1. Introduction

Leishmaniases, protozoan diseases caused by Leishmania species,are transmitted by the female sand flies bites. The diseases are broadly reported in tropical and subtropical areas including Iran[1].It is estimated that more than 1.5 million cases of diseases occur in about 100 countries yearly. Moreover, about 350 million people are at risk of the diseases in the world[1]. Cutaneous leishmaniasis(CL), is more prevalent clinical form of the disease which has been reported from different geographical areas of Iran. The prevalence rates of CL are considerable and more than 30000 new cases annually occur in different parts of the country[2].

例如，在晶状体移植手术中，通常采取PMMA作为移植材料，但这一人工晶状体若与眼角膜上皮细胞接触，将造成角膜的永久性损伤。而通过低温等离子技术中的沉积方法，能够将亲水性的单体如N-乙烯基吡咯烷酮等沉积到PMMA 的表面，从而降低角膜细胞的损伤。通过动物实验发现，利用低温等离子沉积技术处理后的PMMA进行晶状体移植，最低可以将复合表面的细胞损伤控制在10%以下。

酵母菌不服气道：“哼，你们人类一向以貌取人，但可别以‘小’取‘菌’。虽然我们是单细胞的生物，但我们是个大家族，已知的种类就有1 000多种。虽然我们个小，但是繁殖速度快，条件允许时，大概每两个小时就能繁殖一代。所以你们人类1 d，我们酵母菌都好几代同堂了，并且我们有好多好多的兄弟姐妹。我们也不像细菌那样，连个细胞核都没有，有真正的细胞核哟，可是真核生物。”

Among nine recognized genus of Trypanosomatidae family(Leishmania, Trypanosoma, Crithidia, Phytomonas, Endotrypanum,Rhynchoidomonas, Leptomonas, Herpetomonas, and Blastocrithidia),two are known to include species pathogenic for humans and animals(Leishmania and Trypanosoma), and some species of Phytomonas are recognized as pathogens of plants[3]. Crithidia species exclusively parasitize arthropods, mainly insects. The genus is defined by the presence of the choanomastigote (barley corn-like forms) with free flagellum. Also, their cysts forms are able to move from host to host by fecal-oral rout and naturally, the digestive tracts of insects are the developing parasite sites[4]. Besides, Crithidia species make relationship with some parasites of the trypanosomatidae family,and transfer to various hosts along with them[5]. Some molecular investigation on human leishmaniasis revealed that Crithidia species were detected from the human cutaneous leishmaniasis cases in Iran[5]. Recently, prevalence and incidence rates of zoonotic cutaneous leishmaniasis (ZCL) have been increased in southern Iran, especially in Fars province[6]. Crithidia species are not pathogenic for human, but they have been detected and isolated from human CL lesions and it seems that they are able to survey in these circumstances with Leishmania and compete with them[5].Gerbillinae group is one of the most important reservoir host of several pathogens such as leishmania in Iran. Moreover, variant Leishmania species have been reported from rodents in different parts of the Country[6,7]. Among 52 species which have been characterized in Iran[8], Tatera indica (T. indica), commonly known as “The Indian Gerbil” or “Antelope rat”, is considered as an important reservoir host of ZCL in southwest regions of the country[9].

Based on taxonomic studies, 12 species of Tatera were characterized of which only one (T. indica) lives in Asia. This species was first reported from southeastern Turkey by Misonne in 1957[10], and ranges from Indomalayan region throughout the northern Arabia including Iran, and the others (Tatera afra, Tatera bohemi, Tatera brantsii,Tatera guineae, Tatera inclusa, Tatera kempi, Tatera leucogaster,Tatera nigricauda, Tatera phillipsi, Tatera robusta, and Tatera vicina)live in Africa[11]. T. indica is one of the largest species in Gerbillinae group, its color ranges from reddish brown to fawn and has a light brown strip on each side. The soles of their feet are pigmented and bald, and the ears are also nude and elongated. Moreover, ecological studies revealed that T. indica which is nocturnal does not move far from their burrows, and choose sandy plains and grasslands in habitats which are close to agricultural fields[12]. Males and females live alone and their mating place is uncertain. Moreover, their numbers increase between March to September and decrease in January[12,13].

