild and domestic animals. Recent studies have suggested an important role for cytotoxic T cells against Leishmania. Peptide-based vaccines targeting short sequences derived from known immunogenic proteins have been shown to elicit cellular immune responses against disparate pathogens.

Methods

We predicted four HLA-A2 peptides derived from L. mexican/major gp63 and tested these in HHD II mice, as well as four peptides for mouse MHC class I from the same proteins tested in BALB/ mice.

Results

The results revealed immunogenicity for three of the four peptides predicted for HLA-A2. Immunisation with these peptides, along with IFA, induced CTL responses detected by standard 4-hour cytotoxicity assay and significantly upregulated the production of IFN-γ. When HHDII mice were injected IM with L. mexicana gp63 cDNA and splenocytes were restimulated with blasts loaded with the immunogenic peptides, two of the peptides were able to induce significant level of IFN-γ detected by ELISA. None of the peptides predicted for Balb/c mouse MHC class I elicited CTL activity or significantly upregulated the IFN-γ.

Conclusion

The results may help in developing a peptide-based vaccine, which can be applied alone or in combination with drugs against Leishmania.     

Malaria in Mazandaran,Northern Iran: Passive Case Finding During 1997-2012

Background

Malaria is one of the most important parasitic diseases in tropical and temperate regions. The aim of this study was to determine the trend of malaria in Mazandaran Province, northern Iran during 1997-2012.

Methods

This retrospective study was conducted from 1997 to 2012. The population''s study was individuals who registered at health centers of Mazandaran Province. Peripheral blood smear were prepared for each case, stained with Giemsa and examined by light microscope. In addition to demographic data, other parameters including Slide Positive Rate (SPR), Annual Parasite Incidence (API) and Annual Blood Examination Rate (ABER) were analyzed.

Results

In total, 844 cases of malaria were reported. Plasmodium vivax was predominant species with 821 cases (97.4%). The number of malaria cases increased from 1997 to 2005 and then decreased to 3 cases in 2011. Some cities had not reported any cases during last three years. The highest infection rate, 163(20.07%), was seen in 2001-02. The SPR had the highest value (0.54%) in 2004-05. The maximum API and ABER were observed in 2001-02 and 1997-98. 641(75.9%) of cases were imported from hyperendemic areas such as Afghanistan and South-eastern Iran and 94 (11.1%) malaria patients were recorded as introduced cases. The highest infection rate of malaria (21.3%) was seen in Babolsar.

Conclusion

Extensive malaria control should be continued to Mazandaran to become malaria-free region and in prevention of re-introduction stage.     

Inhibition of Murine Systemic Leishmaniasis by Acetyl Salicylic Acid via Nitric Oxide Immunomodulation

Background

The purpose of this study was to evaluate antileishmanial effects of ASA via NO pathway in Leishmania major infected Balb/c mice. Moreover, toxicity and pathological consequences of ASA administration were investigated.

Methods

Balb/c mice were infected with L. major and ASA was inoculated orally after lesion appearance for its ability to modulate NO and to modify Leishmania infection in host, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen, and lymph nodes were also studied as target organs to detect amastigotes. In addition, plasma was investigated for NO induction using Griess microassay.

Results

ASA increased NO production in plasma of both naïve and Leishmania test groups at the ultimate of the experimental period. A decline was observed in proliferation of amastigotes inside macrophages of test group when compared with control one. ASA reduced lesion size, inhibited Leishmania visceralisation in spleen, lymph node, and decreased hepato/splenomegaly in ASA treated animals.

Conclusions

Some antileishmanial effects of ASA by NO-modulation were indicated during systemic leishmaniasis in mice. Despite slight effects on lesion size, ASA decreased parasite visceralization in target organs and declined their proliferation inside macrophages. Therefore, ASA may be indicated to inhibit systemic leishmaniasis via NO pathway in mice model.     

In vivo Susceptibility of Plasmodium vivax to Chloroquine in Southeastern Iran

Background

Plasmodium vivax is the predominant species causes of malaria with about 90% total annual reported malaria in Iran. This study conducted to determine the susceptibility of Plasmodium vivax isolates to chloroquine in Sistan and Balochistan Province, southeastern Iran.

