Thermosensitive and mucoadhesive gels containing solid lipid nanoparticles loaded with fluconazole and niosomes loaded with clindamycin for the treatment of periodontal diseases: a laboratory experiment.

BACKGROUND: Periodontal diseases may benefit more from topical treatments with nanoparticles rather than systemic treatments due to advantages such as higher stability and controlled release profile. This study investigated the preparation and characterization of thermosensitive gel formulations containing clindamycin-loaded niosomes and solid lipid nanoparticles (SLNs) loaded with fluconazole (FLZ), as well as their in vitro antibacterial and antifungal effects in the treatment of common microorganisms that cause periodontal diseases. METHODS: This study loaded niosomes and SLNs with clindamycin and FLZ, respectively, and assessed their loading efficiency, particle size, and zeta potential. The particles were characterized using a variety of methods such as differential scanning calorimetry (DSC), dynamic light scattering (DLS), and Transmission Electron Microscopy (TEM). Thermosensitive gels were formulated by combining these particles and their viscosity, gelation temperature, in-vitro release profile, as well as antibacterial and antifungal effects were evaluated. RESULTS: Both types of these nanoparticles were found to be spherical (TEM) with a mean particle size of 243.03 nm in niosomes and 171.97 nm in SLNs (DLS), and respective zeta potentials of -23.3 and -15. The loading rate was 98% in niosomes and 51% in SLNs. The release profiles of niosomal formulations were slower than those of the SLNs. Both formulations allowed the release of the drug by first-order kinetic. Additionally, the gel formulation presented a slower release of both drugs compared to niosomes and SLNs suspensions. CONCLUSION: Thermosensitive gels containing clindamycin-loaded niosomes and/or FLZ-SLNs were found to effectively fight the periodontitis-causing bacteria and fungi.

Association Between Wolbachia Infection and Susceptibility to Deltamethrin Insecticide in Phlebotomus papatasi (Diptera: Psychodidae), the Main Vector of Zoonotic Cutaneous Leishmaniasis.

Background: Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups. Result: The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi. Conclusion: Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management.

Nanotechnology-Based Drug Delivery Systems in the Transdermal Treatment of Melanoma.

The incidence rate of melanoma is dramatically increasing worldwide, raising it to the fifth most common cancer in men and the sixth in women currently. Resistance generally occurs to the agents used in chemotherapy; besides their high toxicity destroys the normal cells. This study reviewed a detailed summary of the structure, advantages, and disadvantages of nanotechnology-based drug delivery systems in the treatment of melanoma, as well as some nanocarrier applications in animal models or clinical studies. Respective databases were searched for the target keywords and 93 articles were reviewed and discussed. A close study of the liposomes, niosomes, transferosomes, ethosomes, transethosomes, cubosomes, dendrimers, cyclodextrins, solid lipid nanoparticles, and carbon nanotubes (CNTs) was conducted. It was found that these nanocarriers could inhibit metastasis and migration of melanoma cells and decrease cell viability. Conclusively, some nanocarriers like liposomes, niosomes, and transferosomes have been discussed as superior to conventional therapies for melanoma treatment.

Correction to: An outbreak of cutaneous leishmaniasis due to Leishmania major in an endemic focus in central Iran.

[This corrects the article DOI: 10.1007/s12639-022-01474-6.].

An outbreak of cutaneous leishmaniasis due to Leishmania major in an endemic focus in central Iran.

