Experimental Study on Plasmodium berghei, Anopheles Stephensi, and BALB/c Mouse System: Implications for Malaria Transmission Blocking Assays.

BACKGROUND: Plasmodium berghei is a rodent malaria parasite and has been very valuable means in the progress of our understanding of the essential molecular and cellular biology of the malaria parasites. Availability of hosts such as mice and vectors such as Anopheles stephensi has made this parasite a suitable system to study the parasite-host and vector-parasite relationships. METHODS: This study was performed at Medical Entomology and Parasitology laboratories of the School of Public Health, Tehran University of Medical Sciences, Iran in 2016. The investigation was carried out to describe life cycle and parameters influencing maintenance of the parasite within the mice or the mosquito. RESULTS: Results have revealed details and addressed some parameters and points influence maintenance of various life stages of the parasite including merozoites, macrogametocytes, ookinetes, oocysts and sporozoites in the laboratory model P. berghei-A. stephensi-BALB/c mouse. Injection of fresh infected blood results in higher gametocytemia in the animals. The more injected parasites result in earlier and higher parasitemia and exfelagellation centers in the mice blood. However, the highest number of infected mosquitoes and oocysts formation were observed when the parasitemia and exflagellation centers per microscopic field were 9% and 3.6 in the infected mice respectively. The infected mosquitoes should be maintained on 8% (w/v) fructose, 0.05% (w/v) PABA at 20±1 °C and 50%-80% relative humidity. CONCLUSION: This study helps to understand the biology of vertebrate-parasite and mosquito-malaria interactions that may aid in the development of a new generation of drug/vaccine and vector-based measures for malaria control.

Prevalence of Leishmania species in rodents: A systematic review and meta-analysis in Iran.

Leishmaniasis are diverse group of diseases caused by numerous species of genus Leishmania. Herein we have contrived a systematic review and meta-analysis on the prevalence of Leishmania species in rodents of Iran. For this purpose, following the general methodology recommended for systematic reviews and meta-analysis, six English databases (PubMed, Science Direct, Scopus, Ovid, Web of Science and Google Scholar) and four Persian databases (Magiran, SID, Iran Doc and Iran Medex) were explored during January 1995 till June 2015. Papers were selected based on 8 pre-defined inclusion criteria. During the years, a total number of 4485 different rodents were captured; among which 1291 cases were Leishmania positive. The calculated weighted prevalence of Leishmania species in rodents was 23% (95% CI=18-28). Given geographical zones of Iran, the highest and lowest prevalence rate was belonged to North 50% (95% CI=40-61) and West 11% (95% CI=5-17), respectively. Rhombomys opimus (1766), Meriones lybicus (1258) and Tatera indica (488) were the three most abundant captured rodents, while the highest prevalence of Leishmania species was observed in Nesokia indica 48% (95% CI=42-54) and followed by R. opimus 39% (95% CI=30-47). Egger's regression test was performed to detect publication bias, which revealed it may not have a significant influence on overall weighted prevalence estimate (P=0.317). Meta-regression analysis demonstrated that there is no significant relationship between overall prevalence with sample size (P=0.1) and year of publication (P=0.7). The results showed remarkable prevalence of Leishmania species in rodent reservoirs. In future, adopting a suitable strategy for control and combat with rodents is necessary.

Dynamics of Transgenic Enterobacter cloacae Expressing Green Fluorescent Protein Defensin (GFP-D) in Anopheles stephensi Under Laboratory Condition.

BACKGROUND: Enterobacter cloacae bacterium is a known symbiont of the most Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria. However, the population dynamics of this bacterium within An. stephensi and its introduction methods to the mosquitoes have not yet been explored. METHODS: Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages in the laboratory condition. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium. RESULTS: The E. cloacae population remained stable in sugar bait at least for eleven days whereas it was lowered in the insectary larval habitat where the bacteria inadequately recycled. The bacterium was weakly transmitted transstadially from larval to adult stage. The bacterial populations increased smoothly and then dramatically in the guts of An. stephensi following sugar and blood meal respectively followed by a gradual reduction over the time. CONCLUSION: Enterobacter cloacae was highly stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. Sugar bait stations can be used for introduction of the transgenic bacteria in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the semi-field or field conditions.

A novel adjuvant, the mixture of alum and naltrexone, augments vaccine-induced immunity against Plasmodium berghei.