Methods

A total 270 subjects with symptomatic malaria and confirmed P. vivax infection completed the designed 28-day in vivo study. The thick and thin film blood smears were screened for malaria parasites by microscopy. The nested PCR was applied using the Plasmodium 18 subunit ribosomal ribonucleic (Ssr RNA) genes for detecting mixed infections and diagnosis of parasites in the samples with low parasite on days 0, 5, 6, 7, and 28.

Results

P. vivax was cleared in 15%, 50%, 95%, and 100% of patients on days 1, 2, 3, 4 respectively by microscopy assessment. Six patients were exhibited specific P. vivax band in nested PCR on day 5. No recurrence was observed on days 7, 14 and 28. Mean (±standard deviation) parasite clearance time was 2.41 (±0.8) days.

Conclusion

P. vivax is still susceptible to chloroquine in Southeatern Iran. This finding is compatible with results of neighboring countries Pakistan and Afghanistan.     

Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA

Background

Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and AIDS patients, toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is difficult; so detection of parasite or its components could be useful tool for early detection and following treatment of the infection.

Methods

Sixty-three BALB/c mice were injected intra-peritoneal with 5×10³ tachyzoites of Toxoplasma gondii RH strain, nine mice were sacrificed daily for 7 days. Fourteen mice were injected with phosphate buffer saline as control group. Dot–ELISA was performed for detection of T.gondii antigen in mice sera and capture – ELISA was done as golden standard assay too.

Results

Toxoplasma gondii antigen was detected from day 2 in mice sera; 22% of mice sera on day 2, 33% on day 3,77% on day 4 and 100% on day 5 till their death on day 7 had shown antigenemia by dot – ELISA, no positive result was detected in control mice by dot- ELISA.

Conclusion

Dot-ELISA is a sensitive method for diagnosis of T. gondii infection in the animal model; also, this technique is more rapid and easy to perform method in comparison with capture-ELISA.     

Malaria Epidemiology in Mersin Province,Turkey from 2002 to 2011

Background

Malaria is an infectious disease caused by Plasmodium spp. with high morbidity and mortality in human in tropical and subtropical regions. In recent years, number of malaria cases has been significantly reduced because of fight with the disease in Turkey. This study intended to investigate the malaria epidemiology in Mersin Province from 2002 to 2011 using data from the provincial Public Health Directorate.

Methods

Over ten years, 303573 blood samples were taken from the people by active and passive surveillance methods and blood smears were prepared. Smears were stained with Giemsa and examined under the microscope.

Results

Totally, 73 people including 44 male and 29 female were positive in terms of Plasmodium spp. It was determined that P. vivax observed in 67 cases while P. falciparum in 6 cases. Cases were mainly observed in 15 to 44 years old range, showed an increase between June-September periods and a significant decrease after 2006. Out of the 73 malaria cases, 54 cases were from Mersin Province and 13 cases were imported from another province of Turkey. Six cases were transmitted from abroad.

Conclusion

These results provide information about malaria epidemiology in an endemic area in Turkey and contribute its prevention in Mersin Province.     

Comparison of a Commercial ELISA with the Modified Agglutination Test for Detection of Toxoplasma gondii Antibodies in Sera of Naturally Infected Dogs and Cats

Background

Toxoplasma gondii can infect all warm-blooded animals. Modified agglutination test (MAT) and ELISA are widely used for the detection of T. gondii antibodies. However, there is little information on their acceptability for detecting antibodies in companion animals.

Methods

This study compared ELISA and MAT for their ability to detect T. gondii infection in naturally infected dogs and cats. Blood samples were collected from dogs and cats in different areas of Beijing, China and analyzed by ELISA and MAT. The χ² test and κ analysis were used to evaluate their efficiency and agreement.

Results

For dogs, the seroprevalence of T. gondii antibodies detected by ELISA was 34.7%, which was significantly higher than that detected by MAT (P<0.05). There was no significant difference between ELISA and MAT for detecting T. gondii antibodies in cats. Good agreements between MAT and ELISA were seen in both dogs and cats; however, inconsistent results were demonstrated by κ analysis and in MAT titer assay.

Conclusion

Serum-based ELISA may be more satisfactory for screening test of T. gondii infection in dogs, whereas both methods could be acceptable in cats.     

