The experimentally pathogenesis of Aeromonas hydrophila in Astacus leptodactylus with emphasis on temperature in bacterial pathogenesis

In this study, four hundred freshwater Crayfish (A. leptodactylus) with average weight of 25-40g were purchased from Aras dam reservoirs in west Azerbayjan province and transported to Iranian Artemia Research Center of Uromya province in September 2010. (One hundred crayfish extra purchased for probably mortality). Before implement of experiment the Crayfish were acclimated for ten days. These experiments was designed in four group treatments (Number, 1,2,3,4) and one control group (Number 5) in triplicate with 20 Crayfish in each repetition prepared of glass aquarium with size (50x40x30cm). Many of infected Crayfish were used for isolation of bacteria. Haemolymph sample had been gathered from infected Crayfish with cutting their antennules and transferred to TSA medium (tryptic soy agar) and then A. hydrophila were determined in order to biochemical test. The treatments and repetitions has exposed to A. hydrophila. The concentrations of the bacteria in 4 treatments were respectively 3 x 108 (T=10-15°C), 3x106 (T=25°C), 3 x 106 and 3 x 104 Cfu mL-1 (T=10- i5oc) (4, 2, 3 and 1) that were prepared in individual containers for exposure of treatments. The control (5) prepared without any bacteria and disinfected by oxytetracycline antibiotic with concentration 100 ppm for 24 hours. The hemolymph samples were withdrawn from abdominal second segments of Crayfish for measuring of THC and TPC in interval hours (2, 6, 12, 24, 48, 96, 144, 240 and 336). For histopatological studies the crayfish samples fixed in Davidson fixative. The results indicated that interval 2 hours after experiment the difference of THC value between treatment 4 with control and treatments 1,2, and 3 was significant (P< 0.05). After 48 hours of experiment the difference of THC value between control group with treatment 1 ,2 and 3 was significant (P< 0.05). The interval 336 hours after experiment also the difference of THC value between treatment 2 with treatments 1, 3 and 4 was significant (P< 0.05). The finding of TPP value showed that the last time after challenge (336 h) there was significant difference between treatment 2 with treatment 4 and control group (P< 0.05). In histopathology studying, in hepatopancreas observed hemocyte aggregated and necrosis withof peknosis nucleus that with increased concentration of bacteria and temperature, The value of hemocyte has increased. Gill revealed necrosis and cell death especially with increased concentration of bacteria and temperature. In lower concentration of bacteria in heart no difference observed, but with increased concentration of bacteria (3 x 108) the low aggregation of hemocyte observed in heart. In treatment 3 x 106 with high temperature also distributed of high hemocyte in heart was observed. In digestive system didn't appear any difference in treatments land 3 but in concentration of 3 x 108 Cfu m1-1 and 3 x 106 (T=25°C) in digestive system was revealed the low aggregation of hemocyte.

Experimental pathogenesis of Aeromonas hydrophila bacteria in shing Heteropneustes fossilis(Bloch)

Pathogenicity of Aeromonas hydrophila bacteria was tested on the stinging catfish Heteropneustes fossilis. Before artificial infection the morphological, biochemical and physiological characters of Aeromonas hydrophila were studied. The infections were done by two different methods, viz., intramuscular (IM) and intraperitoneal (IP) injection. In infection experiment, each group of 10 fish were injected either intramuscularly or intra peritoneally with one dose higher than the LD50 dose (9.6 x 107 CFU/fish). All the fish tested died within 1 to 9 days. Both in cases of intramuscular and intraperitoneal injection, external pathology were found. Haemorrhagic lesions were evident at the site of injection. The posterior end of the body surface was found to develop greyish-white lesion that was extended up to caudal fin. Hyperemic anal region and the fin bases were also observed. Total bacterial loads in liver, kidney and intestine were determined. Aeromonas hydrophila could be isolated from liver, kidney and intestine of the experimentally infected fish. In case of intramuscular injection the highest and the lowest bacterial load was found to be 2.4 x 107 CFU/g of liver and 2.1 x 102 CFU/g of kidney and in case of intraperitoneal injection they were found to be 3.6 x 106 CFU/g of kidney and 1.2 x 104 CFU/g of kidney respectively. It was concluded that A. hydrophila could cause serious disease condition to Heteropneustes fossilis and its pathogenesis in the fish was also very efficient.

