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In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fsh collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue respon sible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly infuenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius varda rensis ; Karaman, 1928), using for the frst time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fsh under the infuence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fsh from mining afected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems

Mediterranean Sea is the sixth largest area of marine litter accumulation in the world, and plastic pollution is a growing problem in its Adriatic sub-basin. The aim of the present study was to evaluate the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea in comparison with microbiota in seawater and sediment. Plastic litter in the sea contains an autochthonous microbiota that is different from that of the surrounding seawater and sediment. Vibrio abundance was higher on plastic litter than in surrounding seawater and sediment. All isolated Vibrio showing resistance to ampicillin and vancomycin, while resistance to other antibiotics depended on the isolated species. Overall, this study provides for the first time information on the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers and provides a data base for further studies.

Pigmented macrophage aggregates (MAs) are known to change under influence of various factors, such as aging, season, starvation, and/or pollution. In this study, changes in the pigment content of the MAs in the spleen of Vardar chub (Squalius vardarensis, Karaman) (n = 129) collected in spring and autumn, from three rivers with different pollution impact was examined: Zletovska River (metals), Kriva River (metals and municipal wastewater), and Bregalnica River (municipal wastewater). Collected data revealed increased relative volume and number of MAs containing hemosiderin under the influence of metals, significant in autumn (p < .05). In chub exposed to metals combined with municipal wastewater, significant increase of lipochrome accumulation in MAs in autumn, melanin in MAs in fish captured in the spring season, and number of splenic MAs containing combination of melanin and lipochrome was noted. Volumes and number of MAs containing combination of hemosiderin and lipochrome increased in spleen of fish captured in autumn from both Zletovska River and Kriva River, most likely due to the contribution of hemosiderin and lipochrome, respectively. Values measured for the various pigments in splenic MAs in fish captured from Bregalnica River, were overall closer to the values measured for fish captured from Kriva River. Notably, melanin and lipochrome are more likely to be found in fish from waters influenced by municipal wastewater (organic pollution) and hemosiderin in fish spleen from water influenced by mining activity (heavy metals pollution).

Although there are common and well-established bioindicator organisms and tissues, there is still a need for reliable and sensitive bioindicators in aquatic environments. In the present pilot study, calcified structures in fish were applied as indicators of metal exposure in combination with commonly used fish soft tissues and intestinal parasites, therefore comprising short- and long-term indicators. Patterns of metal accumulation and distribution in soft (muscle, liver) and hard (scales, otoliths) tissues of brown trout (Salmo trutta Linnaeus, 1758) and their intestinal parasites, acanthocephalans (Dentitruncus truttae Sinzar, 1955), from the Krka River influenced by industrial and municipal wastewaters were estimated and compared. Most elements had higher levels in acanthocephalans, scales and liver than muscle and otoliths, possibly reflecting differences in metal uptake routes, tissue function and metabolic activity. Despite the recorded differences in metal contents, all applied bioindicators reflected environmental conditions in a similar way, indicating higher levels of most elements in fish from the contaminated rather than from the reference site. Acanthocephalans were confirmed as sensitive bioindicators due to effective metal accumulation capacity, while the combination of soft and hard tissues provided extended temporal information on metal exposure. Wastewater impact was evidenced as moderate metal pollution by all applied indicators and pointed to present but also long-term disturbances in the Krka River and the importance of continuous monitoring and protective actions.

Considering the fact that water is a basic need of every living being, it is important to ensure its safety. In this work, the data on the presence of the opportunistic pathogen P. aeruginosa in drinking water (n = 4171) as well as in pool water (n = 5059) in Primorje-Gorski Kotar County in Croatia in the five-year period (2016–2020) were analysed. In addition, the national criteria were compared with those of neighboring countries and worldwide. The proportion of P. aeruginosa-positive samples was similar for drinking water (3.9%) and pool water (4.6%). The prevalence of this bacterium was most pronounced in the warmer season. P. aeruginosa-positive drinking water samples were mostly collected during building commissioning, while pool samples were from entertainment and spa/hydromassage pools. Outdoor pools showed a higher percentage of positive samples than indoor pools, as well as the pools filled with freshwater rather than seawater. The highest P. aeruginosa load was found in rehabilitation pools. Croatia, Serbia and Montenegro are countries that have included P. aeruginosa in their national regulations as an indicator of the safety of water for human consumption as well as for bottled water, while Slovenia and Bosnia and Herzegovina have limited this requirement to bottled water only. In the case of swimming pool water, this parameter is mandatory in all countries considered in this study.

Europe is one of the leading tourist destinations where tourism is one of the key economic sectors. The quality of bathing waters is a very important factor when choosing a vacation destination. Croatia recognized this early and was one of the first Mediterranean countries to start systematic monitoring of bathing waters. On the other hand, monitoring of inland bathing waters is relatively new and includes a much smaller number of sites (41) compared to coastal waters (894). The aim of this paper was to summarize and analyze the water quality of inland and coastal bathing sites of Croatia, closer regions (non-EU Member States) and in the EU for the last decade. The share of excellent water quality in EU Member States increased by 10.1% and 6.6% for inland and coastal waters, respectively (2011–2020). Germany recorded the highest proportion of excellent water quality for inland waters (92.2%) and Cyprus for coastal waters (99.3%). Looking at the 10-year average of the proportion of bathing waters with excellent quality, the proportion of coastal bathing sites exceeds that of inland waters by 7.1%. It is clear that additional efforts should be made to improve the management and monitoring of inland waters.

Pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even increase their mortality. The fish kidney is one of the first fish organs to be affected by water contamination. Amounts of kidney macrophages (MACs), which are involved in fish immune response, as well as the qualitative and quantitative changes in the pigmented MACs in fish kidney, are used as biomarkers of pollution. Therefore, in this study, we have evaluated relative and total volumes of trunk kidney pigmented MACs, and analyzed the pigments accumulated within them. Fish were sampled from two mining impacted rivers, Kriva and Zletovska, highly contaminated with heavy metals, and from one reference river, Bregalnica, in spring and autumn of 2012. We have observed that main pigments found in kidney MACs of Vardar chub were melanin and lipofuscin/ceroid, as well as that relative volumes of MACs ranged from 0.56 to 1.68%. Moreover, the results showed that relative volumes of pigmented MACs were higher in metal contaminated rivers, especially in autumn season in the Zletovska River, concurrently with extremely high metal exposure. In addition, condition factors and kidney somatic indices were found significantly lower in the Zletovska River in both seasons, autumn and spring, possibly also as a consequence of high water pollution. Our data confirm that increase in relative volumes of pigmented MACs may serve as warning sign of potential heavy metal pollution in aquatic environment.