Monarch-1 Activation in Murine Macrophage Cell Line (J774 A.1) Infected with Iranian Strain of Leishmania major

Background

Leishmania major is an intracellular parasite transmitted through the bite of the female phlebotomine sand flies. Leishmania major is able to escape the host immune defense and survive within macrophages. Modulation of the NF-κB (Nuclear Factor-Kappa B) activation and suppression of the pro-inflammatory cytokines by L. major are the main evasion mechanisms that remain to be explored. This study aims to examine the expression level of the Monarch-1 in L. major-infected macrophages, as a negative regulator of the NF-κB activation.

Methods

Murine macrophage cell line (J774 A.1) was infected by metacyclic form of Leishmania promastigotes at macrophage/parasite ratio of 1:10. After harvesting infected cells at different times, total RNA was extracted and converted to cDNA. Semi-quantitative RT-PCR was performed for Monarch-1 by specific primers. Hypoxanthine Phospho-Ribosyl Transferase (HPRT) was used as an internal control to adjust the amount of mRNA in each sample.

Results

Semiquantitive analysis of Monarch-1 mRNA expression level showed a significant expression increase within 6 to 30 hours after L. major infection of macrophages when compared to the control macrophages.

Conclusion

Monarch-1 expression level reveals a significant increase in the early phase of macrophage infection with L. major, which in turn may suppress IL-12 production in Leishmania infected macrophages and deeply influence the relationship between host and parasite.     

Development of 116 kDa Fraction for Detecting Experimental Toxoplasma gondii Infections in Mice

Background

Serological diagnosis of Toxoplasma gondii infection using crude antigens may not be more accurate. To increase the diagnostic potency of antigens, isolation of their immunogenic fractions could be useful. The current research adopted to obtain an affinity isolated fraction from RH strain using CNBr Sepharose 4B column coupled with infected mice sera helping in detection of IgM and IgG of toxoplasmosis due to RH strain and other strains.

Methods

The isolated fraction was characterized by SDS-PAGE. Moreover, the diagnostic potency of the fraction was assessed by indirect ELISA in mice experimentally infected with RH strain and two other local strains; one of sheep origin and the other of human origin.

Results

The fraction was found to be consisted of a single band of 116 kDa compared with 17 bands ranged from 116 to 16 kDa associated with crude extract. The fraction proved potent diagnostic potentials of acute and chronic mice toxoplasmosis. Where it was detected both IgM and IgG antibodies as early as two days and as late as 2 months post experimental infection with any of the three strains. The level of detected IgM and IgG by RH fraction was higher in mice infected with RH strain than with local strains except IgM due to sheep strain parasite.

Conclusions

The 116 kDa fraction of T. gondii tachyzoites can be considered as a candidate in improving of serodiagnosisof Toxoplasma infections.     

Immunoproteomic Analysis of the Excretory-Secretory Proteins from Spirometra mansoni Sparganum

Background

Sparganosis is caused by the invasion of Spirometra sparganum into various tissues/organs. Subcutaneous sparganosis can be diagnosed by biopsy, while visceral/cerebral sparganosis is not easy to be diagnosed. The diagnosis depends largely on the detection of specific anti-sparganum antibodies. The specificity of the ELISA could be increased by using S. mansoni sparganum excretory–secretory (ES) antigens, but it also had the cross-reactions with sera of patients with cysticercosis or paragonimiasis. The aim of this study was to identify early specific diagnostic antigens in S. mansoni sparganum ES proteins.

Methods

The sparganum ES proteins were analyzed by two-dimensional electrophoresis (2-DE) and Western blot probed with early sera from infected mice at 14 days post-infection. The immunoreactive protein spots were characterized by MALDI-TOF/ TOF-MS.

Results

A total of approximately 149 proteins spots were detected with isoelectric point (pI) varying from 3 to 7.5 and molecular weight from 20 to 115 kDa and seven protein spots with molecular weight of 23-31 kDa were recognized by the infection sera. Three of seven spots were successfully identified and characterized as the same S. mansoni protein (cysteine protease), and the proteins of other 4 spots were not included in the databases.

Conclusion

The cysteine protease from S. mansoni ES proteins recognized by early infection sera might be the early diagnostic antigens for sparganosis.     