Study of bacterial agents on external lesions of cultured Sturgeons in Sngar zone of Gilan Province

This study was carried out to isolate and determine bacterial agents in outer lesions of sturgeons in Shahid Dr. Beheshti sturgeon propagation and rearing center in Gilan province. Five species of sturgeons were studied from viewpoint of lesions. A number of 167 specimens of Beluga, 76 specimens of Persian sturgeon, 27 specimens of Russian sturgeon, 42 specimens of stellate and finally 23 specimens of ship had bacterial lesions in different outer parts of their bodies. After sampling and purification, bacterial cultures and biochemical tests were done. After the isolation of bacteria from lesions, Edwardsiella tarda was selected by means of PCR. To obtain molecular acceptance, a pair of E. tarda special primer, forward primer ETa2-351 and reverse primer (Edwsp-780r) were reproduced. A number of 12 E. tarda DNA sample were identified by PCR. After molecular diagnosis, Persian sturgeon challenged with E. tarda for determination of pathogenesis. Challenge method was done by means of injection of different dilutes of E. tarda into dorsal muscle. Sampling of hematopoietic organs (kidney, spleen and liver) were carried out and located in Boin's fixator to perform pathology survey. Also, in order to survey of existence and effect of E. tarda, sampling of kidney for bacterial culture was done by molecular and biochemical methods. Results showed that the most lesions in all five species belonged to abdominal surface. Skin and scutes of this part were involved in comparison with other parts. Also, It was removed some samples from lesions to pathological survey. Microscopic observations showed some levels of destruction of epidermis layers, necrosis of dermis cells and destruction of muscular layer of skin. On the other hand, invasion of inflammatory cells and haemorrhagic in dermis were clear. Based on biochemical results, Aeromonas sobria, A cavia . A. hydrophila , Acinetobacter lowffii , A.baumanni , A.cakoaceticus, Pseudomonas putida , P fluorescens , P.aeruginosa , Serratia marcescens , Escherichia coli , Enterobacter aerogenes , Edwardsiella tarda , Proteus mirabilis , kelebsiella oxytoca and Staphylococcus sp. were isolated from outer lesions. Results of PCR confirmed that E. tarda was before and after challenge in 200 bp range. LD50, 96h was determined 1.2 x 10^5 (CFU/ml). Pathological experiments showed lesions in the kidney, including hemorrhages, degeneration of glumeruli and tubular epithelia, degeneration and necrosis of interestitium tissue, accumulation of protein casts in the tubular lumen. It was observed haemorhages, engorged blood vessels, congestion of sinusoids, increased of melanine, melano macrophage centers, degeneration and necrosis of hepatocytes in the liver. In the spleen, it was recorded congestion, degeneration, necrosis changes in the white and red pulpa, blood engorged and detachment of ellipsoid wall.

Role of Aeromonas hydrophila in bacterial septicemia of cultured carps in Khouzestan Province and investigation on protectivity of bacterin (s) prepared from acute isolate(s) of it

