WO2008002152A2 - Process for culturing bacteria of the piscirickettsia genus - Google Patents
Process for culturing bacteria of the piscirickettsia genus Download PDFInfo
- Publication number
- WO2008002152A2 WO2008002152A2 PCT/NO2007/000239 NO2007000239W WO2008002152A2 WO 2008002152 A2 WO2008002152 A2 WO 2008002152A2 NO 2007000239 W NO2007000239 W NO 2007000239W WO 2008002152 A2 WO2008002152 A2 WO 2008002152A2
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- WO
- WIPO (PCT)
- Prior art keywords
- bacterium
- bacteria
- vaccine
- piscirickettsia
- genus
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0233—Rickettsiales, e.g. Anaplasma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
Definitions
- the present invention relates to the identification and culturing of particular isolates of bacteria belonging to the Piscirickettsia genus and to vaccines based on these bacterial isolates.
- a main aspect of the present invention pertains to a process for culturing a bacterium belonging to the Piscirickettsia genus wherein said bacterium is cultured in a substantially extracellular environment.
- a general feature of the process is the selection for bacteria that will reproduce in an extracellular environment.
- the invention provides a process for producing a vaccine comprising one or more steps of culturing a bacterium of the Piscirickettsia genus, wherein the bacterium is cultured in a substantially extracellular environment.
- Another aspect of the invention provides a process for obtaining a bacterium belonging to the Piscirickettsia genus, which in some embodiments comprises the steps of: a) providing a sample from a fish, which is infected with bacteria belonging to the Piscirickettsia genus; b) inoculating into an essentially cell free culture medium bacteria from said sample or dilution series hereof, or bacteria from a spread plate culture established from said sample or dilution series hereof; and c) selecting a bacterium that propagate freely in the medium.
- Yet another main aspect of the invention provides a bacterium of the Piscirickettsia genus, said bacterium having the ability to propagate in a substantially extracellular environment.
- the bacterium may be characterised as being one that is obtainable by the described process for obtaining a bacterium belonging to the Piscirickettsia genus.
- a further important aspect pertains to a vaccine comprising a bacterium according to the invention.
- the vaccine is provided in the form of a feed.
- Additional aspects of the invention relate to a bacterium according to the invention or a subunit hereof for use in veterinary medicine.
- a still further aspect relates to the use of a bacterium according to the invention or a subunit of said bacterium in the manufacture of a medicament for the prevention of infections with bacteria of the Piscirickettsia genus.
- the invention provides a method for prevention of infections in fish with bacteria of the Piscirickettsia genus, said method comprising administering to the fish a vaccine according to the invention.
- Bacteria belonging to the Piscirickettsia genus as represented by Piscirickettsia salmonis, have been identified as the causative agent of Salmonid rickettsial septicemia (SRS), or piscirickettsiosis.
- SRS Salmonid rickettsial septicemia
- the bacteria were believed to be highly fastidious obligate intracellular bacteria in agreement with the finding that they replicate within membrane-bound cytoplasmic vacuoles in tissue culture cells or those of the host fish.
- the present invention is based on the surprising finding that it is possible to obtain isolates of bacteria of the Piscirikettsia genus that are capable of growing in an extracellular environment.
- the invention therefore pertains to a process for culturing a bacterium belonging to the Piscirickettsia genus (Piscirickettsia spp. j, and one of the advantages of the present process is that it comprises one or more steps during which bacteria that are capable of growing in an extracellular environment will be selected.
- a selection pressure may be established by culturing the bacteria without any host cells or cells of non-mammalian origin or with a number of host cells that are considered inadequate to sustain intracellular growth of a sufficient number of bacteria.
- further steps selecting for extracellular bacterial growth may be conducted. These may include harvesting the cultured bacteria from the culture medium directly and under non-lysing conditions, i.e. conditions that are not set to cause lysis of cells of non-bacteral origin.
- non-lysing conditions i.e. conditions that are not set to cause lysis of cells of non-bacteral origin.
- the invention pertains to a process for culturing a bacterium belonging to the Piscirickettsia genus (Piscirickettsia spp.) wherein the 5 bacterium is propagated or cultured in a substantially extracellular environment.