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major is an important endemic disease and a major public health concern in Iran. Following an outbreak of leishmaniasis in 2013 in one of the important foci in Iran, the researchers were prompted to determine the underlying causes of the epidemic. Adult sand flies were collected using sticky traps and aspirating tubes and infection with Leishmania parasites was evaluated. Also, rodents were captured using Sherman live traps and stray dogs were hunted and were tested for the presence of leishmanial infection. Active case detection was also conducted and data related to each household were recorded using a researcher-designed form. Nested PCR and PCR-RLFP techniques were employed to determine Leishmania infection in the samples. Phlebotomus papatasi was the most dominant species among the 7 different species of sand flies collected in this study. Sergentomyia clydei, Sergentomyia theodori, and Sergentomyia grekovi were identified for the first time in the study area. 20% of collected Ph. papatasi species and one Meriones libycus were infected with Leishmania major. Stray dogs demonstrated no lesions on different parts of their body. The relative frequency of active lesions and scars on the dogs were respectively 5.49% and 1.23% in 2013 and 5.82% and 0.56% in 2014. The highest number of ZCL cases due to L. major was recorded in Ardestan city. Phlebotomus papatasi is the main vector and M. libycus seems to be the primary reservoir host of ZCL in the suburbs of this city. Furthermore, due to the low density of rodent reservoir hosts in the study area, we support the hypothesis that humans may have had a role in maintaining the parasite cycle in the population.

Bioecological Study on the Sand Flies (Diptera: Psychodidae, Phlebotominae) in Sari County, North of Iran.

Background: Phlebotomine sand flies are vectors of Leishmania species, the causative agents of leishmaniasis in the world. Present study aimed to evaluate the bioecological aspects of sand flies in different ecotopes in Sari County, north of Iran. Methods: Sand flies were collected from four villages in mountainous, forest, plain and peri-urban areas monthly using sticky traps in May-October 2016. Mounted specimens were identified using valid identification keys under optical microscope. The Arc GIS 10.5 software was applied for showing the distribution of sand flies. Shannon-Weiner, Simpson and Evenness species diversity indices were calculated. Results: Generally, 334 specimens were captured and identified, namelly Phlebotomus kandelakii, Ph. papatasi, Ph. major, Ph. sergenti, Ph. longiductus, Ph. halepensis, Ph. tobbi, Sergentomyia dentata, Se. theodori, Se. sintoni, Se. antennata and Se. sumbarica. The most common species was Ph. kandelakii (n= 128, 38.32 %). The highest Simpson index (0.81) and abundance (N= 141) were recorded in the mountaineous area. Shannon diversity index was higher in the forest (H'= 1.53) and the highest evenness index was in the plain area (J'= 0.93). The highest richness (S= 9) and Shannon indices (H'= 1.57) were observed in June. Conclusions: Phlebotomus kandelakii, Ph. sergenti, Ph. tobbi, Ph. longiductus, Se. theodori, Se. antennata and Se. sumbarica were recorded for the first time in the study area. Since some species are incriminated for leishmaniasis transmission, further studies are required in the northern regions of Iran to timely control measures planning.

An Eco-Epidemiological Study on Zoonotic Cutaneous Leishmaniasis in Central Iran.

BACKGROUND: Leishmaniasis is an expanding neglected tropical disease in the world reporting from 98 countries including Iran. This study focused on eco-epidemiological determinants of the disease following a rapid and unexpected increase of leishmaniasis incidence in a strategic residential district in North-East of Isfahan County, Iran. METHODS: This study was accomplished from Apr 2012 to Jan 2014 in a strategic residential zone in North-East of Isfahan County, Esfahan, Iran. Monthly activity, parity, Leishmania infection and susceptibility tests, were determined on sand flies. Some portion of inhabitants and school children were surveyed to find active or passive cases of leishmaniasis and also wild rodents were collected to determine reservoir host. RESULTS: Totally 5223 sand flies belonging to Phlebotomus and Sergentomyia genus were collected and identified; Ph. papa-tasi was the dominant species and started to appear in May and disappeared in Oct. The majority of living dissected sand flies were unfed and parous. Ph. papatasi showed 4.6% Leishmania infection through direct examination and 39.54% by nested-PCR respectively. Phlebotomus papatasi was susceptible against deltametrin 0.05%. Totally 2149 people were surveyed and incidence and prevalence of zoonotic cutaneous leishmaniasis estimated as 45.39 and 314.40 per 1000 population. Rodents showed 73.91% and 80% Leishmania infection by direct examination and nested-PCR respectively. CONCLUSION: Cutaneous leishmaniasis due to L. major has been established in this area. Rodent control operation and personal protection are highly recommended to control the disease in this focus.

