Chemical control of psychrophilic bacterial spoilage of fish 1. Isolation and identification of psychrophilic bacteria from marine fish

Making use of the streak plate technique and low temperature incubation, 28 cultures belonging to six genera namely, Achromobacter, Flavobacterium, Pseudomonas, Micrococcus, Vibrio and Alcaligenes were isolated from different varieties of marine fish. The growth studies indicated that all of them were able to grow between -5 and +5°C within a week's time and none of them showed growth at 37°C. The optimum temperature of growth for all these cultures was in the range 25-28°C. Among these only one, i.e., a Vibrio sp., was found to be an obligate psychrophile.

Isolation and culture in artificial media of Lagenidium from Penaeus monodon larvae

Fungal infection of P. monodon larvae is a problem in hatchery operations. The fungus, which attacks the nauplius to postlarval stages and causes up to 100% mortality, has been tentatively identified as belonging to the genus Lagenidium . This pathogenic organism has recently been isolated and cultured. A description is given of the fungus, and features of its biology and pathology are discussed.

Venomics and biological analysis of Scatophagus argus argus (Spotted scat) venom isolated in Persian Gulf, extraction and purification of hemolytic protein

Green scat namely as Scatophagus argus argus is a venomous aquarium fish belonging to Scatophagidae family. It can induce painful wounds in injured hand with partial paralysis to whom that touch the spines. Dorsal and ventral rough spines contain cells that produce venom with toxic activities. According to unpublished data collected from local hospitals in southern coastal region of Iran, S. argus is reported as a venomous fish. Envenomation induces clinical symptoms such as local pain, partial paralysis, erythema and itching. In the present study green scat (spotted scat) was collected from Persian Gulf coastal waters. SDS-PAGE indicated 12 distinct bands in the venom ranged between 10-250 KDa. The crude venom had hemolytic activity on human erythrocytes (1%) with an LC100 (Lytic Concentration) of about 1.7 μg. The crude venom can release 813 μg proteins from 0.5% casein. Phospholipase C activity was recorded at 3.125 μg of total venom. Our findings showed that the edematic activity remained over 48 h after injection. The purification of the venom was done by HPLC and 30 peaks were obtained within 80 min but only one peak in 68 min retention time showed hemolytic activity at 90% acetonitril was isolated. The area percentage of the hemolytic protein showed that this hemolytic protein consist of 32 percent of total proteins and its molecular weight was 72 KDa in SDS_PAGE. The results demonstrated that crude venom extracted from Iranian coastal border has different toxic and enzymatic activities.

Isolation and recognition Saprolegnia species from fungal infected eggs of the rainbow trout in Mazandaran province farms

Fungal infection in the eggs of freshwater fish is well known as a problematic disease. That isolation and recognition Saprolegnia fungi from fungal infected eggs of the rainbow trout in Mazandaran province was the aim of this research. For this purpose fungal infected eggs were examined from six fish farm in the fall and winter 2005-2006. The eggs with fungi were inoculated on SDA, CMA, GPagar and hemp seed and sesame seed cultures in sterile tap water at room temperature (18-24°C). In this study recognized three genera and six species Saprolegniaceae members, based on morphological characteristics which contain: Saprolegnia, Achlya, Brevilegnia. Four species were identified in the genus Saprolegnia; S.mixta, S.parasitica, S.moniliphera, S.lapponica and one species was identified in the genus Achlya; A.oblongata. S.parasitica was isolated from almost all the farms. In addition, another nine genera and species were identified; Penicillium, Aspergillus, Paeciliomyces, Acremonium, Fusarium oxysporum, F.solani , Alternaria, Helminthosporium, Mucor.

Detection of cryptosporidium oocysts in water and environmental concentrates

Whilst current methods for the isolation and enumeration of Cryptosporidium spp. oocysts in water have provided some insight into their occurrence and significance, they are regarded as being inefficient, variable and time-consuming, with much of the interpretation being left to the expertise of the analyst. Two expectations of novel developments are to reduce the variability and subjectivity associated with the isolation and identification of oocysts. Flocculation, immunomagnetisable and flow cytometric techniques, for concentrating oocysts from water samples, should prove more reliable than current methods, whilst the development of more avid and specific monoclonal antibodies in conjunction with the use of nuclear fluorochromes will aid identification. Further insight into the viability, taxonomy, species identification, infectivity and virulence of the parasite should be forthcoming through the use of techniques such as the polymerase chain reaction, in situ hybridisation and non-uniform alternating current electrical fields. Such information is necessary in order to enable microbiologists, epidemiologists, engineers, utility operators and regulators to assess the safety of a water supply, with respect to Cryptosporidium contamination, more effectively.

Improved handling and preservation of freshwater giant prawn, Macrobrachium rosenbergii for producing safe and wholesome products