(2)创新并未推动制造业内部结构升级。基于经济理论，创新是制造业结构升级的重要影响因素，由于我国当前科技体制不合理、研发效率和科技成果转化率低等问题的存在，使得创新对制造业结构升级的促进效应也没有发挥，由此应加大实施创新驱动发展战略，推动制造业结构升级。

高温试验过程中，观察到以下现象：炉温约200℃时，明显见水蒸气从炉膛排气孔中冒出；继续升温达到400℃时，大量水蒸气冒出，之后逐渐变少；接近600℃时，水蒸气基本消失。由此可知，200℃时试件内部混凝土游离水与结晶水开始丢失，400℃左右时，游离水与自由水丢失严重。

Based on molecular categorizations, Tatera genus is considered as polyphyletic taxon, so, T. indica was divided to Tatera sensu stricto and other African spp. located in Gerbilliscus Thomas genus[14].Among identified various subspecies of T. indica, four of them are reported from Iran, including T. indica scansa (in Kerman), T. indica persica (in Sistan), and T. indica bailwardi and T. indica monticola (in Fars and Khuzestan)[15].

Conflict of interest statement

2. Materials and methods

2.1. Study area

6月下旬至7月上旬核桃当年生新梢半木质化时进行芽接，由于嫁接时间有限，对拜城县核桃实生树改接带来了制约。在核桃夏季当年生新梢5月嫩枝嫁接、6月在2年生以上枝条上采用方块芽接等嫁接技术的试验成功，不仅拉长了核桃夏季嫁接时间，而且可以有效的在2年生枝条上进行芽接，打破了传统的在当年生新梢半木质化期芽接核桃新品种的限制，为拜城县乃至南疆地区加快核桃优良品种的发展提供质量保证。

2.2. Rodent collections

Rodents’ active colonies were identified in several parts of Shiraz and Kharameh cities. Afterwards, they were caught alive via wire cages located at entrances of burrows in 10 different locations of each city from March to October 2016. Traps were set in the evening and were checked the next morning. In cases of unsuccessful trapping,checking was continued at 4 hours intervals and it was conducted on average twice per week. Each time an average of 8 traps was used[1]. Rodents which were caught alive were carried to the animal laboratory cages. For recording the morphological characteristics(such as species, and sex), and finding any ulcers in their body,they were anesthetized with chloroform slightly. In this study, all applicable international, national, and/or institutional guidelines for the care and use of animals were followed, and ethical permission number IR.SUMS.REC.1395.S475 was granted for animal studies through the science and ethics committee of Shiraz University of Medical Sciences held on 20th August 2016. All performed health procedures were in accordance with the ethical standards of the Iranian institutional and/or national research committee guidelines and with the 1964 Helsinki declaration. Accordingly, rodents were euthanized by deep anesthesia and impression smears and culture sampling were prepared from ears, spleen, feet, nose, and liver. For culturing, the sampling surfaces were sterilized using 70% ethanol,Thereafter, surface cuts of 2 to 3 mm were prepared and impregnated into liquid phase of modified Novy Mac Neal Nicole (NNN)medium culture near to the flame and finally were placed in culture incubator. At least ten samples, two from each part of their bodies,were prepared for molecular, microscopic, and culture methods.

During study on rodents from March to October 2016, 115 rodents of five species; including T. indica (85), Rattus rattus (R. rattus) (12),Meriones libycus (M. libycus) (9), Mus musculus (M. musculus) (7),and Rattus norvegicus (R. norvegicus) (2), were caught alive from different regions, of which, 67 (58.3%) were male and 48 (41.7%)were female.