Insecticide Resistance Status of Malaria Vectors in a Malarious Area, Southeast of Iran.

Background: Malaria continues to be the main vector-borne disease in Iran. The endemic foci of malaria are in Sistan and Baluchistan Province, the borderline of Iran and Pakistan. By the year 2020 the program of the country is malaria elimination. The main vector control is using insecticide as Indoor Residual Spraying. The aim of the study was to evaluate the susceptibility of main malaria vectors to different insecticides recommended by WHO. Methods: All the insecticides papers supported by WHO and evaluation of insecticide resistance of Anopheles stephensi, Anopheles culicifacies, Anopheles superpictus to different chemical groups of imagicides including DDT 4%, malathion 5%, propoxur 01.%, lambdacyhalothrin 0.05%, deltamethrin 0.025% and permethrin 0.75% were followed by the WHO guideline. Results: Results of the susceptibility test against different insecticides revealed that An. stephensi and An. culicifacies are resistant to DDT and susceptible to other insecticides. An. superpictus is susceptible to all groups of pesticides. Conclusion: Knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes in local and global scales.

Evaluation of Susceptibility Status of Phlebotomus papatasi, the Main Vector of Zoonotic Cutaneous Leishmaniasis, to Different WHO Recommended Insecticides in an Endemic Focus, Central Iran.

Background: Among neglected zoonotic diseases, leishmaniases caused by Leishmania parasite through infected female sand fly bite, are a group of diseases found in 98 countries and territories representing a critical burden of disease worldwide. Vector management plays a crucial role in reducing the burden of vector-borne diseases by WHO's global plan. The objective of the current study was to assess the susceptibility status of wild phlebotomine sand flies from Esfahan Province, central Iran, to the recommended insecticides by WHO. Methods: Sand flies were collected by mouth aspirator in Matin Abad desert Eco-resort and were tested using WHO adult mosquito test kit against Dichlorodiphenyltrichloroethane (DDT) 4%, Deltamethrin 0.05%, Malathion 5% and Propoxur 0.1%. The number of knockdown sand flies were recorded during exposure time in ten minutes interval for DDT and Deltamethrin and they were allowed to recover for 24 hours. Knockdown Time50 (KD50) and KD90 were generated for them using Probit software. They were mounted and identified by valid keys. Results: Among the tested insecticides against female Phlebotomus papatasi, DDT, Deltamethrin, and Malathion recorded the highest mortality rate of 100%, followed by Propoxur with 92.2% mortality for a one-hour exposure. For DDT, KD50 and KD90 were calculated 21.87 and 42.93 and for Deltamethrin, they were 23.74 and 56.50 minutes respectively. Total sand flies exposed with DDT and Deltamethrin shed their leg(s). Conclusion: It is concluded that Ph. papatasi from central Iran is susceptible to DDT, Deltamethrin, Malathion, and Propoxur.

Prevalence and distribution of resistance and enterotoxins/enterotoxin-like genes in different clinical isolates of coagulase-negative Staphylococcus.