- the term "in a substantially extracellular environment” further refers to a culture of bacteria, wherein at least 10%, such as at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, 99.5 or 100% of the bacteria propagate freely in the medium.
- the bacterium is propagated (cultured) substantially in the absence of cells of non-bacterial origin.
- cells of non-bacterial origin comprise vertebrate cells, such as cells that would otherwise be suitable as host cells for a bacterium belonging to the Piscirickettsia genus.
- the terms "in a substantially extracellular environment” and “substantially in the absence of cells of non-bacterial origin” define a culture of bacteria, wherein bacteria are cultured to a TCID 50 titre of at least 1 x 10 4
- bacteria/ml medium such as at least 1 x 10 4 , at least 5 x 10 4 , at least 1 x 10 5 , at least 5 x 10 5 , at least 1 x 10 6 , at least 5 x 10 6 , at least 1 x 10 7 , at least 5 x 10 7 , at least 1 x 10 8 , least 5 x 10 8 , least 1 x 10 9 , at least 2 x 10 9 , at least 3 x 10 9 , at least 4 x 10 9 , at least 5 x 10 9 , at least 6 x 10 9 , at least 7 x 10 9 , at least 8 x 10 9 , at least 9 x 10 9 , at least 1 x 10 10 , at least 2 x 10 10 , at least 3 x 10 10 , at least 4 x 10 9 , at least 5 x 10 9 , at least 6 x 10 9 , at least 7 x 10 9 , at least 8 x 10 9 , at least 9 x 10
- 25 10 10 or such as at least 5 x 10 10 bacteria/ml medium in the presence of at the most 10 s cells of non-bacterial origin/ml culture medium, such as at the most 2 x 10 5 , such as at the most 3 x 10 5 , at the most 4 x 10 s , at the most 5 x 10 5 , at the most, 6 x 10 s , at the most 7 x 10 5 , at the most 8 x 10 s , at the most 9 x 10 5 , or such as in the presence of at the most 1 x 10 6 cells of non-bacterial origin/ml
- the culture may be established by inoculating a culture with a volume of a stock solution corresponding preferably to 0.25 to 4%, more preferably 0.5 to 2%, most preferably 1% of the of the final volume of medium in said culture and having an optical density of between 2.5 and 3.5, such as an optical density of 2.8 or 2.9.
- the bacteria may be cultured in ventilated spinner flasks, preferably at 30-200 RPM and a temperature of 18.5 to 19.5 0 C for a period of from 1-10 days, such as from 1 to 8 days, such as from 2 to 5 days, such as from 2 to 4 days, such as from 2 to 3 days.
- P.salmonis can be grown in shaker flasks or in static cultures.
- the process is one for culturing a bacterium belonging to the Piscirickettsia genus, wherein a substantial amount of the bacteria are propagated/cultured in an extracellular environment
- An initial step of the process according to the invention may involve establishing a stock suspension of bacteria from a selected bacterial isolate. It is contemplated that the stock suspension may be established by selecting bacteria from primary isolates that are capable of propagating in an extracellular environment.
- the stock suspension may contain bacteria of the Piscirikettsia genus in amounts of 10 5 , 10 6 , 10 7 , 10 s , 10 9 , 10 10 , 10 11 TCID 50 AnI.
- the TCID 50 unit is defined as that dilution of a virus required to infect 50% of a given batch of inoculated cell cultures.
- the stock suspension can be frozen with a cryoprotectant such as DMSO, glycerol or others, and be kept for prolonged times at -80 0 C.
- the process according to the invention thus comprises establishing a stock suspension by a process comprising the steps of: a) providing a sample from a fish, which is infected with bacteria belonging to the Piscirickettsia genus; b) inoculating into a culture medium, which is preferably essentially cell free, bacteria from said sample or dilution series hereof, or bacteria from a spread plate culture established from said sample or dilution series hereof; and c) selecting bacteria that propagate freely in the medium.
- the bacterium belonging to the Piscirickettsia genus is a bacterium that has the ability to introduce the symptoms and disease of piscirickettsiosis in a fish.
- External symptoms of piscirickettsiosis include: dark colour, anorexia and lethargia, erratic swimming due to infections in the brain, skin lesions that can progress to shallow ulcersas well as pale gills resulting from significant anaemia.