Karst rivers are of great interest for commercial fishing and aquaculture, yet they are quite vulnerable aquatic environments because the permeable karst rocks do not effectively filter out contaminants. To understand the current state of karst rivers water quality, we analysed the physico-chemical and microbiological parameters, focusing on antibiotic pollution and the emergence of antibiotic- resistant microbes of three such rivers in Croatia. Water quality varied between classes I and II across sampling sites, and the numbers of total coliforms, enterococci and heterotrophic bacteria varied substantially among sites. Swabs from fish gills, spleen, liver and kidneys were cultured and 94 isolates identified by MALDI-TOF mass spectrometry. The predominant genus was Aeromonas (42.5% of all identified isolates), known for its adaptability to polluted environments and its frequent association with antibiotic resistance. Of the selected Aeromonas isolates known as most pathogenic, half were resistant to at least three antibiotic categories. The Enterobacteriaceae family was represented by the greatest number of genera, most of which are pathogenic for humans and animals and are spoilage bacteria for fish. The results of this study highlight the extent of antibiotic contamination in aquatic environments and the increasing threat of pathogenic and spoilage bacteria in traditionally high-quality karst rivers.

Seasonal sampling of the seawater column and sediment in Adriatic coastal areas affected by various anthropogenic activities, primarily aquaculture, was conducted during 2017. In total, 32 samples from two sites (central and southern Adriatic) were analysed by 16S rRNA amplicon sequencing. This approach was selected to test the possibilities of using metabarcoding in studying marine cyanobacteria, exploring their ecology and potential as an indicator group in anthropologically stressed coastal environments. Additionally, physicochemical water column parameters, sediment granulometry and composition were assessed. Water column revealed a seasonal variation of amplicon sequencing variants (ASVs) closely related to Cyanobium PCC-6307, Prochlorococcus MIT9313 and Synechococcus CC9902, as well as seasonal grouping of physico-chemical parameters in PCA analysis. Sediment analysis uncovered greater community richness of 13 cyanobacterial genera and two uncultured groups. The most abundant in sandy gravels and gravelly sand type of sediments were ASVs closely related to Pleurocapsa PCC-7319 and Xenococcus PCC-7305. Furthermore, identified cyanobacterial ASVs predominantly displayed similarity to isolates from tropical areas (e.g. Neolyngbya, Chroococcidiopsis, Trichodesmium, etc.), which could indicate the tropicalization process already ongoing in the fish fauna of the Adriatic Sea.

During the last years, the report of the occurrence of waterborne disease symptoms related to non-enteric pathogens has increased, without any record of higher levels of indicator bacteria (Escherichia coli and intestinal enterococci). Therefore, the use of current indicators is not always adequate when assessing the overall potential health risk and the inclusion of additional parameters needs to be examined. This paper reports on the incidence and levels of Staphylococcus aureus at 258 locations in Primorje-Gorski Kotar County (Croatia) recorded by official bathing water quality monitoring, as well as supplemental monitoring carried out at the two most frequented beaches in the City of Rijeka. The number of bathers was found to be the main factor affecting S. aureus levels (r = 0.321, p &lt, 0.05). The share of S. aureus positive samples from the official monitoring was significantly lower, when compared to the share of samples from supplemental monitoring (2.2% and 36.3%, respectively, p &lt, 0.01). Besides the number of bathers, one of the main reasons is likely the higher sampling frequency. No correlation was found between S. aureus levels and the indicator bacteria. The results indicate that the determination of S. aureus and increased sampling frequency is recommended for overcrowded beaches.

Many natural freshwater ecosystems, especially in the north eastern Macedonia, are polluted with heavy metals, which are released by active mines. Long-term exposure to high levels of dissolved metals might result in increased metal bioaccumulation in organs of aquatic organisms, and consequently might cause various sub-toxic and toxic effects. The aim of this study was to assess the health of Vardar chub (Squalius vardarensis) inhabiting mining impacted rivers Zletovska and Kriva, in comparison with chub from the reference Bregalnica River. It was done by use of indicators of tissue damage (histopathology of liver and gonads) and general indicators of exposure to environmental stressors (condition factor, organo- somatic indices and external/internal macroscopic lesions). Histological assessment of gonads revealed good reproductive health in all three rivers, indicating high tolerance of gonads to contaminant exposure. Contrary, several external/internal lesions were more pronounced in chub from severely metal contaminated Zletovska River. Prevalence of hepatic lesions was also higher in mining impacted rivers (in Kriva, 70% ; in Zletovska, 59%) compared to Bregalnica River (38%). The spectrum of histological lesions observed in chub liver varied from non-specific minor degenerative conditions, such as lymphocyte infiltration, fibrosis, parasites, granulomas and lipidosis, to extensive and/or more severe changes such as bile duct proliferation, necrosis, megalocytosis, light-dark hepatocytes and hepatocytes regeneration. The results of histopathological investigation for all three rivers showed clear signs of water contamination, especially prominent in mining influenced rivers. More research efforts should be devoted to study of environmental conditions and metal contamination in the mining impacted rivers worldwide, especially of their effects on health of local ichtyofauna.