BACKGROUND: Coagulase-negative staphylococcus (CoNS) is considered to be the major reservoirs for genes facilitating the evolution of S. aureus as a successful pathogen. The present study aimed to determine the occurrence of genes conferring resistance to fluoroquinolone, determining of the prevalence of insertion sequence elements IS256, IS257 and different superantigens (SAgs) among CoNS isolates obtained from various clinical sources. MATERIALS AND METHODS: The current study conducted on a total of the 91 CoNS species recovered from clinical specimens in Hamadan hospitals in western Iran in 2017-2019. The antimicrobial susceptibility testing was performed using disk diffusion method and the presence of the IS256 and IS257, genes conferring resistance to fluoroquinolone and enterotoxins/enterotoxin-like encoding genes were investigated by polymerase chain reaction (PCR) method. RESULTS: Among genes encoding classic enterotoxins, sec was the most frequent which was carried by 48.4% of the 91 isolates, followed by seb in 27.5% of the isolates. None of the CoNS isolates was found to be positive to enterotoxin-like encoding genes. In 11(12%) of all isolates that were phenotypically resistant to levofloxacin, 9 isolates (81.8%) were positive for gyrB, 8 isolates (72.7%) were positive for gyrA, 8 isolates (72.7%) harbored grlB and 7 isolates (63.6%) were found to carry grlA. The IS256 and IS257 were identified in 31.8% and 74.7% of the isolates, respectively. The results of statistical analysis showed a significant association between the occurrence of staphylococcal enterotoxins (SEs) encoding genes and antimicrobial resistance. CONCLUSION: Antimicrobial resistant determinants and SEs are co-present in clinical CoNS isolates that confer selective advantage for colonization and survival in hospital settings. The coexistence of insertion elements and antibiotic resistance indicate their role in pathogenesis and infectious diseases.

Identification of hemolysin encoding genes and their association with antimicrobial resistance pattern among clinical isolates of coagulase-negative Staphylococci.

OBJECTIVE: Coagulase-negative staphylococci (CoNS) are as considered opportunistic pathogens which capable of producing several toxins, enzymes and resistance genes. The current study aimed to determine the occurrence of different hemolysins genes and patterns of antibiotic resistance among CoNS species. RESULTS: The highest frequency of antibiotic resistance was observed against cefoxitin in 49 isolates (53.8%), and the lowest resistance was against novobiocin in 5 isolates (5.5%). None of the isolates was resistant to vancomycin. The prevalence of hla, hla_yidD, hld, and hlb genes was: 87.9%, 62.6%, 56%, and 47.3%, respectively. The hla/yidD and hld genes were detected in 69.4% of S. epidermidis and the hla gene in 94.6% of S. haemolyticus isolates; the hlb gene was detected in 53.1% of the S. epidermidis isolates. The mecA gene was identified in 50 (55%) of the CoNS isolates. In conclusion, the results of statistical analysis showed that the hld gene had a significant association with resistance to levofloxacin and erythromycin antibiotics, the hlb with clindamycin resistance and the hla/yidD with rifampicin and novobiocin resistance. The results of this study showed that there is a significant relationship between hemolysin encoding genes and antibiotic resistance patterns; therefore, detection of virulence factors associated with antibiotic resistance has become a significant issue of concern.

Epidemiological survey on Cutaneous Leishmaniasis in southwestern Iran.

BACKGROUND & OBJECTIVES: Cutaneous leishmaniasis (CL) is still a public health threat in many rural areas of Iran. It causes considerable morbidity among a huge number of people in the endemic areas. Khuzestan is considered as a CL focus in Iran and recently several cases have been reported from Shush County. The aim of the current study was to determine different aspects of CL epidemiology in this County. METHODS: The study was conducted in four villages in the rural district of Shush County. In order to survey the scar and acute rate of the disease, 100 inhabitants were questioned in each village. In addition, to determine the reservoir host and vector of the disease, rodents were captured using Sherman traps, and sandflies were collected from around the rodent burrows using sticky paper traps. RESULTS: The scar and acute lesion rates were reported as 5.26 and 0.57% among inhabitants, respectively. Fifty-four rodents including Tatera indica (55.5%), Nesokia indica (33.3%), Mus musculus (9.3%) and Rattus norvegicus (1.9%) were captured and identified. Nested PCR showed 3 out of 54 rodents infected with Leishmania major. In total, 1122 sandflies were collected, and Leishmania major detected in Phlebotomus papatasi (4.35%), Sergentomyia dentata (23%), and Sergentomyia clydei (10%). INTERPRETATION & CONCLUSION: Zoonotic cutaneous leishmaniasis due to L. major prevailed in this area. The common and the main vector was Ph. papatasi and, T. indica seems to be the main while N. indica as a secondary reservoir host of the disease in this area.