Motile Aeromonas are the most common bacteria of freshwater in the world that cause disease in fish and other cold-blooded and warm-blooded hosts. Among this group of bacteria, Aeromonas hydrophila is important in causing complications such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. Several virulence factors involved in the pathogenesis of Aeromonas hydrophila, including extracellular enzymes (protease, lipase, elastase, gelatinase and nuclease) and toxins. From the exotoxins, hemolysin, aerolysin and cytolytic enterotoxin play an important role in pathogenesis. Detection of virulence markers by PCR as a key component of determining the pathogenesis of the bacteria and using indigenous vaccines for better immunization against this disease is important. In this study, a total of 200 fanned carps (126 common carp. 39 silver carp and 35 of grass carp) with symptoms suspected aeromonas septicemia were isolated from Khouzestan province farms. 125 bacteria belong to Aeromonas genus detected by biochemical and PCR methods. 31 of all isolates recognized as Aeromonas hydrophila with biochemical methods, I6srRNA detection and Lipase genes. Results showed that the role of Aeromonas sp. and Aeromonas hydrophila in fish with disease symptoms were 62.5% and 15.5% respectively. By using specific primers, three virulence genes including hemolysin, aerolysine and cytolytic enterotoxin were detected in these confirmed isolates, that 18 isolates (58/06%) hemolysin positive (hlyA +), 16 isolates (51/61%) aerolysine positive (aerA+) and 23 isolates (74/19%) for cytolytic enterotoxin gene (act+) were positive. The result of present study showed that most of the confirmed isolates genotype was hlyA+ act- with frequency equal to 51/61%. For investigating the protection effect of acut strain of bacteria, UV inactivated bacterin was used.

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]

Bacterial flora of the gut of the African snakehead, Channa obscura (Pisces: Channidae)

A qualitative and quantitative investigation of the bacterial flora of the gut of the African snakehead, Channa obscura was undertaken. The types of bacteria isolated from the different parts of the gut of C. obscura include Pseudomonas, Streptococcus, Citrobacter and Proteus. The coliform (Escherichia coli, Enterobacter) and some other Enterobacteriaceae such as Salmonella were also present. The stomach and intestine were found to have a preponderance of Pseudomonas and Vibrio species. Klebsiella sp. and Bacillus sp. (only in the pyloric caeca) were also isolated. On the whole, the correlation coefficients of the two incubation temperatures showed a high statistical significance. Thus the bacterial load of the gut of C. obscura has been shown as a function of temperature

Distribution and occurrence of bacterial fish diseases in the different cultural facilities of Lake Kainji area

Fish farming practices in the Lake Kainji Area of Nigeria are categorized under seven main cultural facilities, namely, earthen ponds/reservoirs, indoor/outdoor concrete tanks, plastic tanks, floating cages/hapas, aquaria, sewage and feral conditions. The presence of Bacteria isolates associated with diseased fish conditions varied significantly (P<0.05) with different cultural facilities. The highest bacteria isolates and bacterial disease incidence, 33% and 46% respectively, was associated with diseased fish in the indoor/outdoor concrete tanks. The least incidence of bacteria isolates (3.5%) and blue bacterial disease (3%) was associated with diseased fish in the aquaria and feral conditions. Nine Gram-negative and two Gram-positive bacteria genera were isolated during this investigation. Pseudomonas spp. (23.6%) and Staphylococcus spp. (14.3%), were the predominant Gram-negative and Gram-positive bacteria genera in the different cultural facilities, respectively. This paper highlights the relevance of occurrence and distribution of bacteria isolates associated with diseased fish to bacterial fish diseases under different cultural facilities

Watershed monitoring and the TMDL modeling to assess bacterial loading in estuarine environments to improve management

Shellfish bed closures along the North Carolina coast have increased over the years seemingly concurrent with increases in population (Mallin 2000). More and faster flowing storm water has come to mean more bacteria, and fecal indicator bacterial (FIB) standards for shellfish harvesting are often exceeded when no source of contamination is readily apparent (Kator and Rhodes, 1994). Could management reduce bacterial loads if the source of the bacteria where known? Several potentially useful methods for differentiating human versus animal pollution sources have emerged including Ribotyping and Multiple Antibiotic Resistance (MAR) (US EPA, 2005). Total Maximum Daily Load (TMDL) studies on bacterial sources have been conducted for streams in NC mountain and Piedmont areas (U.S. EPA, 1991 and 2005) and are likely to be mandated for coastal waters. TMDL analysis estimates allowable pollutant loads and allocates them to known sources so management actions may be taken to restore water to its intended uses (U.S. EPA, 1991 and 2005). This project sought first to quantify and compare fecal contamination levels for three different types of land use on the coast, and second, to apply MAR and ribotyping techniques and assess their effectiveness for indentifying bacterial sources. Third, results from these studies would be applied to one watershed to develop a case study coastal TMDL. All three watershed study areas are within Carteret County, North Carolina. Jumping Run Creek and Pettiford Creek are within the White Oak River Basin management unit whereas the South River falls within the Neuse River Basin. Jumping Run Creek watershed encompasses approximately 320 ha. Its watershed was a dense, coastal pocosin on sandy, relic dune ridges, but current land uses are primarily medium density residential. Pettiford Creek is in the Croatan National Forest, is 1133 ha. and is basically undeveloped. The third study area is on Open Grounds Farm in the South River watershed. Half of the 630 ha. watershed is under cultivation with most under active water control (flashboard risers). The remaining portion is forested silviculture.(PDF contains 4 pages)