This research presents a comprehensive study of bacterial assemblages within the water column and in the surface sediments in the zone of two European sea bass cage farms. By the application of the high-throughput amplicon sequencing of 16S rRNA gene, and further implementing microbial ecology tools, a bacterial segment from cage culturing systems and their respective controls were analyzed, with special reference to potential impact on animal, human and environmental health. Samples of seawater and sediments were collected seasonally, at locations situated in the central and southern Adriatic Sea. Bacterial composition was significantly different in the seawater vs. sediment. No significant differences in alpha diversity in sediments were indicated between aquaculture and control sites, and it appears that it is not affected by farming practices. Control sediments have higher relative abundance of aerobic and facultative anaerobic bacteria, while aquaculture sediments are markedly anaerobic. Sediments largely contain functional groups for respiration of sulfate and sulfur compounds, though doubly more in aquaculture sites. Seasonal groupings of bacterial assemblages were confirmed in the seawater, with higher relative abundance of known aquaculture pathogens (except Photobacterium in the winter samples) detected in the winter and summer, opposed to other two seasons. Rare taxa were analyzed in the sediment and in the water column in the search for known fish pathogens, with five genera detected: Vibrio, Pseudomonas, Photobacterium, Tenacibaculum and Mycobacterium. Biomarkers important for the impact of aquaculture on the environment were identified, e.g. Blastopirullela, Sva0081, Suflurovum, Spirochaeta 2, etc., as well as human and fish potential pathogens: Vibrio ichtyoentery, V. harvey, Acinetobacter lwoffi, A. johnsonii, Clostridium perfringens, etc. Chemoheterotrophy has emerged as the dominant functional group in both environments. Regarding priorities for aquaculture microbial management, seawater seems to contain a higher percentage of taxa connected to health-related functional groups.

Freshwater contamination with mining waste can result with high concentrations of toxic metals in the water and in fish organs. In North-Eastern Macedonia, several rivers (e.g., Zletovska, Kriva) are exposed to acid mine drainage from active Pb/Zn mines. Previous studies confirmed high concentrations of dissolved metals in their water. This study was performed in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928) from three Macedonian rivers (Bregalnica, Kriva and Zletovska) in spring and autumn 2012. The aim was to establish if increased exposure to certain metals have resulted with their increased bioaccumulation. The concentrations of 19 elements were measured in cytosolic tissue fractions, to obtain information on metabolically available metal species. The following ranges of cytosolic concentrations of highly toxic elements were measured in the Vardar chub liver (in μg/L): Cd, 1.18–184; Cs, 0.25–25.4; Tl, 0.02–5.80; Pb, 0.70–61.1. Their ranges measured in the gills (in μg/L) were the following: Cd, 0.24–59.2; Cs, 0.39–24.4; Tl, 0.01–1.00; Pb, 0.65–87.2. Although the water of the mining impacted Zletovska River was highly contaminated with several essential metals, especially with Mn and Zn, the majority of essential elements (Na, K, Ca, Mg, Co, Cu, Fe, Mn, Mo, and Zn) did not reflect the exposure level. In contrast, seven nonessential elements reflected the level of exposure in the water. Significantly increased hepatic and gill concentrations of Cs, Rb, Sr, and Tl were detected in Vardar chub from the Zletovska River compared to the other two rivers, of Cd and Pb in the Zletovska and Kriva River compared to Bregalnica, and of V in the Bregalnica River compared to Zletovska and Kriva rivers. Observed significant metal bioaccumulation, in particular of highly toxic elements, as a consequence of exposure to water contaminated with mining waste points to necessity of intensified supervision of mining impacted rivers.

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) were studied in the period from April 2015 to May 2016 at two sampling sites on Croatian river Krka, to establish if river water contamination with metals/metalloids downstream of Knin town has influenced metal bioaccumulation in S. trutta liver. Differences were observed between two sites, with higher concentrations of several elements (Ag, As, Ca, Co, Na, Se, Sr, V) found downstream of Knin town, whereas few others (Cd, Cs, Mo, Tl) were, unexpectedly, increased at the Krka River spring. However, total metal/metalloid concentrations in the liver of S. trutta from both sites of the Krka River were still mainly below previously reported levels for pristine freshwaters worldwide. The analysis of seasonal changes of metal/metalloid concentrations in S. trutta liver and their association with fish sex and size mostly indicated their independence of fish physiology, making them good indicators of water contamination and exposure level. Metal/metalloid concentrations in the metabolically available hepatic cytosolic fractions reported in this study are the first data of that kind for S. trutta liver, and the majority of analyzed elements were present in the cytosol in the quantity higher than 50% of their total concentrations, thus indicating their possible availability for toxic effects. However, the special attention should be directed to As, Cd, Cs, and Tl, which under the conditions of increased exposure tended to accumulate more within the cytosol. Although metal/metalloid concentrations in S. trutta liver were still rather low, monitoring of the Krka River water quality and of the health status of its biota is essential due to a trend of higher metal/metalloid bioaccumulation downstream of Knin town, especially taking into consideration the proximity of National Park Krka and the need for its conservation.

Exceptional natural value of karst rivers, as well as their potential as a source of drinking water supply, contributes to the importance of developing strict environmental protection. Although most of its watercourse is proclaimed national park, Krka River is impacted by technological and municipal wastewaters, which are released without proper purification only 2 km upstream of the park border. In order to assess water ecological status of the Krka River, vulnerability of karst ecosystem and potential threat to the Krka National Park, spatial and temporal variability of physico-chemical and microbiological water parameters, as well as concentrations of 25 total dissolved metals/metalloids were evaluated. All parameters indicated deteriorated water quality near the wastewater influences, which was for certain metals and bacterial counts even comparable to other world rivers of technological/rural catchment. Downstream location pointed to purification processes in karst river, although nutrients and conductivity were still in a range below good water quality and Al, Co, Fe, Li, Mn, Ni, Sr, Ti, and Zn levels remained comparable to their levels in the pollution impacted area, especially in autumn, indicating season-dependent processes. Presented data provided background status and identified pollution influences in the karst riverine system which indicated potential risk for protected area and a need for comprehensive water quality assessment.

Pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even increase their mortality. The fish kidney is one of the first fish organs to be affected by water contamination. Amounts of kidney macrophages (MACs), which are involved in fish immune response, as well as the qualitative and quantitative changes in the pigmented MACs in fish kidney, are used as biomarkers of pollution. Therefore, in this study, we have evaluated relative and total volumes of trunk kidney pigmented MACs, and analyzed the pigments accumulated within them. Fish were sampled from two mining impacted rivers, Kriva and Zletovska, highly contaminated with heavy metals, and from one reference river, Bregalnica, in spring and autumn of 2012. We have observed that main pigments found in kidney MACs of Vardar chub were melanin and lipofuscin/ceroid, as well as that relative volumes of MACs ranged from 0.56 to 1.68%. Moreover, the results showed that relative volumes of pigmented MACs were higher in metal contaminated rivers, especially in autumn season in the Zletovska River, concurrently with extremely high metal exposure. In addition, condition factors and kidney somatic indices were found significantly lower in the Zletovska River in both seasons, autumn and spring, possibly also as a consequence of high water pollution. Our data confirm that increase in relative volumes of pigmented MACs may serve as warning sign of potential heavy metal pollution in aquatic environment.