The Potential Role of Humans in the Transmission Cycle of Leishmania major (Kinetoplastida: Trypanosomatidae), the Causative Agent of the Old World Zoonotic Cutaneous Leishmaniasis.

Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major (Yakimoff and Schokhor, 1914) is known as a neglected tropical disease, transmitted by Phlebotomus papatasi (Scopoli, 1786) in North Africa and the Middle East. The main reservoirs of ZCL are desert gerbils and the role of humans as a reservoir host of the disease is not clearly defined and is therefore investigated in this study. In order to achieve this objective, Ph. papatasi sand flies were allowed to take blood through indirect blood sources (human, Rhombomys opimus (Lichtenstein, 1823) (great gerbil), and BALB/c mice) using artificial feeding or direct xenodiagnoses from ZCL patients. To detect Leishmania promastigotes inside digestive canal, blood-fed sand flies were dissected and examined under light microscope and then confirmed by nested-PCR. In indirect xenodiagnoses, promastigote forms of Leishmania parasite were observed in 7.1%, 12%, and 13.6% of sand flies which fed on Rh. opimus, BALB/c mice, or human blood sources, respectively. In direct xenodiagnoses, among 93 female lab-reared sand flies which fed on active L. major lesion(s), 2.1% Leishmania infection was subsequently observed in the sand flies. No infection was detected in those sand flies that fed on nonlesioned skin of CL patients. Humans can serve as a reservoir of ZCL since the data indicate that Ph. papatasi is able to acquire L. major parasites from an active lesion of ZCL patients and the parasites can complete metacyclogenesis in the sand fly.

Aquatic Insect from Iran for Possible Use of Biological Control of Main Vector-Borne Disease of Malaria and Water Indicator of Contamination.

Iran has a wide variety of zoogeographical regions and different seasons. Here are some important mosquito-borne diseases. Mosquitoes normally live in waters. Its aquatic insect fauna is highly unexplored. To being resolved this faunal gap, a variety of literature records from previous century in different parts of Iran was reviewed. In some southern and southeastern foci in Iran, Malaria is still a main endemic disease which is unstable with two seasonal spring and autumn peaks even though Iran is lunching Malaria elimination. This review article showed the wide variety of aquatic insects throughout the country. Researchers can discuss water pollutant and its quality by using aquatic insect fauna as well as biological control for vectors. Types of aquatic insects and macroinvertebrates sampling can be useful for water quality monitoring as indicators. Looking at aquatic insects' life in water could be one of the most cost-effective and the easiest method to assess the water contaminations by different pollutants and will provide a guideline for scientific communities and environmental agencies for decision making.

Designing and Introducing a New Artificial Feeding Apparatus for Sand Fly Rearing.

BACKGROUND: Due to strict ethical rules, the risk of accidental disease transmission and the most importantly, inconvenience regarding using of live animals, artificial feeding apparatus has been developed for colonization of haematophagous insects. Rearing of sandfly is more difficult than other haematophagous insects. METHODS: In the current study, a new apparatus for membrane feeding of Phlebotomus papatasi was designed, made and compared with available apparatus in Sand Fly Insectary, Tehran University of Medical Sciences, Tehran, Iran, in 2014. RESULTS: In comparison to other apparatus designed for artificial feeding of other arthropods, our designed apparatus had the highest performance which after up to 1h, the majority of sand flies landed and took blood and among tested membranes, chicken skin was proved the most efficient membrane. CONCLUSION: Sand fly artificial feeding apparatus can be used at least for rearing of Ph. papatasi.