- Internal symptoms include swollen and discoloured kidney, an enlarged spleen, ascites in the peritoneum, haemorrhages on the visceral fat, stomach, swim bladder, and body musculature, as well as whitish or yellow, multifocal, coalescing, pyogranulomatous nodules found in the liver.
- Further internal symptoms in the liver include multifocal necrosis of hepatocytes, accompanied by a chronic inflammatory infiltrate of mononuclear cells, vascular and perivascular necrosis and intravascular coagulation resulting in fibrin thrombi within major vessels.
- vascular changes similar to those in the liver may also be observed, and granulomatous inflammation also occurs in the interstitium and parenchyma of the kidney and spleen, respectively. Meningitis, endocarditis, peritonitis, pancreatitis, and branchitis may be observed with accompanying chronic inflammatory and vascular changes similar to those in the liver and haematopoietic organs.
- a presumptive diagnosis of fish which are infected with bacteria belonging to the Pisci ⁇ ckettsia genus, may be by visualization of the causative agent within macrophages or hepatocytes in histological sections or tissue imprints.
- PCR assays can also be conducted directly on isolated tissues (Marshal et a!., 1998, Mauel et al. 1996, both incorporated herein by reference in their entirety), and thus PCR assays on tissues along with the observation of suspect organisms within macrophages or hepatocytes are suitable methods for confirmatory diagnosis.
- P.salmonis will usually appear as whitish streaks on the plate, and may also form single colonies.
- Piscirickettsia spp. can be detected with Giemsa-stained tissue smears, followed by indirect immunofluorescent antibody test (IFAT), polymerase chain reaction (PCR) with relevant primers and/or nucleic acid sequencing for positive identification.
- IFAT indirect immunofluorescent antibody test
- PCR polymerase chain reaction
- Infected fish tissues suitable for examination in IFAT, PCR, tissue imprints and histology are kidney, liver and blood, collected from diseased fish during either overt or covert infections. Due to sensitivity of P. salmonis to antibiotics in vitro, none should be used in media during collection of tissue or the culture of cells.
- the tissues mentioned above provide suitable sources for samples to be used in the process of isolating a bacterium of the Piscirickettsia genus and for establishing a stock suspension of Piscirickettsia spp. according to the present invention. Accordingly, preferred embodiments of the invention pertains to a process, wherein said sample is taken from a body tissue, a body fluid, a secretion or, preferably, from a tissue homogenate. In a further preferred embodiment the tissue homogenate has been clarified by centrifugation or filtration.
- the selected bacterium is further cultured over at least 1, such as at least 2, at least 3, at least 4, at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 passages before a stock suspension is established.
- the present invention pertains to a process of culturing a bacterium which is capable of introducing the symptoms and disease of piscirickettsiosis.
- a bacterium belonging to the Piscirickettsia genus may cause the disease and symptoms of piscirickettsiosis.
- experiments may be conducted in which Atlantic salmon weighing 20-40 grams are challenged intraperitoneally in fresh water with 10-fold dilutions of Piscirickettsia spp. cultures.
- the cultures should have a titre of at least 10 s TCID 50 /ml Piscirickettsia spp.
- the fish should be observed daily for the next five weeks, and should display the above-mentioned symptoms of piscirickettsiosis within this time.
- bacteria of the species Piscirickettsia salmonis have been identified as causative agents of piscirickettsiosis. Accordingly, for the purpose of the present invention it may be preferred that the bacterium cultured in the process of the invention is of the species Piscirickettsia salmonis.
- said bacterium belonging to the Piscirickettsia genus thus has genotypic and/or phenotypic characteristics related to those of any of the bacteria which are deposited in the European Collection of Cell Culture (ECACC) Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 OJG UK on the 9 june 2006 under the following provisional accession numbers: 06050901 (isolate AL10005), 06050902 (isolate AL10007), 06050903 (isolate AL10008) or to those of the bacteria deposited on 21 March 2007 under the accession number 07032110 (isolate ALlOO 14).
- ECACC European Collection of Cell Culture
- ITS internal transcribed spacers
- the ITS are non-coding regions of DNA sequence that separate genes coding for the 16S and 23S ribosomal RNAs.