Mine waste is recognized as being one of the most serious threats for freshwater ecosystems, and it still represents one of the greatest environmental concerns in Macedonia. The aim of our investigation was to obtain an in-depth understanding of mining influence on freshwater systems from water contamination to effects on aquatic organisms. In this study, we assessed the impact of active lead (Pb)/zinc (Zn) mines Zletovo and Toranica on the water quality of three rivers in northeastern Macedonia (Bregalnica, Zletovska, and Kriva rivers) based on data collected in spring and autumn of 2012. The Bregalnica River, near Shtip, was characterized mainly by weak contamination with arsenic, barium, iron, molybdenum, titanium, uranium, vanadium, nitrate, and phosphate, as well as critical faecal pollution, which alltogether could be connected to agricultural activities; however, an impact of the mines was not observed. Contrary, both the Zletovska and Kriva rivers showed a clear impact of Pb/Zn mines on water quality. In the Zletovska River, increased concentrations of cadmium (Cd), cobalt, cesium, copper, lithium, manganese (Mn), nickel, rubidium, tin, strontium, thallium, Zn, sulphates, and chlorides were found, especially in autumn (e.g., Cd 2.0 μg L(-1); Mn 2.5 mg L(-1); Zn 1.5 mg L(-1)). In the Kriva River, increased Cd (0.270 μg L(-1)) and Pb (1.85 μg L(-1)) concentrations were found only in spring, possibly due to sediment resuspension during greater water discharge. The selected sampling sites on the Bregalnica, Zletovska, and Kriva rivers were confirmed as being appropriate locations for further studies of mining waste's impact on freshwater ecosystems, the first one as a nonimpacted site and the other two as possible areas of increased exposure of aquatic organisms to metals.

Vardic I., D. Kapetanovic, Z. TeskeredIic, E. TeskeredIic: First Record of Infectious Haematopoietic Necrosis Virus in Rainbow Trout Fry in Croatia. Acta Vet Brno 76, 2007: 65-70. The paper describes the first diagnosis of infectious haematopoietic necrosis virus (IHNV) in Croatia. The viral causative agent was detected in pooled organ samples from the imported rainbow trout fry on the fish farm. Reverse transcriptase - semi-nested polymerase chain reaction (RT- snPCR) was applied directly on the infected tissue for rapid virus detection. After isolation on cell cultures, IHNV isolate was characterised on the basis of the 303 nt region of the glycoprotein gene (Mid-G) sequence. Phylogenetic comparison to North American and European IHNV isolates revealed that this Croatian isolate belongs to the M genogroup, confirming the prediction of the M genogroup origin of European IHNV isolates. The introduction of the virus presents a threat of further spreading of the disease in Croatia, as the infected farm is in a direct contact with the open waters. Rainbow trout, IHNV, RT-snPCR, molecular epidemiology Infectious haematopoietic necrosis (IHN) is a world-wide disease problem in farmed rainbow trout and other cultured salmonid species. The disease is caused by a fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), which belongs to the genus Novirhabdovirus (Fauquet et al. 2001). IHN is typically characterized as an acute viremia with resulting haemorrhage and necrosis of major organ systems, although infected fish may not show clinical signs. Juvenile fish are more susceptible to the disease, and their mortality can reach over 90%, especially in hatcheries where the disease breaks out for the first time (Bootland and Leong 2003). The transmission of IHNV between fish is primarily horizontal, and the major cause of a wide geographical spreading of the virus is the movement of infected eggs and/or fry. IHNV was first identified in western North America, but soon spread to Japan and Europe. Although IHNV has been detected in neighbouring countries, for example Italy in 1987 (Bovo et al. 1987), and Slovenia in 1993 ( Jenaia et al.2002), there were no reported cases in Croatia previously (Orai c and Zrnaic 2005). Fast IHNV spreading and virus establishment among carrier fish require a rapid and reliable diagnostic method. Reverse transcriptase-polymerase chain reaction (RT- PCR), which detects specific parts of the IHNV genome, has been described (W inton and Einer-Jensen 2002) and approved by OIE as a valuable confirmation method for the IHNV diagnosis (OIE 2003). Further molecular characterisation, based on the sequence comparison of the glycoprotein (G) and non-virion (NV) genes from the different IHNV isolates proved to be useful in understanding and explanation of IHNV spreading, evolution and origin (Troyer et al. 2000; Kurath et al. 2003; Troyer and Kurath 2003; Garver et al. 2003). Phylogenetic analysis of partial G gene sequences (Mid-G, 303 nucleotide region coding antigenic determinants) from the North American IHNV isolates revealed 3 major virus genogroups. Those groups are designated U, M and L for the upper, middle and lower portions of IHNV

Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex.

Data on the microbiological quality of the freshwater systems under the anthropogenic influence, such as the Sava River, are of the major importance for the water resource management. Furthermore, analyses of the microbial quality of fish meat provide information of the fish as a valuable food resource from the investigated river basin. The health status of the fish, including dynamics of infection and biodiversity of endoparasites, is important bioindicator of changes in the ecosystem structure and function. For the ecosystem-based approach to the Sava River management, investigations of microbiological quality of the Sava River water and the meat of the European chub as the bioindicator organism, as well as dynamics of infection/biodiversity of intestinal parasites Acanthocephala, were performed. The survey comprised the data collected in periods 2005, 2006 and 2012. Microbiological investigation of water was performed in 2006 and 2012, while microbiological analyses of fish meat and ichthyo-parasitological investigation took place during 2005–2006. A high number of heterotrophic bacteria were recorded during 2006 survey, confirmed by the distinctly higher values of the three faecal indicators (total coliform, E. coli and enterococci), and indicated poor water quality downstream of the cities Zagreb and Velika Gorica, as a result of the municipal sewage outlets. The results from 2012 survey indicated the existence of moderate to critical faecal and organic pollution in all samples. Accumulation of the bacteria in the European chub meat was mainly uniform along the watercourse within standards and limitations for the human consumption. Sampling sites downstream cities of Zagreb and Velika Gorica were characterised with the lower prevalence and abundance of two common species of the chub intestinal acanthocephalan parasites, Pomphorhynchus laevis and Acanthocephalus anguillae. Poor microbiological quality of the water and lower distribution of chub intestinal parasites were related to the anthropogenic influence, downstream of the urban areas. Milačić R, Ščančar J, Paunović M, editors. The Sava River. Berlin, Heidelberg: Springer-Verlag; 2015. p 201-28.