Assessing the Ovarian Accessory Glands to Determine the Parity of Phlebotomus papatasi, Vector of Zoonotic Cutaneous Leishmaniasis, under Laboratory Condition.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease prevailed in many rural areas of 17 out of 31 provinces in Iran. The main vector of the disease is Phlebotomus papatasi and the causative agent is Leishmania major in ZCL foci of Iran. In the current study we investigated the validity of accessory glands secretions as an indicator to recognize parous from nulliparous Ph. papatasi females under laboratory conditions. METHODS: Over 235 laboratory-reared females of Ph. papatasi were dissected in 6 groups including: newly emerged, one hour, one day, two days, and three days after blood feeding and also after oviposition under stereo microscope for their parity in 2014-2015. RESULTS: Transparent glands were compatible with nulliparous only in newly emerged sand flies. In sand flies dissected after oviposition, accessory glands were rather large as a result of oviposition though they were transparent. CONCLUSION: The accessory glands secretions could not be as an indicator for distinguishing parous from nulliparous of Ph. papatasi females.

Assessing the insecticide susceptibility status of field population of Phlebotomus papatasi (Diptera: Psychodidae) in a hyperendemic area of zoonotic cutaneous leishmaniasis in Esfahan Province, Central Iran.

Leishmaniasis is a Neglected Tropical Disease (NTD) and emerging parasitic infection that affect mainly poor regions around the world. This study aimed to determine the baseline susceptibility of Phlebotomus papatasi to commonly used insecticides in a hyper endemic area using WHO standard procedure in central Iran. A total of 4-5 replicates containing 120-200 sand flies were used for each insecticide. Baseline susceptibility to DDT and pyrethroids was assessed on 5326 specimens collected from the study area. The LT50 and LT90 values were measured according to the World Health Organisation test using probit analysis and regression lines. The test results against males P. papatasi revealed that LT50 values to DDT 4%,Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15% and Lambdacyhalothrin 0.05% were 564.07, 38.08, 1.95, 0.60 and 9.78s and the figures for females were 584.44, 110.10, 11.64, 1.53 and 16.91s, respectively. Our results indicated that P. papatasi as the main cutaneous leishmaniasis vector was susceptible to Cyfluthrin 0.15%, Lambdacyhalothrin 0.05%, Permethrin 0.75% and Deltamethrin 0.05% and tolerant to DDT 4%. This study was carried out in one out of many Leishmaniasis foci in Iran. We recommend that future studies incorporate other regions and use the same procedure for monitoring and evaluating sand fly resistance. Also, WHO can provide a specific guideline and create a test kit for sand fly resistance monitoring and for applying susceptibility test because the tubes prepared for mosquitoes are not actually fit for sand flies.

Rearing and Biology of Phlebotomus sergenti, the Main Vector of Anthroponotic Cutaneous Leishmaniasis in Iran.

BACKGROUND: Establishment of sand flies laboratory colonies is essential to understand various biological aspects of Phlebotominae sand flies. The aims of the current study were to establish the colony of Phlebotomus sergenti Parrot (1917), the main vector of anthroponotic cutaneous leishmaniasis in old world, and to study biological parameters of this species. METHODS: The sand flies were reared at 26-28 °C temperature, 14:10 (light: dark) photoperiod and 70-80% relative humidity. Larval diet was a composted mixture of rabbit faces and rabbit pellets which is prepared through a special process. First to fifth generations of P. sergenti were used to define biological parameters. RESULTS: Results showed that, P. sergenti blood feeding percentage were 42% on chicken, 21% on BALB/c and 37% on golden hamster. Average time of blood digestion, egg incubation, 1st instar larva, pupa and adult emerging was recorded at 3.4, 8.7, 15, 33.3 and 41.2 days after blood feeding, respectively. Mean number of laid eggs was 55.1 and retained eggs were 35 per a female. Fecundity and production rate were 61.6%, and 42.2% respectively. Average longevity recorded at 15.2 days for females and 14.8 days for males. CONCLUSION: Colony of P. sergenti has been established for the first time in Iran. Average interval time from egg to adult of this species was 32.5 days. Chicken and golden hamster were recommended as a blood source for colony initiation and routine blood feeding, respectively.

