Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

Separation and identification fresh water leeches of waters resources in Kordistan Province

In the primary studying, known leeches have included into free living and parasitic which the parasitic group, besides of direct impacts like: growth detraction, anemia, making wound in the connecting part with the skin, with making plat for secondary bacterial and molding infections in the toll place, is able to cause to transfer blood flagellate and virus to the fish too. Therefore, by accusing information related to fauna leeches of each area a risk factor from the viewpoint of the possibility of being or accusing transferred diseases by these leeches, one can predict and forehand about them. Freshwater leeches of Iran to present accurate recognition (morphological, molecular) have not been, and there are some limited reports from different parts of the country about them. One of the areas that its leeches have not been identified yet is Kurdistan, By having five latrines and big permanent rivers and 32000 springs and a lot of deep and semi-deep wells and this province is a convenient bed for growing aquatics in the country. Therefore, identifying risk factors for development of aquaculture on water resources is one important factor to access achieving development goals. For recognizing leeches of this province, some samples from 10 stands were token. Samples from under stones, sticking to the fish, turtles, plants and solid substances in the water were separated and after recording their physical characteristics, calming with 10% ethylic alcohol with 10% formalin become fixed and after painting with Carmen acetic acid by standard keys for 7 species of Helobdella stagnalis, Placobdella costata, Hemiclepsis marginata, Erpobdella octoculata, Hirudo medicinalis, Dina lineate lineata have been identified and described. Which Helobdella stagnalis has the highest distribution in the province and the minimum one is Hirudo medicinalis. However, that the data obtained in leeches in Kurdistan is a relatively complete collection in this research, recognizing fauna of these areas needs more studying. The Placobdella costata and Hemiclepsis marginata sticking to the fish were separated among identified species which showed that these are parasites for the fish. The sticking area of those leeches to the skin was accompanied with scales cast, damage to mucous membranes beneath the parasite and bleeding Was associated with Histopathology studying effect includes observing break and disconnection in the leech connecting place to the epithelial layer of epidermis in the skin, destroyed nucleus in skin Epithelial cells with observing necrosis in ulcerative place become of the leech and the sub acute inflammated penetration until acute necrosis with opening in Dermis layer is observable. Kidney of this fish have changes such as: proliferation, like proliferative kidney disease with increasing proliferative glomerular cells and increasing in membranous cells in Capillary corpuscle, observing necrotic cells in haematopoietic tissue of kidney along with increasing in infiltration of leukocyte's cells generally mono nucluars such as lymphocytes and less poly morpho nucluars such as neutrophiles that are symptoms of disorders causing anemia become of nourishing and sucking blood by the leech and creating a chronic kidney infection that originally root is in another place like the skin. Also Hemorrhagic anemia causes losing RBC's is because of using the host blood by the leech. (In this situation, one can see immature RBC red cells in Peripheral blood. To identify potential carriers of the leech to the viruses, after finding them in recorded stands and putting them in 75% ethanol for viruses cause IPN, VHS, IHN, they were tested by PCR that the conclusion of these experiments approved IPN virus in Hemiclepsis marginata and Hirudo medicinalis. This kind of leeches can act like a mechanical carrier and causing spreading the agent of this disease. It is worth mentioning that studying the pathogenicity of this virus for aquaculture sources, mentioned before needs more research. During the study of infected fish with leeches that was done after preparing bloody slides and staining them, no case blood parasites was observed. During a research about infecting fish experimentally to known leeches it become clear that 5 days after being in aquarium including leeches, samples of sticking Hirudo medicinalis leech to the golden carp with scales cast were observed. Including leeches to the fish started with molting the scales in the sticking area in the fish and fish become too uneasy and by rubbing themselves to the malls and things inside the aquarium, tried to separate them. Finally, after around 30 hours, leeches penetrate the skin, feeding from blood and tissue liquids and cause mortality the fish and then they become separated from them. If the corpse of these fish stayed in the aquarium, the Helobdella stagnalis and Erpobdella octoculata would start feeding them.

Protection of white leg shrimp (Litopenaeus vannamei) against white spot disease virus using oral recombinant bioencapsulated VP28

Aquaculture, is perceived as having the greatest potential to meet the growing demand for aquatic food. Crustaceans form one of the main value added components in aquaculture and among them, shrimp aquaculture is the predominant one. Industrial shrimp fanning, in combination with poor management in shrimp aquaculture, has quickly led to severe pollution in shrimp ponds, thereby creating a suitable environment for development of bacterial and virus diseases. White spot disease is one of the most deadly diseases that are caused heavy loss in all Penaeid shrimps family. In Iran during 2002 to 2004 in the Kuzestan province and in 2005 in Bushehr province, the most ponds and farms infected with white spot and the entire industry was facing threat of closure. Owing to the impact of WSSV infection to shrimp aquaculture, there is an urgent need to develop suitable strategies to protect cultured shrimps and make aquaculture more sustainable. Therefore, this study aimed to examine the possibility of protecting shrimp against white spot syndrome virus using bioencapsulated Anemia with E. coil containing the recombinant protein VP28, designed. Virus genome was extracted from naturally infected Litopenaeus vannamei in the Choebdch farms and VP28 gene by designed primers was amplified, extracted, purified and cloned in E. coli TGI. Protein expression evaluated and inactivated bacteria containing recombinant protein encapsulated in Artemia nauplii. White shrimp post larvae stage 5 were fed for 5 days with recombinant nauplii and twice on days 7 and 25 after feeding with Artemia nauplii were challenged with white spot virus. The results of the first experiment revealed that cumulative mortality percent in the group receiving the bacteria containing recombinant plasmid (pMal + VP28) was %14.44±1.11 and the relative percent survival %80.30±1.51. In this group the mortality rates in the various repetitions varied from the 13.33% to 16.66% and relative percent survival of 77.27% to 81.81%. in the Non-recombinant plasmid group (pMal) Mean percent mortality was% 33.33±3.84 and the Relative Percent Survival %54.54±5.24 and in the group that received bacteria contained no recombinant plasmid the Mean cumulative mortality percent was%48.88 ± 5.87 and Relative Percent Survival%33.33± 8.01.