On methods of evaluation of bacterial production. [Translation from: Lietuvas TSR Mokslu Akademijos Darbai, C Serija 2(55) 3-9, 1971.]

By now a great deal of work is known concerning the methods of determining the production of bacteria or similar questions; among these the problems of a common terminology is discussed. The article discusses formulae of production of bacterial populations over time.

Bacterial production of nitrate in aquatic systems: progress report. Nitrate-loss processes in raw water

This short interim progress report builds on previous progress reports which have described the quantification of the process both within and between lakes of different degrees of eutrophication. These data indicated that slight changes in methodology, particularly when investigating sediment deposits, could grossly affect the measured activity. The aim of the present research was an attempt to rationalize these differences. If this could be achieved it would enable meaningful interpretation of published data obtained using different methods and therefore enlarge the available database. In addition some observations have been made on the production of nitrite by Grasmere profundal sediment slurries sampled during the circulation period.

Variation in bacterial populations in time and space

Obtaining a reliable estimate of the bacterial population is one of the main problems facing the bacterial ecologist. The author discusses the various methods available and concludes that the observed variability in bacterial populations depends on the sampling interval used.

Decomposition in lake sediments: bacterial action and interaction

This review discusses the processes involved in the decomposition of organic carbon derived initially from structural components of algae and other primary producers. It describes how groups of bacteria interact in time and space in a eutrophic lake. The relative importance of anaerobic and aerobic processes are discussed. The bulk of decomposition occurs within the sediment. The role of bacteria in the nitrogen cycle and the iron cycle, and in sulphate reduction and methanogenesis as the terminal metabolism of organic carbon are described.

Surveying bacterial human pathogens in shrimp culture in Thailand

Any presence of bacterial human pathogen in shrimp products may be of public health concern. This note concludes that Salmonella do not appear to constitute a part of the microbial flora where shrimp culture is practiced in Thailand. Vibrio cholerae 01, the cause of cholera are rarely recovered from the environment with no isolates containing genes encoding cholera toxin. Further studies are needed to describe the prevalence of bacterial human pathogens in shrimp culture, especially determination of possible postharvest cross-contamnation with these pathogens

Using wildlife activity and antibiotic resistance analysis to model bacterial water quality in coastal ponds

Models that help predict fecal coliform bacteria (FCB) levels in environmental waters can be important tools for resource managers. In this study, we used animal activity along with antibiotic resistance analysis (ARA), land cover, and other variables to build models that predict bacteria levels in coastal ponds that discharge into an estuary. Photographic wildlife monitoring was used to estimate terrestrial and aquatic wildlife activity prior to sampling. Increased duck activity was an important predictor of increased FCB in coastal ponds. Terrestrial animals like deer and raccoon, although abundant, were not significant in our model. Various land cover types, rainfall, tide, solar irradiation, air temperature, and season parameters, in combination with duck activity, were significant predictors of increased FCB. It appears that tidal ponds allow for settling of bacteria under most conditions. We propose that these models can be used to test different development styles and wildlife management techniques to reduce bacterial loading into downstream shellfish harvesting and contact recreation areas.