In order to determine an unknown fish population from the Vrana Lake, mitochondrial cytochrome b gene and non-coding nuclear region Cyfun P were investigated. Stabile population of Bulldog rudd, Scardinius dergle Heckel & Kner, the endemic Croatian freshwater fish in the Krka River, was genetically characterized with the same markers in order to compare it with the material from the Vrana Lake. Genetic markers were sequenced and aligned with the similar ones obtained from the GenBank in order to determine taxonomic and phylogenetic position of these two species. A significant discrepancy between nuclear genetic markers of our specimens and the sequence from the GenBank was found. Phylogenetic analysis suggested that the specimens from the Vrana Lake belong to the species S. hesperidicus. Morphometric characteristics, the maximum length and body mass showed new maximum values for both S. dergle and S. hesperidicus.

In the present study, anisakids from: tuna (Thunnus thynnus) fattened in the Croatian farm in middle Adriatic Sea, three different feral fish species caught near tuna farm (Trachurus trachurus, Scomber japonicus and Oblada melanura) and fish marketed in Croatia (T. trachurus) were analysed by morphology and molecular methods. Larvae were identified to the species level by PCR restriction fragment length polymorphism and characterised by sequencing of nuclear (internal transcribed spacer) and mitochondrial (cytochrome c oxidase subunit 2) markers. The results revealed diverse Anisakidae community consisting of: Anisakis pegreffi, Anisakis simplex (s.s.), Anisakis typica and Hysterothylacium aduncum. This is the first report of A. typica in Adriatic Sea, and also the first record of this species in T. thynnus as host in Mediterranean Sea. Molecular identification of H. aduncum found in co-infection with Anisakis larvae type I expands our knowledge of the occurrence of these taxa in the Adriatic Sea. Zoonotic Anisakidae worms found in fish from the Adriatic Sea could represent a risk to acquire parasitic infection/allergies in Croatia.

The study on medium size river Sutla in Croatia indicated considerable water contamination at specific sites during the baseflow period, probably associated to low flow-rate (0.73-68.8 m3 s(-1)), and consequently low dilution capacity of this river. Various aspects of contamination were observed: increased conductivity to 1,000 microS cm(-1), decreased dissolved oxygen level to 50%, 4-5 degrees C increased water temperature, increased concentrations of several dissolved trace elements (e.g., maximal values of Li: 45.4 microg l(-1); Rb: 10.4 microg l(-1); Mo: 20.1 microg l(-1); Cd: 0.31 microg l(-1); Sn: 30.2 microg l(-1); Sb: 11.8 microg l(-1); Pb: 1.18 microg l(-1); Ti: 1.03 microg l(-1); Mn: 261.1 microg l(-1); and Fe: 80.5 microg l(-1)) and macro elements (e.g., maximal values of Na: 107.5 mg l(-1); and K: 17.3 mg l(-1)), as well as moderate or even critical fecal (E. coli: 4,888 MPN/100 ml; total coliforms: 45,307 MPN/100 ml; enterococci: 1,303 MPN/100 ml) and organic pollution (heterotrophic bacteria: 94,000 cfu/ml). Although metal concentrations still have not exceeded the limits considered as hazardous for aquatic life or eventually for human health, the observed prominent increases of both metal concentrations and bacterial counts in the river water should be considered as a warning and incentive to protect the small and medium size rivers from the future deterioration, as recommended by EU Water Framework Directive.

This report describes the first clinical investigation of furunculosis affecting Arctic charr (Salvelinus alpinus), occurred in April 2008 on the salmonid fish farm, Croatia. On this fish farm beside Arctic charr, brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) were also reared but none of these species were affected. Specimens obtained from eight Arctic charr were investigated by culture for the presence of bacteria. Microbiological analysis of these fish revealed the presence of Aeromonas salmonicida in pure culture from examined organs. This represents the first isolation of Aeromonas salmonicida from the Arctic charr and particularly the first report of furunculosis in the Arctic charr in Croatia.

The karyotype and major ribosomal sites as revealed by silver staining and CMA3-fluorescence of Croatian leuciscine cyprinid endemic to Cetina River, Telestes ukliva were studied. The diploid chromosome number was invariably 2n = 50. Karyotype consisted of eight pairs of metacentric, 13 pairs of submetacentric and four pairs subtelocentric chromosomes. The largest chromosome pair of the complement was subtelocentric, which is a characteristic cytotaxonomic marker for all representatives of the cyprinid lineage Leuciscinae. The nucleolar organizer regions (NORs) were detected in the telomeres of two pairs of medium-sized submetacentric chromosomes. Staining with CMA3 revealed four positive signals that corresponded to NOR sites. No heteromorphic sex chromosomes were found. The karyotype pattern of T. ukliva is nearly identical to that found in most other representatives of the Eurasian leuciscine cyprinids, while the multiple NOR phenotype appears to be more derived as opposed to a uniform one NOR-bearing chromosome pair, ubiquitous in this group.