2.3. Parasites culturing

Sterilized surface cuts of ear, feet, nose, liver, and spleen were collected and transferred to the modified NNN culture medium tubes. NNN medium were slightly modified and consisted of two phases of an overlay Locke’s solution and a horse blood agar base.The specimens were inoculated into the liquid phase of the biphasic medium and incubated at (24±2) ℃. Every 2 to 3 days, the liquid phases of cultures were examined under invert microscopy. After detecting the motile promastigotes, cultures were transferred in RPMI-1640 media (Gibco, Frankfurt, Germany) containing 15% heat inactivated FCS (Sigma, cat. No. 308056), 2 mM L-glutamine, 100 μg/mL streptomycin (Gibco, Frankfurt, Germany), and 100 U/mL penicillin for mass cultivation. At the early stage of stationary phase,approximately 5 × 106 promastigotes/mL were harvested and used for more tests. Some of parasites were carried to Semi-solid Lockeblood agar medium and transported to 4 ℃ for more adaptation and investigation in prolonged time. Furthermore, some of the promastigotes were transferred to cryo tubes containing 5% dimethyl sulfoxide and stored in -70 ℃ and liquid nitrogen. Additionally, some promastigotes were harvested by centrifugation (10000 g for 10 min) and washed twice in cold sterile PBS (pH 7.2). Parasites pellets were stored at -20 ℃ until used[18].

2.4. Microscopic studies

Smear samples prepared from rodents liver, ears, feet pads, nose,and spleen were fixed by methanol, stained by 5% Giemsa, and microscopy checked for detection of Leishmania and/or Crithidia in at least 40 min times.

子曰:“书不尽言,言不尽意。”然则圣人之意,其不可见乎?子曰:“圣人立象以尽意,设卦以尽情伪,系辞焉以尽其言，变而通之以尽利，鼓之舞之以尽神”。 [7](P563)

2.5. PCR assays

2.5.1. DNA extractions

The scraped smears of the glass slides and culture sediments of rodents were extracted by kit extraction (YTA genomic DNA extraction mini kit, Cat no. YT9030) processor; briefly, samples transferred to micro-tubes, 20 μL Proteinase K and 200 μL Lysis buffer were added to the samples, then mixed and incubated at 60 ℃for 15 min to be completely lysed. Afterward, 200 μL absolute ethanol were added to the samples and mixed by pluse-vortexing for 30 s.Afterward, the samples mixtures containing some precipitates were carefully transferred to column micro-tubes, centrifuged for 1 min at 8000 rpm and washed by buffers several times to remove impurities from column micro-tubes. Finally, 100 to 200 μL of elution buffer or ddH2O were added to the membrane center of the column tubes and after 3 min, they were centrifuged for 2 min at 14000 rpm to elute the DNA and stored at -20 ℃ for PCR assays[19].

2.5.2. Leishmania and Crithidia detections

Extracted DNAs of smears and collected culture sediments were used for Leishmania species detections by a sensitive modified PCR method[20]. Each 25-μL reaction mixture (total volume) contained 12 μL Ampliqon taq DNA polymerase master mix buffer (Cat No. A180301), 1 μL of each primers of forward LINR4 (5’-GGG GTT GGT GTA AAA TAG GG-3’) and reverse LIN17 (5’- TTT GAA CGG GAT TTC TG-3’) (with 10 pico mol concentrations),3 μL DNA sample and 8 μL double distilled water. For PCR,Thermocycler (Eppendorf AG Master-cycler Gradient, Germany)programmed for an initial temperature of 94 ℃ for 5 min (one cycle), continued by temperatures of 94 ℃, 52 ℃, 72 ℃, for 30 s,30 s, and 1 min, respectively (30 cycles), and followed by a final temperature of 72 ℃ for 5 min (one cycle). Finally, 5 μL of PCR products were run in 1.2% electrophoresis agarose gel which stained with ethidium bromide, and visualized with UV trans-illuminator[1].Reference strains of Leishmania major (L. major) (MHOM/IR/54/LV39), with a band of 650 bp, would have shown the existence of L.major kDNA.