In Vivo Antimalarial Effects of Iranian Flora Artemisia khorassanica against Plasmodium berghei and Pharmacochemistry of its Natural Components

Background

The aim of this study was to evaluate the antimalarial effects of Iranian flora Artemisia khorassanica against Plasmodium berghei in vivo and pharmacochemistry of its natural components.

Methods

The aerial parts of Iranian flora A. khorasanica were collected at flowering stage from Khorassan Province, northeastern Iran in 2008. They were air-dried at room temperature; powder was macerated in methanol and the extract defatted in refrigerator, filtered, diluted with water, then eluted with n-hexane and finally non-polar components were identified through Gas Chromatography and Mass Spectroscopy (GC-MS). Toxicity of herbal extracts was assessed on naïve NMRI mice, and its anti-malarial efficacy was investigated on infected Plasmodium berghei animals. This is the first application on A. khorssanica extract for treatment of murine malaria. The significance of differences was determined by Analysis of Variances (ANOVA) and Student''s t-test using Graph Pad Prism Software.

Results

The herbal extract was successfully tested in vivo for its anti-plasmodial activity through artemisin composition, which is widely used as a standard malaria treatment.

Conclusion

Although, this study confirmed less anti-malarial effects of A. khorssanica against murine malaria in vivo, however there are some evidences on reducing pathophysiology by this medication. In complementary assay, major components were detected by GC-MS analysis in herbal extract including chrysanthenone (7.8%), palmitic acid (7.4%) and cis-thujone (5.8%). The most retention indices of the component are given as n-eicosane, palmitic acid and n-octadecane.     

Immunogenicity of HLA-DR1 Restricted Peptides Derived from Leishmania major gp63 Using FVB/N-DR1 Transgenic Mouse Model

Background

Leishmaniasis is a worldwide disease prevalent in tropical and sub-tropical countries. In the present study the immunogenicity of three human HLA-DR1 restricted peptides derived from L. major gp63 protein was evaluated using FVB/N-DR1 transgenic mouse model.

Methods

The immunity generated by three MHC class II – restricted peptides with the sequence of AARLVRLAAAGAAVT (AAR), AAPLVRLAAAGAAVT (AAP) and SRYDQLVTRVVTHE (ASR) derived from L. major gp63 protein were predicted using a web-based software (SYFPEITHI) and tested in FVB/N-DR1 transgenic mice.

Results

Immunization of FVB/N-DR1 transgenic mice with one of the three predicted peptides (AAR) resulted in high levels of Th1-type immune response as well as significant levels of IFN-γ detected by Proliferation assay and ELISA.

Conclusion

The results indicate a high level of immunogenicity for AAR, which can be a potent candidate for peptide vaccine in Leishmania infections.     

Larva Migration and Eosinophilia in Mice Experimentally Infected With Gnathostoma spinigerum

Background

Gnathostoma spinigerum causes larva migran in human which is endemic in Southeast Asia. Information regarding larva migration is limited. In this study, we investigated the parasite migration by recovery of worms from the whole body of mouse after oral infection with advanced third stage larvae (AL3). The percentage of blood eosinophils was examined in parallel.

Methods

Mice were orally infected with AL3 and histological study of organs was investigated in order to study the migration of AL3, along with blood eosinophilia.

Results

At 1 hr post infection (PI), the larvae remained in the stomach, thereafter at 3, 5, 7, 10 and 24 hr PI; they were recovered from various organs including liver, mesentery, esophagus, diaphragm, lung, heart and dorsal fat. At day 15 PI, they were mostly found in muscles (76.47%). The average worm recovery (5 months) was 78.03%. The worms were found in the liver at every time point. Larva encystment was detected. There was a significant difference in blood eosinophils between the 8 larvae- (average 9.33% + 6.25%) and the 15 larvae-infected groups (average 22.66% + 11.03%). Surprisingly, the blood eosinophils (average 19.00% + 2.92%) were not higher in the higher infective dose- group (25 larvae).

Conclusion

Liver was involved by G. spinigerum throughout the study. We detected larva encystment which had never been reported in human gnathostomiasis. The highest percentage of eosinophil occurred during the invasive stage.