Studies were undertaken to evaluate the quality changes in freshwater giant prawn, Macrobrachium rosenbergii during various storage conditions of handling and preservation and producing safe and quality products. The samples kept in ice immediately after catch with head-on and head-less condition were found to be acceptable for 6 days and 7 days, respectively. Delaying of icing considerably shortened the shelf-life. The pH value increased from 6.36 to 8.0 after 10 days in ice. The initial average TVB-N value of sample increased from below 10 mg/100 g to 25 mg/100 g with the lapse of storage period. The Ca++ ATPase activity in presence of 0.1M KCl slightly decreased at the end of 10 days of ice storage. Immediately after harvest, initial aerobic plate count (APC) was 2.88x10^6 CFU/g which gradually increased to 1.12x10^8 CFU/g after 6 days in ice storage and showed early signs of spoilage. Initial bacterial genera in the prawn iced at 0 hours were comprised of Coryneform (22.21 %), Bacillus (7.40%), Micrococcus (11.11 %), Achromobacter (48.14%), Flavobacterium/Cytophaga (7.40%), Pseudomonas (3.70%) and Aeromonas (3.70%). During ice storage Coryneforms and Bacillus were always dominating along with less prominent ones - Micrococcus, Achromobacter and Flavobacterium. Studies were conducted on the stability of myofibrillar protein of M. rosenbergii under different storage and pH conditions. The influence of a wide range of pH on the remaining Ca++ ATPase activity of M. rosenbergii muscle myofibrils after storage at -20°C for 2 days, at 0°C for 2 days and at 35°C for 30 minutes demonstrated that ATPase activities were lower in acidic and alkaline pH regions and the activity remained relatively high. Mg++ ATPase activities both in presence and absence of Ca++ remained high at neutral pH compared to those of acidic and alkaline region. The solubility of myofibrillar protein decreased gradually both in acidic and alkaline pH regions. The study also examined the bacteriological quality of freshly harvested M. rosenbergii, pond sediment and pond water from four commercial freshwater prawn farms at Fulpur and Tarakanda upazilas in the district of Mymensingh. The study included aerobic plate count (APC), total coliform count, detection, isolation and identification of suspected public health hazard bacteria and their seasonal variation, salt tolerance test, antibiotic sensitivity test of the isolates and washing effect of chlorinated water on the bacterial load in the prawn samples. APC in sediment soil and water of the farm and gill and hepatopancreas of freshly harvested prawns varied considerably among the farms and between summer and winter season. The range of coliform count in water, gill and hepatopancreas ranged between 6 - 2.8x10^2 CFU/ml, 1.2x10^2 - 3.32x10^2 CFU/g and 1.43x10^2 - 3.89 x10^3 CFU/g, respectively. No coliform was detected in pond sediment sample. Suspected health hazard bacteria isolated and identified from pond sediment, water, gill and hepatopancreas included Streptococcus, Bacillus, Escherichia coli, Klebsialla, Salmonella, Staphylococcus, Pseudomonas and Aeromonas. Bacillus, Salmonella and Staphyloccus [sic], and were found to be highly salt tolerant and capable of growing at 10% NaCl. The antibiotic discs with different concentration of antibiotics were used for the sensitivity test. The organisms were found to be most sensitive against Tetracyclin and Gentamycin.

Vibrio parahaemolyticus and the seafood industry

The role of Vibrio parahaemolyticus in food borne gastroenteritis outbreaks associated primarily with the consumption of contaminated seafoods has been well documented. Information pertaining to various aspects of its occurrence in seafoods, procedures for isolation and identification, generation time and inactivation profiles is discussed. Emphasis has been given to the response of V. parahaemolyticus to low temperatures, heating and antibacterial agents. The public health hazard posed by the pathogen is outlined and the guidelines for control are reviewed in detail.

Bacterial and pathological study of caudal fin rot in brood stocks of Salmo trutta caspius from the propagation and breeding center of Shahid Bahonar of Kelardasht region

In order to study caudal fin rot with emphasis on Aeromonas hydrophila and Pseudomonas fluorescens in Salmo trutta caspius from the salmonids propagation and breeding center of Shahid Bahonar of kelardasht region, One hundred and eighty brood stocks having fin damage symptoms were chosen. Two bacterial samples from each fish were cultured on Aeromonas and Pseudomonas specific media. Biochemical tests, API2OE identification system and antibiogram test using six antibiotic disks were performed for diagnosing isolates bacteria and finding suitable antibiotic. Thirty samples from caudal fin of damaged fishes were fixed in 10% formalin and 51.tm microscopic sections were prepared using standard scatological methods and then stained by Haematoxylin-Eosin staining method to observe the pathological changes and also Maccallum-Goodpasture staining method to observe the bacterial colonies. In second stage of the study, bacterial samples were taken from thirty brood stocks using similar method at the first stage of sampling. For isolation and biochemical diagnosis of Aeromonas and Pseudormonas genus, the samples were analyzed by molecular research included PCR amplification (using 16S rDNA genes of the genus pseudomonas and 16S-23S rDNA intergenic spacer of the genus Aeromonas) and restriction analysis by four restriction enzymes for each genus. The results of biochemical tests showed that isolated bacteria were belonged to Aeromonas caviae and Aeromonas hydrophila (subspecies anaerogenes), Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas alcaligenes while the results of API2OE identification system showed that the isolated bacteria belonged to Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas aeruginosa. Restriction analysis of Aeromonas samples with Hin6l, Csp6I, Taql, and Tasl revealed three samples were different from others while restriction analysis of Pseudomonas samples with Alul, Hinfl, Rsal, and Trull showed at least five species or biovars. The results of antibiogram test showed all Aeromonas samples were sensitive to Trimethoprim, Chloramphenicol and Nitrofurazone, mostly to Nalidixic acid and Chloramphenicol, while most of samples were resistant to Erythromycin and Oxytetracycline. Pseudomonas samples were only sensitive to Nitrofurazone and mostly resistant to Oxytetracycline, Nalidixic acid, Erythromycin, Trimethoprim and Chloramphenicol. The results of light microscope study showed hyperplasia and spongiosis of the malpigian cells of epidermis, increasing of melanin pigments underlying epidermis; sever necrosis in both epidermis and dermis and also sloughing the epidermis in some cases. Occurrence of clefts through the epithelium, neovascularization, hyperemia and mild inflammatory response in dermis and separation of the fin rays also were observed. No bacterial colonies were found in the sections.

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.