All extracted DNAs of rodent’s tissues were selected for Crithidia detection, using minicircle kDNA sequences of internal transcribed spacer (ITS) gene. The specific primers were designed by GenScript online PCR primers designs tool by using gene bank information(Unpublished document). Each 25-μL reaction mixture (total volume) contained 12 μL Ampliqon master mix buffer, 1 μL of each primers of 5’-TCCATGTGCGAGGACAACGTGCT-3’and 3’-CGCGTCGTTGATGAAGTCGCT-5’ (with 10 pico mol concentrations), 5 μL DNA sample and 6 μL double Distilled water. Eppendorf Master-thermocycler programmed for an initial temperature of 94 ℃ for 5 min (one cycle), followed by temperatures of 94 ℃, 55 ℃, 72 ℃, for 30 s, 1 min, and 1.5 min, respectively(30 cycles), and continued by a final temperature of 72 ℃ for 5 min (one cycle). Afterward, 5 μL of PCR products were used for electrophoresis assay. Reference strain of Crithidia fasciculata was used and a band of 800 bp would have shown the existence of Crithidia in the tested smears.

3. Results

3.1. Rodent identifications

晟图机械是一家自1998年起就开始潜心研制、开发适用于精装印后装订，以及精品礼品盒包装皮壳科技的民营企业，伴随着中国改革开放的浪潮，晟图机械目前已经成为这一领域领先的民族品牌，而其背后成功的秘诀、一路走来的感悟，就这样在庹明珠的款款道来中徐徐展开……

3.2. Microscopic findings

Totally, 490 slide samples from five different tissues belonging to 115 rodents (115, 100, 85, 100, and 90 samples of ears, liver,nose, feet, and spleens, respectively) were checked for Leishmania and/or Crithidia detections. As there were no reliable differential criteria between Leishmania and Crithidia detections in microscopic method, consequently, the slides checked for both parasites together,and finally positive slides were confirmed by PCR assays. The results revealed that 5.3% of slides (26/490) were found positive in microscopic method, of which 24.7% (21/85) of the T. indica and 11.1% (1/9) of the M. libycus were microscopic positive for Leishmania and/or Crithidia parasites (Figure 1). Furthermore, none slide smears of R. rattus, R. norvegicus, and M. musculus were found positive in microscopic studies.

Overall, 24 out of 490 positive slides were belonged to the T. indica organs, of which 3.5% of ears, 6.0% of feet, 3.5% of noses, 6.0% of livers, and 7.8% of spleens were microscopic infected to parasites.In addition, 2 slides of one M. libycus belonged to Kharameh focus(1% of each organs of ear and liver) were positive in microscopic investigations (Table 1).

3.3. Culture findings

Because of no accurate differential criteria between Leishmania and Crithidia detections and due to preparation of the same culture media for both parasites, they were checked together in cultivation method,and finally were confirmed by molecular methods. Some cultures media were infected, so they were removed from the study. Totally,37 out of 227 cultures media (16.3%) were positive, 33 samples belonged to the T. indica, of which 14.3% of ears, 9% of feet, 5.7%of noses, 9% of livers, 18% of spleens were cultural infected to parasites. Furthermore, 2 positive media were isolated from organs of ear and liver (1% of each organ), and were belonged to two of M. libycus caught from Kharameh focus. Overall, 41.17% (35/85)and 22.2% (2/9) of T. indica and M. libycus were found positive in microscopic and cultivation methods, respectively (Table 1).

Figure 1. Leishman bodies detected on impression smears prepared from spleen (A) and liver (B) of T. indica, and ear (C) and liver (D) of M. libycus caught from Kharameh, 2016.

3.4. PCR findings

3.4.1. Leishmania detection

The authors declare that there is no conflict of interest.

3.4.2. Crithidia detection

Crithidia kDNA was detected in foot-pad, and spleen samples of 2.35% (2/85) of T. indica caught from Kharameh focus (Figure 2B).In addition, M. libycus, M. musculus R. rattus and R. norvegicus were checked for Crithidia, but the samples prepared from these species were negative for the parasite (Table 1).