Razvoj akvakulture, pa tako i tova tuna, u posljednjim je desetljećima, povećao zanimanje javnosti za moguće utjecaje na okoliš. Istraživanja utjecaja uzgoja gospodarski važnih ribljih vrsta su brojna, ali ne i u pogledu procjena mikrobioloških utjecaja, a napose ne u kontekstu tova tuna. Cilj ovog istraživanja bio je procijeniti utjecaj tova tuna (Thunnus thynnus) na okoliš na temelju praćenja mikrobioloških pokazatelja kakvoće morske vode na tovilištu tuna u srednjem Jadranu, te ostvarene rezultate usporediti s rezultatima istraživanja na tovilištu tuna u Mediteranu. Kakvoća morske vode analizirana je na dva lokaliteta, pri čemu je jedan lokalitet bio na tovilištu tuna, a drugi na udaljenosti oko 500m od tovilišta i poslužio je kao tzv. kontrolna točka. Uzorkovanje je provedeno u dvije sezone (proljeće/jesen), u 2007. i 2008. godini. Tijekom ovog istraživanja, analizirani su osnovni fizikalno-kemijski (temperatura, salinitet, prozirnost) i mikrobiološki parametri (ukupan broj bakterija, Vibrio, ukupni koliformi, Escherichia coli, fekalni koliformi, enterokoki) kakvoća morske vode. Za mikrobiološku analizu uzorci morske vode serijski su razrijeđeni s PBS otopinom (Merck). Za određivanje ukupnog broja heterotrofnih bakterija, uporabljene su metodom širenja razmaza dvije podloge, Marine agar (BBL) i Trypticase soy agar (BBL) s dodatkom 1% NaCl-a (Kemika), te supstrat metoda s uporabom SimPlate testa (IDEXX), sve u duplikatu. Za određivanje broja ukupnih koliforma i E. coli, odnosno enterokoka uporabljeni su supstrat testovi Colilert (IDEXX), odnosno Enterolert (IDEXX) u duplikatu. Utvrđene vrijednosti broja heterotrofnih bakterija, koje ukazuju na utjecaje tovilišta u vidu eutrofikacije mora, ukupno su manje od vrijednosti utvrđenih u istraživanjima poduzetim na tovilištima tuna u Mediteranu. Broj E. coli i enterokoka nije značajno različit između tovilišta i kontrolne točke, ali su te vrijednosti veće u odnosu na dosadašnja istraživanja na sličnim tovilištima u Mediteranu. Povećani broj enterokoka u uzorcima mora, i oko tovilišta, najvjerojatnija je indirektna posljedica nepravilne tehnologije hranidbe, koja privlači kolonije galebova. Ovi rezultati ukazuju da promjene kakvoće morske vode, a u svezi s tovom tuna, ovise o širem ekološkom kontekstu. Stoga, smještaj i procjenu utjecaja tova na okoliš valja razmatrati u sklopu šireg ekološkog konteksta područja., Aquaculture including tuna fattening, increased public interests in the possible impact on the environment in recent decades. Influences of cultivation of economically important fish species are numerous, but not in regard to assessment of microbial influences, particularly in the context of tuna. The aim of this study was to assess the effects of tuna fattening (Thunnus thynnus) in the sense of the environmental monitoring of microbial indicators of seawater quality at tuna farm in the central Adriatic, and to compare the achieved results with the results of research on a tuna farm in the Mediterranean. The quality of sea water was analyzed at two sites; one site was at the tuna farm, and the other at a distance of about 500m from the farm, the control point. Sampling was conducted in two seasons (spring / autumn), in 2007 and 2008. During this study, the basic physico-chemical (temperature, salinity, transparency) and microbiological parameters of seawater quality (total number of bacteria, Vibrio, total coliforms, Escherichia coli, faecal coliforms, enterococci) were analyzed. For microbiological analysis of seawater; samples were serially diluted with PBS solution (Merck). To determine the total number of heterotrophic bacteria, spread plate method was used on Marine agar (BBL) and Trypticase soy agar (BBL) supplemented with 1% NaCl-a (Kemika), as well as substrate method using SimPlate test (IDEXX), all in duplicate. To determinate total coliforms and E. coli, and enterococci substrate tests Colilert (IDEXX) and Enterolert (IDEXX) were used in duplicate. Number of heterotrophic bacteria, which indicate the effects of the farm on the eutrophication, was less than the number determined in similar study in the Mediterranean. Number of E. coli and enterococci were not significantly different between farm and control point, but these values are higher than those from research in the Mediterranean. Increased numbers of enterococci, on the farm and control point, were indirect consequence of improper nutrition technology, which attracted a colony of seagulls. These results suggest that changes in seawater quality in relation to tuna fattening, depend on the broader ecological context. Therefore, location and environmental impact of tuna fattening on the environment must be considered within the broader context of ecological areas.

Voda kao životni okoliš organizama koji u njoj žive svojom kvalitetom utječe na njihovo zdravstveno stanje. U dostupnoj je literaturi malo podataka o pokazateljima zdravstvenoga stanja klena, osobito iz rijeke Save. Zbog toga je svrha ovog istraživanja bila procijeniti zdravstveno stanje klena kao pokazatelja kvalitete vode, te odrediti odnos histoloških i parazitoloških pokazatelja prema broju bakterija u vodi. Istraživanje je provedeno na ukupno 289 klenova s pet postaja od slovensko–hrvatske granice do utoka rijeke Une u Savu. Uzorci organa klena uzeti su za parazitološku i histološku pretragu. Istodobno je uzorkovana voda i određen broj heterotrofnih i koliformnih bakterija. Razlike u količini bakterija u vodi bile su izražene među pojedinim postajama. Najviša je vrijednost utvrđena na postaji Oborovo, a najniža na postaji Otok Samoborski. Intenzitet histoloških promjena bio je u korelaciji s mikrobiološkom kakvoćom vode. U svim uzorkovanim organima utvrđen je barem jedan histološki pokazatelj u visokoj korelaciji s mikrobiološkom kakvoćom vode. Od parazitarnih invazija samo su ihtioftirijaza i trihodinijaza pokazale visoku negativnu korelaciju s mikrobiološkom kvalitetom vode. U sklopu parazitološke pretrage primijenjivane su i molekularne metode u svrhu identifikacije crijevnih parazita iz grupe Acanthocephala., Water quality has infuence on the health status of the organisms living in it. In the available literature, there is only a few reports on the health status of chub, particularly in the Sava river. The aim of this study was to estimate health status of chub as indicator of water quality. Furthermore we aimed at relating fish histology and parasitology to the number of bacteria in the water. We sampled 289 chubs for parasitological and histological examination. Fish were sampled on five sites from slovenian–croatian border through to the inflow of river Una in Sava. At the same time, water was sampled for the counting of coliforms and heterotrophic bacteria. The difference in the number of bacteria in the water was pronounced at different locations, with Oborovo and Otok Samoborski having the highest and the lowest values respectively. The intensity of histological parameters was in correlation with the number of bacteria in water. At least one histological parameter highly correlating with the water quality was found in every organ. Regarding parasite invasions, only ichthyophthiriasis and trichodiniasis showed high negative correlation with microbiological quality of the water. As part of parasitological examination, molecular methods were used for the identification of Acanthocephala.