Differential expression profiles of the salivary proteins SP15 and SP44 from Phlebotomus papatasi.

BACKGROUND: Sand fly saliva has been shown to help parasite establishment and to induce immune responses in vertebrate hosts. In the current study, we investigated the pattern of expression of two Phlebotomus papatasi salivary transcripts in specific physiological and seasonal conditions at a hyperendemic area of zoonotic cutaneous leishmaniasis (ZCL) in Iran. METHODS: Sand flies were collected during 2012-2013, and grouped according to physiological stages such as unfed, fed, semi-gravid, gravid, parous, nulliparous, infected or non-infected with Leishmania major and also based on the season in which they were collected. Quantitative Real-Time PCR was applied for assessment of the expression of two relevant salivary transcripts, PpSP15 and PpSP44, associated to protection from and exacerbation of ZCL, respectively. RESULTS: The expression of PpSP15 and PpSP44 transcripts was significantly up-regulated (1.74 and 1.4 folds, respectively) in blood fed compared to unfed flies. Among four groups of fed, unfed, semi-gravid and gravid flies, the lowest levels of PpSP15 and PpSP44 expression were observed in gravid flies. Additionally, the expression levels of both PpSP15 and PpSP44 transcripts in P. papatasi collected during summer were significantly up-regulated (3.7 and 4.4 folds, respectively) compared to spring collections. In addition, the PpSP15 transcript exhibited a significant up-regulation (P < 0.05) in non-infected flies compared to those infected with L. major. CONCLUSIONS: This study contributes to our knowledge of the differential expression of salivary genes among different groups within a P. papatasi population under natural field conditions. Cutaneous and visceral leishmaniasis are of public health importance in many parts of Iran and neighbouring countries where P. papatasi is the proven and dominant sand fly vector for ZCL, the most prevalent and endemic form of the disease in Iran. Therefore, the current study could be helpful in understanding the influence of salivary genes on Leishmania transmission by phlebotomine sand flies. Our findings demonstrate the differential expression of salivary transcripts under various physiological conditions potentially influencing the sand fly capacity for parasite transmission as well as the outcome of disease.

Seasonal and Physiological Variations of Phlebotomus papatasi Salivary Gland Antigens in Central Iran.

BACKGROUND: Sand fly saliva helps parasite establishment and induce immune responses in vertebrate hosts. In the current study, we investigated the modulation of Phlebotomus papatasi salivary gland antigen expression by seasonal and biological factors. METHODS: Sand flies were grouped according to physiological stages such as unfed, fed, semi-gravid, gravid, parous, nulliparous, infected or non-infected with Leishmania major and based on the season in which they were collected. Salivary gland antigens (SGAs) were analyzed using SDS-PAGE and the antibody response against SGAs in Rhombomys opimus was determined by ELISA and Western blot. RESULTS: The highest protein content was found in the salivary glands of unfed sand flies. The saliva content was higher in parous compared to nulliparous, in summer compared to spring, and in Leishmania-infected compared to non-infected flies. The salivary gland lysate (SGL) electrophoretic pattern variations were observed among sand flies with various physiological stages particularly from 4-9 protein bands of 14-70 kDa. The SGL of unfed and gravid flies had extra protein bands compared to fed and semi-gravid sand flies. There was missing protein bands in SGL of parous compared to nulliparous; and in summer compared to spring collected flies. Rhombomys opimus serum reacted strongly with an antigenic band of around 28 kDa in the SGL of all sand fly groups. CONCLUSION: Certain biological and environmental characteristics of wild populations of vector sand flies affect the protein content and antigenicity of saliva. This might have an important implication in the design of vector-based vaccines.