In vitro phagocytic study of blood leucocytes and peritoneal macrophages of walking catfish Clarias batrachus ( Ham.) against Aeromonas hydrophila and Escherichia coli.

In observation of in vitro phagocytic activity against Aeromonas hydrophila isolate 34k (a virulent form) and Escherichia coli (an avirulent bacteria) of neutrophil- and monocyte-like cells of walking catfish Clarias batrachus showed phagocytosis. N eutrophils and monocytes phagocytized the avirulent form of bacterial isolate more than the virulent one. Other blood leucocytes did not show phagocytosis. Peritoneal macrophage of the fish were separated by glycogen elicitation and the macrophages were being adhered on plastic cover slips for studying their in vitro phagocytic activity. Most of the cells were alive after adherence and showed phagocytosis against the virulent and avirulent bacteria. The percent phagocytosis and phagocytic index were higher against the avirulent E. coli than the virulent A. hydrophila.

Control of bacterial disease in small scale fresh-water aquaculture: Project R0754. Project completion report December 1998 - May 1999

The work presented here represents an 18-month study to examine the relationship between environmental conditions, bacterial load in the water and bacteria levels in tissue macrophages of a range of clinically healthy freshwater fish species, farmed in a range of culture systems in Thailand and Vietnam. Preliminary assessment was made of the clinical significance of the macrophage bacterial load. The aim of this work was to improve production in fresh-water aquaculture through the control of clinical bacterial disease and subclinical infection, and to identify management practices most effective in promoting fish health. [PDF contains 37 pages]

Vertical movement patterns of skipjack tuna (Katsuwonus pelamis) in the eastern equatorial Pacific Ocean, as revealed with archival tags

Thirty-three skipjack tuna (Katsuwonus pelamis) (53−73 cm fork length) were caught and released with implanted archival tags in the eastern equatorial Pacific Ocean during April 2004. Six skipjack tuna were recap-tured, and 9.3 to 10.1 days of depth and temperature data were down-loaded from five recovered tags. The vertical habitat-use distributions indicated that skipjack tuna not associated with floating objects spent 98.6% of their time above the thermocline (depth=44 m) during the night, but spent 37.7% of their time below the thermocline during the day. When not associated with floating objects, skipjack tuna displayed repetitive bounce-diving behavior to depths between 50 and 300 m during the day. The deepest dive recorded was 596 m, where the ambient temperature was 7.7°C. One dive was particularly remarkable because the fish contin-uously swam for 2 hours below the thermocline to a maximum depth of 330 m. During that dive, the ambient temperature reached a low of 10.5°C, and the peritoneal cavity temperature reached a low of 15.9°C. The vertical movements and habitat use of skipjack tuna, revealed in this study, provide a much greater understanding of their ecological niche and catchability by purse-seine fisheries.

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 .

Effect of organophosphate, diazinon on some immunophysiological, haematological and histological variable of Rutilus frisii kutum (Kamensky,1901)