In this paper we describe the bacterial community associated with salmonids from the Krka River. Diversity analysis demonstrated that majority of the recovered bacteria were related to Aeromonadaceae group. Bacterial analysis also revealed the presence of Shigella spp. and Pseudomonas fluorescens. Isolation of Aeromonas salmonicida from trout, presents first isolation of this bacteria Croatian rivers.

Od 24. do 27. rujna 2008. u hotelu Donat u Zadru održan je Četvrti hrvatski mikrobiološki kongres s međunarodnim sudjelovanjem. Kongresu je prethodila radionica »Adenoviruses — basic biology to gene therapy«, a organizator obaju skupova bilo je Hrvatsko mikrobiološko društvo.

Rast akvakulturne proizvodnje potiče i povećano zanimanje i brigu za njezin utjecaj na okoliš. Tijekom salmonidne proizvodnje na ribogojilištu u vodu se unose određene količine organske tvari. Količina visokoproteinske hrane i metabolički riblji proizvodi utječu na kakvoću vode koja se koristi u uzgoju riba. Bakterije imaju ključnu ulogu u razgradnji organske tvari. Prekomjerno obogaćivanje nutrientima dovodi do osiromašenja kisikom kroz mikrobiološku oksidaciju organske tvari i takvi uvjeti nepovoljno utječu na ukupnu kakvoću vodenog ekosustava. Cilj ovih istraživanja bio je procijeniti utjecaj salmonidnog uzgajališta na kakvoću vode na osnovi praćenja dinamike bakterijske populacije i fitobentosa u odnosu na fizikalno-kemijske parametre vode (temperatura, kisik, pH itd.). Istraživanja su provedena na pastrvskom ribogojilištu s godišnjom proizvodnjom oko 100 t ribe, kod protoka vode od oko 300 L/s. Uzorci vode za mikrobiološku obradu i uzorci fitobentosa sakupljeni su na 8 lokaliteta uz istovremeno mjerenje fizikalno-kemijskih parametara elektrosondom. Lokaliteti uzorkovanja odabrani su tako da se obuhvate sve uzgojne faze salmonidnog ribnjaka: ulaz u ribnjak, 4 lokaliteta na ribnjaku po pojedinim fazama uzgoja, na izlazu iz ribnjaka, te uzvodno i nizvodno od ribnjaka. Za određivanje ukupnog broja bakterija (cfu) uzorci vode su serijski razrijeđeni sa sterilnom Ringerovom otopinom pH 6,0 (Pliva) i inokulirani metodom razlijevanja po podlozi na mediju za izolaciju sukladno EN ISO 6222:1999. Nakon inkubacije do 5 dana na 22°C, prebrojane su izrasle bakterijske kolonije i rezultati su izraženi za 1 ml vode (cfu/ml). Dobiveni uzorci bentosa stavljeni su u boce i konzervirani s 4 %-tnim formalinom. Mikroskopska obrada uzoraka obavljena je u laboratoriju, pomoću mikroskopa «Opton» povećanja 12,5 x10; 12,5 x 25 i 12,5 x 40. Relativna zastupljenost vrsta fitobentosa određena je po Knöpp-u, od 1 do 7, (1954), a saprobne vrijednosti indikatorskih vrsta po Wegl-u (1983). Indeks saprobnosti na osnovi indikatorskih biljnih vrsta određen je prema Pantle-Buck-u (1955), te na osnovi dobivenih vrijednosti pokazatelja kakvo_e vode, istraživani lokaliteti svrstani su u određenu vrstu, prema Uredbi o klasifikaciji voda (1998). Fizikalno-kemijske karakteristike vode na istraživanim lokalitetima ne pokazuju u pravilu značajnije razlike. Gustoća populacije aerobnih bakterija pokazuje progresivne promjene na lokacijama unutar uzgajališta, a sukladno očitovanju obogaćenja nutrijentima. Tijekom istraživanja u fitobentosu najbrojnija skupina bile su dijatomeje ili Bacillariophyceae, tipični oblici fitobentoske zajednice. Od ukupnog broja utvrđenih vrsta, više od 50% pripadalo je indikatorima saprobnosti. Indikatorske vrste pripadale su oligosaprobnom i betamesosaprobnom stupnju. Vrijednosti izračunatog indeksa saprobnosti bile su 1,7 na lokalitetima prije i nakon izlaska iz ribogojilišta, te 1,8 na izlazu iz bazena II i III, gdje se nalazi konzumna riba. Vrijednosti P-B indeksa saprobnosti upućuju na kakvoću vode istraživanih lokaliteta na I odnosno II vrstu. Ova mikrobiološka i fitocenološka istraživanja upućuju na zaključak da sam proces uzgoja riba nema većeg utjecaja na kakvoću vode. Utvrđeno je da je voda koja izlazi iz ribnjaka bolje kakvoće od vode recipijenta., Aquaculture production is undergoing a rapid expansion including an increasing interest and concern for their potential impact on the environment. Salmon fish farming releases a substantial amount of organic matter in to the water. High protein fish food and fish excretion affecting the quality of water used in fish farming. Bacteria play the major role in decomposition of the organic matter. Excessive enrichment of nutrients leads to oxygen depletion in the oxidation of the organic matter. Such conditions have negative effects on aquatic environment. The aim of this study was to estimate the impact of salmon farming on the water quality, on the basis of bacterial population and phytobenthos monitoring in relation to physico-chemical water characteristics (temperature, dissolved oxygen, pH etc.). The investigation was carried out on a rainbow trout fish farm with an annual trout production of 100 tons, and water flow of 300 l/s. Water samples for microbiological and phytobenthos analyses were collected at eight locations. Physico-chemical parameters were measured by an electrosonde at the same time. Sampling locations were determined to include all stages of salmon production: entry to the fish farm, four locations on the fish farm at different stages of salmon production, outflow from the fish farm, and locations upstream and down stream from the fish farm. Water samples for the heterotrophic bacteria enumeration (cfu) were serially diluted with Ringer solution pH 6.0 (Pliva) and inoculated by spread plate method on the solid medium according to EN ISO 6222:1999. After incubation at 22 °C for five days colonies were counted and results expressed for 1 ml of water (cfu/ml). Samples for benthos analysis were collected in the bottles and conserved with 4 % formaldehyde. Samples were examined under ‘’Opton’’ light microscope with magnifications 12.5x10; 12.5 x 25 and 12.5 x 40 in the laboratory. Relative phytobenthos assemblage determination from 1 to 7 was performed according to Knöpp (1954), and saprobity measures of indicators species according to Wegl (1983). Index of saprobity was determined according to Pantle-Buck (1955). Examined locations were classified according to ‘’Uredba o klasifikaciji voda’’ (1998) on the basis of the obtained water quality values. Physico-chemical water characteristics were without significant differences on examined locations. Density of the heterotrophic bacteria showed progressive changes on the locations within the fish farm which was consistent with nutrient enrichment. The most frequent species in the phytobenthos were diatoms and Bacillariophyceae, which are typical forms of phytobenthic community. More than 50 % of all determined species belonged to saprobity indicators. Indicator species were characteristic for oligosaprobic and betamesosaprobic water. Saprobity indexes were 1.7 at locations upstream and downstream of the fish farm. At the escit from 2nd and 3rd pool, with consumer fish, saprobity indexes were 1.8. These the values of P-B saprobity index indicate first and second degree water quality of the examined locations. The results of these microbiological and phytocenological examinations lead to the conclusion that fish production has insignificant local impact on the water quality.