We previously showed that the mixture of naltrexone (NLT), a general opioid antagonist, and alum, acts as an effective adjuvant in enhancing vaccine-induced T helper 1 (TH1) humoral immune responses against Toxoplasma gondii. Here, we tested the efficacy of the mixture of NLT and alum in the induction of immunity in response to blood stages of Plasmodium berghei (BSPb) as a model vaccine. BALB/c mice were divided into five vaccination groups. Mice in the experimental groups received the BSPb vaccine alone or in combination with the adjuvant alum, NLT or the alum-NLT mixture. Mice in the control group received PBS. All mice were immunized on days 0, 7 and 14. Two weeks after the last immunization, immune responses to Plasmodium berghei were assessed. Our results indicated that including the alum-NLT mixture as an adjuvant during vaccination increased the ability of the BSPb vaccine to enhance lymphocyte proliferation, shifted the immune response towards a TH1 profile and increased Plasmodium berghei-specific IgG2a. This resulted in improved protective immunity against Plasmodium berghei. In conclusion, administering alum-NLT mixture in combination with the BSPb vaccine enhanced the vaccine-induced immunity, and shifted the immune response toward TH1 pattern.

A novel adjuvant, mixture of Alum and naltrexone, elicits humoral immune responses for excreted/secreted antigens of Toxoplasma gondii tachyzoites vaccine in Balb/c murine model.

OBJECTIVE: The excreted-secreted antigens (ESA) from the tachyzoites seem to play a key role in immunity against Toxoplasma gondii. The aim of this study is to investigate whether Alum-NLT mixture, as a new adjuvant, can induce humoral immunity in response to excreted secreted antigens (ESA) of Toxoplasma gondii as a model vaccine or not. METHODS: Six- to eight-week-old female Balb/c mice were divided into five groups. Mice in the experimental groups received either ESA vaccine alone or in combination with the adjuvant Alum, NLT or Alum-NLT mixture; Mice in the negative control group received phosphate buffered saline (PBS). All mice were immunised, three times subcutaneously (s.c.) with a total volume of 150µl each with a 10-day interval. Ten days after the final immunisation, immune response to Toxoplasma gondii was assessed. RESULTS: Our results revealed that Alum-NLT mixture as an adjuvant during vaccination boosts the efficacy of the ESA vaccine by means of increasing Toxoplasma gondii-specific IgG, IgG2a production and the ratio of IgG2a/IgG1 (P-value < 0.05). The use of this adjuvant mixture improved the protective immunity against Toxoplasma gondii. CONCLUSION: Administration of the Alum-NLT mixture as an adjuvant in ESA vaccine enhances humoral immunity.

Identification of Echinococcus granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan - Iran.

BACKGROUND: The aim of this study was DNA extraction from protoscolecses of Echinococcus granulosus and identification of these strains in West-Azerbaijan Province, north western Iran. METHODS: Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restriction enzymes of Taq 1, Rsa 1 and Alu 1. RESULT: Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex). The results of RFLP analysis also were the same for all isolates. PCR-RFLP patterns restriction enzymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approximately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples. CONCLUSIONS: Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex) is dominant genotype in this province.

Effect of different concentrations of hypertonic saline at different times on protoscoleces of hydatid cyst isolated from liver and lung.

OBJECTIVE: Most surgeons inject scoloidal materials into the cyst before or after its removal, since any contamination to normal sites will cause re-growth of the same cyst. The aim of this study was to determine the lethal effect of hypertonic saline at different doses and different times on protoscolexes of lung and liver. METHODS: The livers and lungs of killed animals with hydatid cyst disease were gathered from Urmia Industrial Abattoirs. They were transferred to the university parasitological lab immediately. The hydatid cyst fluid was aspirated with a 10 mm syringe and poured into a 15 cc tubes. The movement of protoscoleces and staining with 0.1% eosin was the test to determine viability of protoscoleces. Those with color absorption were those which were not viable. Different concentrations of hypertonic saline were given at different time. RESULTS: The results showed that in 20% of hypertonic saline in the 4th minute, 80% of protoscoleces were alive while in the 5th minute 50% were alive, in the 7th minute 20% and 8th minute 5%, 9th minute all of them were dead. In the 10% concentration, at up to 9 minutes 50% were alive, in the 18th minute 20% and in 30 minutes 10% of protoscoleces were alive. In the 5% concentration at up to 10 minutes 90% were alive while in the 22nd minute 80% and in 30 minutes 70% of protoscoleces were alive. CONCLUSION: When we inject 20% hypertonic saline into the cyst cavity there is aprobability that the cyst contaminates the bile duct and liver through the small hole we made. This material may cause widespread necrosis of the liver. We should use 10% hypertonic saline minimally for 45 minute before surgery and after cyst removal, since the hypertonic saline itself may cause injury to the biliary system.