J. P. Nielsen

The microflora of rainbow trout intestine : a comparison of traditional and molecular identification

Abstract The culturability of the intestinal microflora of 48 rainbow trout was detected by comparing direct microscopic counts (4′,6-diamidino-2-phenylindole, DAPI) with plate counts (tryptone soya agar, TSA). In general, a high percentage (average 50%) of the microflora could be cultured. The counts of the intestinal microflora varied 3–5 log units between fish within the same sampling point. A total of 504 bacteria were identified by physiologic criteria and 153 strains also by partial sequencing of the 16 S rRNA gene. High agreement was found between classical and molecular identification. The dominant intestinal microflora was identified as bacteria belonging to the gamma subclass of Proteobacteria (of the genera Citrobacter , Aeromonas and Pseudomonas ), to the Gram-positive bacteria with low G+C-content (of the genus Carnobacterium ) and as bacteria belonging to the beta subclass of Proteobacteria . However, the composition of the intestinal microflora showed high variation among three investigated fish farms and also at different time points within one fish farm.

Selection and identification of autochthonous potential probiotic bacteria from turbot larvae ( Scophthalmus maximus ) rearing units

The purpose of this study was to select, identify and characterise bacteria as a disease control measure in the rearing of marine fish larvae (turbot, Scophthalmus maximus). Thirty-four out of 400 marine bacterial strains exhibited in vitro anti-bacterial activity against three fish larval pathogens. Two strains originated from culture collections and thirty two strains were isolated directly from turbot larvae rearing units using a pre-selection procedure to facilitate detection of antagonists. Approximately 8,500 colonies from colony-count plates were replica-plated on agar seeded with Vibrio anguillarum, and 196 of them caused zones of clearing in the V. anguillarum agar layer. Of these, 32 strains exhibited reproducible antibacterial properties in vitro when tested against the fish pathogens V. anguillarum 90-11-287, V. splendidus DMC-1 and a Pseudoalteromonas HQ. Seventeen antagonists were identified as Vibrio spp. and four of twelve tested were lethal to yolk-sac larvae. The 15 remaining strains were identified as Roseobacter spp. based on phenotypic criteria and 16S rDNA gene sequence analysis of two strains representing the two major RAPD groups. Most of the remaining 164 strains selected in the initial replica plating were identified as Vibrionaceae or Pseudoalteromonas. Roseobacter spp. were not lethal to egg yolk sac turbot larvae and in two of three trials, the mortality of larvae decreased (p > 0.001) in treatments where 10(7) cfu/ml Roseobacter sp. strain 27-4 was added, indicating a probiotic potential.

In vitro antagonism of the probiont Pseudomonas fluorescens strain AH2 against Aeromonas salmonicida does not confer protection of salmon against furunculosis

Pseudomonas fluorescens strain AH2 which acts as a probiont in rainbow trout reducing vibriosis-caused mortality has a strong in vitro antagonism against Aeromonas salmonicida. Strain AH2 inhibited the growth of A. salmonicida in defined glucose-casamino acid media in both agar-well-diffusion assays and in broth cultures. The inhibition was significantly enhanced by iron limited conditions as compared to iron surplus conditions (0.1 mM). The possible probiotic activity of strain AH2 against furunculosis in salmon (Salmo salar L.) infected by co-habitant infection (10% or 20% co-habitants each i.p. infected with 104 cfu) with A. salmonicida was investigated with three levels of probiont added to the tank water: (a) 105 cfu/ml added three times every other day, (b) 105 cfu/ml added three times per day, and (c) 106 cfu/ml added twice per day. With a water flow of 0.8 l/kg/min in tanks of 145 l, the levels of probiont were estimated to 103–105 cfu/ml during days of addition in experiments (a) and (b) both with 2 kg fish diluting to <50 cfu/ml overnight. In experiment (c) with 2.5 kg fish, levels of probiont were estimated to 103–106 cfu/ml during day time diluting to <50 cfu/ml overnight. Co-habitants died within 3–5 days after infection and furunculosis appeared in non-co-habitants at day 11 after start of the trial. Accumulated mortality stabilised at approx. 70–80% after 3 weeks. In neither of the trials did the treatment with P. fluorescens result in any effect on furunculosis-related mortality. The difference in in vivo effect in the vibriosis-rainbow trout system as compared with the furunculosis–salmon system shows that the selection and application of probiotic cultures must be tested for each individual host–pathogen combination and any in vivo activity cannot be predicted based on in vitro testing.

Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils

A PCR for the detection of Actinobacillus pleuropneumoniae was evaluated. All of 102 field, isolates of A, pleuropneumoniae reacted in the PCR by amplification of a 985 bp product. No PCR amplification product was observed when examining strains of A. ureae, A. capsulatus, A. hominis, A. equuli, A, rossii, A. suis, Escherichia coli, Bordetella bronchiseptica. Streptococcus suis, Pasteurella haemolytica, Pasteurella multocida, Haemophilus parasuis, Haemophilus taxon Minor group, Haemophilus taxon D/E and haemophilus taxon F. Amplification of a 985 bp product was, however, observed when testing strains of A. lignieresii. The lower detection limit of the PCR test was 10(3) A. pleuropneumoniae CFU/PCR test tube and was not affected by addition of 10(6) E. coli CFU/PCR test tube. Mixed bacterial cultures from tonsils of 101 pigs from 9 different herds were tested by culture and by PCR using four different bacteriological media. While 65% reacted positive in the PCR only 23% were positive by culture, thereby suggesting a superior sensitivity of the PCR test to that of culture. The use of selective media, large inoculum and incubation for 48 h gave the highest number of positive PCR reactions from mixed bacterial cultures. Tonsil cultures from 50 pigs from an A. pleuropneumoniae-negative herd did not react in the PCR. The results show that PCR on mixed bacterial cultures from tonsils may be a highly sensitive method for the detection of A. pleuropneumoniae in pig herds.

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes - evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.