Table 1 Leishmania/Crithidia infections of the examined samples consistent with the investigation methods and examined organs of rodents in Shiraz and Kharameh, Fars province, southern Iran.

*Some slides were missed during the investigation. **Some cultures media were infected, so removed from the culture study.

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Figure 2. Gel electrophoresis of samples prepared from the rodents’ organs caught from urban areas of Shiraz and Kharameh during 2016.

A: PCR based products of Leishmania detections; The bands correspond to molecular weight marker (Lanes 1 and 9), Reference strains of L. major (650 bp) (Lane 2), Leishmania tropica (760 bp) (Lane 3), Leishmania infantum (720 bp) (Lane 4), foot, and liver sample of T. indica, respectively (Lanes 5 and 6), ear sample of M. libycus (Lane 7), and double distilled water as a negative control (Lane 8), in Kharameh focus, liver and spleen sample of M. musculus,respectively (Lanes 10 and 11), in Shiraz focus. B: PCR based products of Crithidia detections; double distilled water as a negative control (Lane 1), molecular weight marker (Lane 2), Reference strains of Crithidia sp. (Lane 3), foot and spleen of T. indica, respectively(Lanes 4 and 5).

4. Discussion

In Iran, Fars province is a considerable endemic focus of ZCL and new studies indicated that the prevalence of the disease has increased recently[21,22]. In this study, five species of rodents were caught (T.indica, R. rattus, M. libycus, M. musculus, and R. norvegicus). The largest number of infested rodents was related to T. indica in several villages of Kharameh, which the largest number of T. indica was captured within or out of human residual places. Their breeding places consisted of agricultural lands which products such as wheat,corn, or alfalfa were planted. This species was mostly found in countryside’s regions, especially in rural regions of Kharameh, and was not trapped in Shiraz focus. The outcomes of the microscopic and/or cultural, and molecular assays revealed that 41.17% and 45.3% of T. indica were positive for L. major, respectively. It should be noted that this study included the first detection of Crithidia sp.from Iranian T. indica caught in Kharameh region, where ZCL has recently occurred.

In Asia, T. indica is recorded from Iran, Iraq, Turkey, Pakistan, Syria,Afghanistan, Sri Lanka, India, Kuwait, Nepal, and Bangladesh[10].However, other genus of Tatera, such as Tatera lataste, has been reported and widespread from some parts of Middle East and Pakistan subcontinent[23]. In Iran, T. indica, Rhombomys opimus, M. libycus,and Meriones hurrianae have been reported as the main “reservoir”hosts of ZCL in several endemic foci of Iran[6]. In addition, Gerbillus nanus, and Nesokia indica have been reported as the accidental, main,or probable “reservoir” hosts of ZCL in different parts of Iran[1,24].In Fars province, T. indica, Meriones persicus, and M. libycus are reported positive for L. major in different foci of Marvdasht, Zarqan,Fasa, Estahban, and Jahrom[25-27]. In addition, M. musculus, R.rattus, and R. norvegicus have also been found positive to L. major in the province, which may explain the considerable increase of ZCL in some urban areas of the country in the recent years[28,29]. T. indica along with other rodents (such as Rhombomys opimus, Meriones persicus, and M. libycus) served and confirmed as Leishmania spp.reservoir hosts in different regions of the country. Furthermore, they were found to be infected with L. major in different provinces of Iran including Khuzestan, and Fars[15,26].

Acknowledgments

In the current study, Crithidia sp. was detected in foot-pad and spleen of T. indica, which was introduced as a new reservoir host of ZCL in southern regions of Iran. However, the presence and role of Crithidia in leishmaniasis infection in human has remained considerable[5,30].

In this investigation, M. libycus and M. musculus were also found infected with L. major in Kharameh and Shiraz rural areas,respectively. M. libycus mostly were captured on the edge of agricultural lands, where the man-made canals carried out water to lands. This species was not trapped in Shiraz, but was founded in Shiraz countryside villages. Living places of M. musculus was limited to human residual places, and only captured within the houses in both foci of Shiraz and Kharameh.

