AEROMONAS SPP BACTEREMIA OF RAINBOW TROUT FRY (ONCORHYNCHUS MYKISS): BIOCHEMICAL CHARACTERIZATION OF THE CAUSATIVE ORGANISM AND ANTIMICROBIAL SUSCEPTIBILITY

Aeromonas hydrophila i druge bakterije roda Aeromonas ubikvitarne su u vodenom okolišu i dio su normalne bakteriološke flore mlađi kalifornijske pastrve. Infekcije uzrokovane tim bakterijama registrirane su u mnogim zemljama svijeta. U ovom su radu prikazana naša iskustva i spoznaje s aeromonasnom bakterijemijom, kao uzrokom uginuća mlađi kalifornijske pastrve. U studenome 2003. pastrvska mlađ u dobi od 7 mjeseci počela je iznenada ugibati. U Laboratorij za istraživanje i razvoj akvakulture dostavljeno je 17 uzoraka riba, uginulih i moribundnih pastrvica, koje su odmah i pregledane. Primjenom Api 20 NE testova identificiran je Aeromonas hydrophila / caviae tip I s 99,9 % točnosti (1 test protiv), odnosno Aeromonas hydrophila / caviae tip II s 99,5 % točnosti (1 test protiv) iz uzoraka uzetih iz jetre, slezene, bubrega, crijeva i očnih lezija. Svi izolirani i identificirani sojevi testirani su na osjetljivost prema antibioticima disk difuzijskom metodom. Utvrđena je najveća osjetljivost na flumekvin, a relativno slabija prema kloramfenikolu i enrofloksacinu. Liječenje je uspješno provedeno Flubactinom®.Aeromonas hydrophila and other members of Aeromonas genus are ubiquitus in aquatic environment and make part of normal bacterial flora of rainbow trout. Aeromonas spp. infections are worldwide registered. Here we present our experience and knowledge on Aeromonas bacteremia, which causes mortality of rainbow trout fry. Rainbow trout fry, 7 month old, started to die in November 2003. Fish samples (17 samples) of dead and moribund fish were delivered to the Laboratory for aquaculture. With Api 20 NE tests Aeromonas hydrophila / caviae type I was identified with an average probability of 99.9 % (one test against), as well as Aeromonas hydrophila / caviae type II with an average probability of 99.5 % (one test against) from liver, spleen, kidney, intestines and damaged eye. All of isolated and identified samples were tested for antibiotic susceptibility by disc diffusion method. The test showed that specimens were most sensitive on flumequin, and relatively less sensitive on chloramphenicol and enrofloxacin. Therapy was successfully applied with Flubactin®

AEROMONAS SPP BACTEREMIA OF RAINBOW TROUT FRY (ONCORHYNCHUS MYKISS): BIOCHEMICAL CHARACTERIZATION OF THE CAUSATIVE ORGANISM AND ANTIMICROBIAL SUSCEPTIBILITY

Aeromonas hydrophila i druge bakterije roda Aeromonas ubikvitarne su u vodenom okolišu i dio su normalne bakteriološke flore mlađi kalifornijske pastrve. Infekcije uzrokovane tim bakterijama registrirane su u mnogim zemljama svijeta. U ovom su radu prikazana naša iskustva i spoznaje s aeromonasnom bakterijemijom, kao uzrokom uginuća mlađi kalifornijske pastrve. U studenome 2003. pastrvska mlađ u dobi od 7 mjeseci počela je iznenada ugibati. U Laboratorij za istraživanje i razvoj akvakulture dostavljeno je 17 uzoraka riba, uginulih i moribundnih pastrvica, koje su odmah i pregledane. Primjenom Api 20 NE testova identificiran je Aeromonas hydrophila / caviae tip I s 99,9 % točnosti (1 test protiv), odnosno Aeromonas hydrophila / caviae tip II s 99,5 % točnosti (1 test protiv) iz uzoraka uzetih iz jetre, slezene, bubrega, crijeva i očnih lezija. Svi izolirani i identificirani sojevi testirani su na osjetljivost prema antibioticima disk difuzijskom metodom. Utvrđena je najveća osjetljivost na flumekvin, a relativno slabija prema kloramfenikolu i enrofloksacinu. Liječenje je uspješno provedeno Flubactinom®.Aeromonas hydrophila and other members of Aeromonas genus are ubiquitus in aquatic environment and make part of normal bacterial flora of rainbow trout. Aeromonas spp. infections are worldwide registered. Here we present our experience and knowledge on Aeromonas bacteremia, which causes mortality of rainbow trout fry. Rainbow trout fry, 7 month old, started to die in November 2003. Fish samples (17 samples) of dead and moribund fish were delivered to the Laboratory for aquaculture. With Api 20 NE tests Aeromonas hydrophila / caviae type I was identified with an average probability of 99.9 % (one test against), as well as Aeromonas hydrophila / caviae type II with an average probability of 99.5 % (one test against) from liver, spleen, kidney, intestines and damaged eye. All of isolated and identified samples were tested for antibiotic susceptibility by disc diffusion method. The test showed that specimens were most sensitive on flumequin, and relatively less sensitive on chloramphenicol and enrofloxacin. Therapy was successfully applied with Flubactin®

BACTERIAL FLORA OF RAINBOW TROUT LARVAE AND FRY (ONCORHYNCHUS MYKISS)

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

BACTERIAL FLORA OF RAINBOW TROUT LARVAE AND FRY (ONCORHYNCHUS MYKISS)

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

DETEKCIJA RENIBACTERIUM SALMONINARUM U TKIVU POTOČNE ZLATOVČICE (SALVELINUS FONTINALIS) METODOM »NESTED« RT–PCR

Pathogenic bacterium Renibacterium salmoninarum causes kidney disease with high mortality rate and considerable economic losses in salmonid farming. Thus, application of fast and sensitive method for R. salmoninarum diagnosis is of great importance. This paper describes detection of R. salmoninarum in brook trout tissue with gross clinical signs of disease by nested RT–PCR. Determination of partial sequence of bacterial msa gene was done prior to comparison with similar sequences from different R. salmoninarum isolates. Nested RT–PCR proved to be a rapid and valuable diagnostic tool for R. salmoninarum detection, and sequence analysis confirmed previously reported genetic uniformity of this bacteria.Bakterija Renibacterium salmoninarum uzrokuje bakterijsku bolest bubrega karakteriziranu visokom stopom smrtnosti i značajnim ekonomskim gubitcima u uzgoju salmonidnih riba. Zato je primjena brze i osjetljive metode za detekciju R. salmoninarum veoma važna. U radu je opisan nalaz R. salmoninarum u tkivu potočne zlatovčice s karakterističnim kliničkim znakovima bolesti metodom »nested« RT–PCR. Određen je nukleotidni slijed dijela bakterijskog gena msa, koji je zatim uspoređen sa sličnim sljedovima iz različitih izolata R. salmoninarum. »Nested« RT–PCR pokazao se brzom I korisnom dijagnostičkom metodom u detekciji R. salmoninarum, a analiza nukleotidnoga slijeda msa potvrdila je prije uočenu genetičku jednolikost ovih bakterija