Information on the bacterial population associated with cultured tuna are scarce, particularly for bluefin tuna (Thunnus thynnus). The aim of this work was to investigate the initial microflora of bluefin tuna in the Adriatic Sea. Four bluefin tunas weighing 6.5 to 15.0 kg were obtained from tuna farm in the Middle Adriatic. Fish samples were taken in spring and autumn from 2003 to 2005. Samples for bacterial isolation were collected from skin, gills, spleen, liver and kidney, and cultured on Tryptic soy agar (BBL) supplemented with 1 % NaCl. Isolated strains were further characterized using the API kits (bio Merieux, France) and identified by the APILAB Plus identification software (bio Merieux, France). Gram-negative bacteria from genera Moraxella, Pasteurella, Brevundimonas, Weeksella, Klebsiella and Pseudomonas dominated the microflora on examined tuna, whereas Staphylococcus was the only one Gram- positive isolated genus.

U dostupnoj literaturi nema podataka o strukturi bakterijske flore kod ličinki i mlađa kalifornijske pastrve u prvim danima života. Zadatak je našeg rada bio pratiti bakterijsku floru od 3. do 8. tjedna života. Tijekom 35 dana pokusa utvrđivana je bakterijska flora kalifornijske pastrve uz praćenje fizikalno–kemijskih parametara kvalitete vode i njezina utjecaja na zdravlje. Uzorci za bakteriološku pretragu uzimani su sa škrga, srca i područja bubrega i inokulirani na podloge. Bakterijske su kolonije pregledane makroskopski, razmazi su obojeni po Gramu, a zatim su načinjeni biokemijski testovi. Pri identifikaciji primjenjivan je APILAB Plus program (bio Mérieux, France). Bakterijska flora ličinki i mlađa kalifornijske pastrve mijenjala se je sa starošću. Fizikalno–kemijske karakteristike vode kretale su se unutar optimalnih vrijednosti. Od inokuliranog materijala najviše je izraslo bakterijskih kolonija s izolata škrga (64,4 %), zatim iz srca (21,8 %) i iz područja bubrega (13,8 %). Bakterijsku floru ličinki u inkubatoru većim su dijelom tvorile gram–pozitivne bakterije (64,7 %), i to vrste: Renibacterium salmoninarum (23,5 %), Lactobacillus spp. (11,8 %), Staphilococcus spp. (17,6 %) i Corynebacterium aquatile (11,8 %). Premještanje ličinki iz inkubatora u bazene reduciralo je bakterijsku floru (–66,7 %) nakon 45 minuta boravka u bazenu. Gram–negativne bakterijske vrste koje su u ličinki u inkubatoru bile zastupljene u niskom postotku (35,3 %), premještanjem ličinki u bazene postale su dominantne i činile su više od 95 % bakterijske flore ličinki, odnosno mlađa kalifornijske pastrve. Flavobacterium, Acinetobacter i Yersinia bili su pretežiti gram–negativni rodovi u ličinki u inkubatoru, dok su Aeromonas, Pseudomonas, Flavobacterium i Pasteurella glavni izolati iz ličinki, odnosno mlađa u bazenima do kraja pokusa. Bakterijska flora ličinki u inkubatoru većinom se sastoji od gram–pozitivne flore, a u ličinaka i mlađa kalifornijske pastrve u bazenima od gram–negativne bakterijske flore., There are no information in available literature about the structure of bacterial flora in rainbow trout larvae and fry in the first days of their lives. The objective of our work has been to follow bacteroflora between the third and the eighth week of their lives. During 35 days of experiment bacteroflora of rainbow trout has been examined, along with following physico–chemical characteristics of water quality as well as it’s influence on health. Samples for bacteriological examination were taken from gill, heart and kidney areas and innoculated on the plates. Bacterial colonies were examined macroscopically, slides with Gram staining, and afterwords biochemical tests were performed. For identification, APILAB Plus programme (bio Mérieux, France) was used. Bacterial population of rainbow trout larvae and fry changed in dependence with their age. Physico–chemical characteristics of water ranged within optimal values. Most of bacterial colonies originated from gill isolates (64,4 %), than from heart (21,8 %) and kidney areas (13,8 %). The bacterial flora of larvae in incubator was composed mostly of Gram–positive bacteria (75,1 %), genera: Renibacterium (25 %), Lactobacillus (16,7 %), Staphilococcus (16,7 %) and Corynebacterium (16,7 %). The transfer of larvae from incubator into the pools resulted in reducing bacterial flora (–66,7 %) after 45 minute stay in the pool. Gram–negative bacteria, which have been represented in larvae in incubator with low percent (24, 9 %), after the transfer of larvae to the pools became dominant and represented more than 95 % of rainbow trout larvae and fry bacterial flora. Flavobacterium, Acinetobacter and Yersinia were the predominant Gram–negative genera in larvae in incubator, whereas Aeromonas, Pseudomonas, Flavobacterium and Pasteurella were the main isolates from rainbow trout larvae and fry until the end of experiment. Bacterial flora of larvae in incubator mostly consists of Gram–positive bacteria, whereas in rainbow trout larvae and fry in pools Gram–negative bacterial flora prevail.