R. rattus and R. norvegicus were trapped in Shiraz city and mostly captured on the sidelines of sewages, gardens and parks.These species were not found in rural areas of Kharameh. In our laboratorial studies, no Leishmania and/or Crithidia parasites were detected in Rattus species in Shiraz foci.

2013年，马云卸任阿里巴巴集团CEO，从那时起，阿里已经经历了多次内部的交接。2013年陆兆禧接任阿里巴巴集团CEO，2015年张勇接任CEO，标志着阿里“70后”合伙人全面掌权；2016年井贤栋出任蚂蚁金服的CEO，并于2018年4月接任董事长。显然，在阿里巴巴合伙人机制下，轮换交接是常态。不仅是阿里和蚂蚁，阿里云、菜鸟等阿里体系的其他重要板块也都完成了至少一次的管理团队更替。外界能够清晰地感受到，阿里巴巴的战略从未因人事上的变动而发生变化，而阿里巴巴的增长势头也始终强劲。马云的接班人张勇，彭蕾的接班人井贤栋，都在合伙人群体这个人才储备库中诞生的。

Different experimental methods, such as biochemical, molecular,and immunological methods, have been used for leishmania detection, but, accurately detection of parasites species on the smears of rodent tissues needs a specific and sensitive PCR. In this investigation, using the PCR method was very helpful to confirm the parasitological and/or cultural finding. Besides, the missed parasites,which were not recognized by microscopic (because of low parasite rates) and/or cultivation methods (un-adaptation or unavoided infection in culture media), were detected in PCR assays.

In conclusion, considering the role of rodents in ZCL epidemiological aspects, the present study results revealed that T. indica was a main host of L. major in the ZCL focus of Kharameh County. Additionally, M.libycus and M. musculus played significant roles in the maintenance of the leishmania agent in urban areas of Kharameh and Shiraz.Also, Crithidia parasite was isolated from Iranian T. indica for the first time. Therefore, further studies are required to be conducted on the role of this species along with Leishmania species in the maintenance, transmission, and epidemiologic cycle of ZCL.

Because of the proper ecological circumstances caused between reservoir hosts and vectors of Leishmaniasis in Iran, the present investigation was performed to identify Leishmania and Crithidia species involved in rodents populations of Shiraz and Kharameh (its north countryside) cities in Fars province, southern Iran, by using and comparing microscopic, cultivation and molecular methods(PCR) to have a better understanding of T. indica role as reservoir host of ZCL pathogens, and also develop new environmental control strategies in CL[16,17].

Fars province is located in south of Iran, which includes 23 counties with an area of 122400 km². Shiraz (the capital city of the province) and Kharameh (80 kilometers northeastern of Shiraz) are situated at 29°59’18” North, 52°58’ 37” East, and 29°50’20” N, 53°°31’24” E, and about 1200-1500 m above the sea level. Recently, Shiraz and Kharameh are considered as the most important foci of cutaneous leishmaniasis in Fars province, southern Iran.

In PCR assays, L. major kDNA was detected in rodents’ organs of T. indica, M. libycus, and M. musculus. The samples of the R. rattus and R. norvegicus were negative for Leishmania spp. parasite (Figure 2A). Of 49.56% (57/115) PCR positive samples, 63.53% (54/85)samples belonged to the T. indica, of which 61.18% (52/85)) were infected to L. major (Table 1).

In the recent investigation, the most number of trapped rodents(73.9%) were T. indica. Based on findings obtained in other regions of Fars province, this result was predictable. Besides, despite the wild life, this species is able to adapt itself to live in/out of human residential houses, consequently, the transmission cycle of the ZCL infection was facilitated in the studied regions.

This research was financially supported by the Research Vicechancellor of Shiraz University of Medical Sciences and extracted from the results of an approved Ph.D. student thesis (No: 94-01-104-10873) conducted by the author, Mr. Mohsen Kalantari.

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