The acute toxicity and effects of diazinon on some haematological parameters of kutum (Rutilus frisii kutum, Kamensky, 1901) weighing 613.33 g±157.06 g were studied under static water quality conditions at 15°C ± 2ºC in winter and spring 2009. The effective physical and chemical parameters of water were pH= 7-8.2, dh= 300mg/L (caco3), DO= 7 ppm and T= 15°C±2ºC. The first test was primarily to determine the effects of acute toxicity (LC5096 h) of the agricultural toxicant diazinon (emulsion 60%) on kutum male brood stocks. For this purpose, 4 treatments were used to test toxicity; each treatment was repeated in 3 tanks with 9 fish per treatment and with 180 litres water capacity. After obtaining the final results, the information was analysed statistically with Probit version 1.5 (USEPA, 1985), and we determined the LC10, LC50 and LC90 values at 24 hours, 48 hours, 72 hours and 96 hours; the maximum allowable concentration value (LC5096 h divided by 10) (TRC, 1984); and the degree of toxicity. The second stage of testing consists of four treatments: LC0= 0 as experimental treatment, treatment A with a concentration of LC1= 0.107 mg/L, treatment B with concentration of LC5= 0.157 mg/L, treatment C with concentration of MAC value= 0.04 mg/L. Male brood stocks of kutum were treated with these concentrations for 45 days. Experiments were carried out under static conditions based on the standard TRC, 1984 method over 45 days. Our results show that long-term exposure to diazinon causes a decrease in the erythrocyte count (RBC), haemoglobin (Hb), haematocrit (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leucocyte count (WBC), lymphocyte, testosterone, iron (Fe), sodium (Na), lactate dehydrogenase (LDH), and cholinesterase (CHeS). In addition, diazinon also causes an increase in prolymphocyte, aspartate aminotransferase (AST), cholesterol, alkaline phosphatase (ALP) and adrenaline (P<0.05). There are no significant effects on monocyte, eosinophil, magnesium (Mg), chloride (Cl), glucose (BS), urea (BUN), uric acid (U.A), triglyceride (TG), calcium (Ca), albumin (Alb), total protein (TP), cortisol, noradrenaline and high density lipoprotein (HDL) levels in kutum male brood stocks (P>0.05). Pathology results showed toxin diazinon no effect on average weight and fish body length, the average weight of heart, brain, spleen, liver, kidney and liver index but caueses decrease of gonad weigth and gonad index and also, cause complications of tissue necrosis, vascular congestion, inflammation in the liver, a sharp reduction in the number of glomeruli, necrosis, vascular congestion and haemorage in the kidney, capsule thickening and fibrosis, atrophy, vascular congestion, macrophages release increased, increasing sediment Hemosiderine and thickening of artery walls in the spleen, atrophy, fibrosis and necrosis in testis , vascular congestion, increased distance between the myocardium and fibrous string in heart and neuronal loss, vascular congestion and edema in the brain of kutum male brood stocks.

Effects of toxins induced by phytoplankton bloom on physiological function of indicator animals in Anzali Lagoon

The present overview summarizes data from one year's study during the period of 1379 to 1380 in the regions by "Anzali" lagoon called "Abkenar" and "Hendkhale". Specimens from this lagoon obtained weekly during mordad and shahrivar mounths (July 21 to september 21). The study included 67 types of 5 phytoplanktonal phylum. In "Abkenar" region Cyanobacters with maximum of 97% and minimum of 64.5% of total combination of phytoplanktones made the dominating combination during the period of study , while in "hendkhale" chyrsophyta with maximum of 89% and minimum of 38.7% of total phytoplanktonal was the dominating figure at the same period of time. Researches on ecological parameteres showed that the avarage dissolved oxygen in -Abkenar" and "hendlchale" regions was 10.7 and 8.0 mg/lit respectivly, also total rate of Phosphat in these regions was 0.085 and 0.15 mg/lit respectivly. This study showed that the rates of Nitrat and Amonium in "Abkenar" region was 0.043 and 0.79 mg / lit while for the same substances in "Ilendkhale" measured 0.08 and 0.7 mg I lit respective. Also the avarage rate of chlorophyll a in these two areas measured 58,38 and 40.45 j.un /ht respectivly. Depending on results of correlation cofficient in "Abkenar" region we had Cyanobacters , water and air temperature , Chlorophyll a and total amount of Phosphat as a poitive correlation with transparency while Amonium and Nitrat showed , a negative correlation , EC and finally dissolved oxygen showed a very low rate of correlation coffiocient. To perform this research 5 genus of Cyanobacteres horn "Anzali" lagoon have been isolated and cultured in a laboratorial conditions Later by using Mouse Bioassay method one of these genus identified as a toxic algae. Levels of LD50 with intra peritoneal injection of toxin on mouse in 24 hours was 660 mg/kg and Levels of LC50 by using Bioassay method on Artemia and Daphnia has been shown 618 and 1000 mg kg respectively. Also the physiological effects of were investigated. algae on two types of Cypronides family called Cyprinus carpi° and Hypophrthabnichthys Resultes of blood analyses of Cypronides who were feeded by toxic algae showed a significant decline (P < 0.05) in white and red blood cells and their hematocrites. Levels of LDH , SGOT and SGPT in their blood serum had a significant increase in porportion to control group (P < 0.05) but there was no evidance a differences in Total Protein levels. Pathological studies show damage and destruction of hepato pancreas and kidney of these fishes. Signs and symptoms of intoxication caused by Cyanobacter called Planktohrix agardlltii in mouse and fish show heptotoxic character. This toxin belongs to cyclicpeptides of microcystines group.