- rRNA ribosomal RNA
- a preferred embodiment of the invention relates to a process, wherein said bacterium belonging to the Piscirickettsia genus is characterised by having an internal transcribed spacer region which comprises a nucleic acid sequence which is more than 95% identical to the sequence:
- said bacterium belonging to the Piscirickettsia genus is characterised by having an 16S rRNA gene which comprises a nucleic acid sequence which is at least 97% identical to the sequence:
- sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to give the best possible fit, allowing the insertion of gaps or, alternatively, truncation at the ends of the polypeptide sequences or
- sequence identity can be calculated as Nnf , wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences.
- Ndif is the total number of non-identical residues in the two sequences when aligned
- Nref is the number of residues in one of the sequences.
- the percentage of sequence identity between one or more sequences may also be based on alignments using the clustalW software
- nucleotide sequences may be analysed using programme DNASIS Max and the comparison of the sequences may be done at http://www.paraliqn.org/. This service is based on the two comparison algorithms called Smith-Waterman (SW) and ParAlign. The first algorithm was published by Smith and Waterman (1981) and is a well established method that finds the optimal local alignment of two sequences. The other algorithm, ParAlign, is a heuristic method for sequence alignment; details on the method is published in Rognes (2001). Default settings for score matrix and Gap penalties as well as E-values were used.
- SW Smith-Waterman
- ParAlign ParAlign
- said bacterium belonging to the Piscirickettsia genus is characterised by having an internal transcribed spacer region which comprises the sequence of SEQ ID NO: 1.
- the bacterium belonging to the Piscirickettsia genus may be further characterised by having a 16S rRNA gene which comprises a nucleic acid sequence which is at least 95,0% identical to the sequence of SEQ ID. NO. 2, such as at least 95,1%, at least 95,2%, at least 95,3%, at least 95,4%, at least 95,5%, at least 95,6%, such as at least 95,7%, at least 95,8%, at least 95,9%, at least 96%, at least 97%, at least 98%, at least 99%, or such as at least 99.5% identical to the sequence of SEQ ID. NO. 2.
- the 16S rRNA gene comprises a sequence which is identical to that of an isolate of obligate intracellular bacteria of the Piscirickettsia salmonis species. Accordingly, in an embodiment of the process according to the invention, said bacterium belonging to the Piscirickettsia genus may be further characterised by having a 16S rRNA gene, which comprises the sequence
- sequences of the 16S rRNA of a bacterium belonging to the Piscirickettsia genus may be amplified in a nested PCR procedure using primers according to Mauel et al, 1996. These primers amplify a conserved region of the Piscirickettsia salmonis 16S rRNA gene. For amplification of a 283 base pair sequence from the ITS region, primers according to Marshall et al., 1998 are used. Other specific genotypic characteristics comprise a Pulsed Field Gel
- Specific phenotypic characteristics comprise a band pattern similar to that seen with AL 10005 (accession number 06050901) when performing Western blotting with an ant ⁇ -P.salmonis antibody.
- said bacteria belonging to the Piscirickettsia genus is any of the bacteria, which are deposited deposited under the Budapest Treaty with the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 OJG UK on the 9 June 2006 under the following accession numbers: 06050901 (isolate AL10005), 06050902 (isolate AL10007) and 06050903 (isolate AL10008) or the bacteria which are deposited on 21 March 2007 under the accession number 07032110 (isolate AL10014).
- a bacterial stock suspension as described above provides a source of bacteria for the bacterial culture that is eventually established for production of the bacterium belonging to the Piscirickettsia genus.
- this is relevant when production of the bacteria is performed in large scale, such as in industrial scale.
- the bacterium is propagated at a temperature of between 10 and 25 0 C, preferably at a temperature of between 12 and 23 0 C, more preferably of between 15 and 22 0 C, even more preferably of between 17 and 21 0 C, still more preferably of between 18 and 2O 0 C and most preferably at a temperature of between 18.5 and 19.5°C.
- the bacterium is propagated at a pH of between 5.5 and 8.0, more preferably of between 5.7 and 7.0, even more preferably of between 5.9 and 6.8, still more preferably of between 6.1 and 6.6, and most preferably between 6.2 and 6.5.
- Suitable means for controlling the pH include automatic addition of acid or base or Na 2 HCO 3 and CO 2 .
- Various different culture media may be used in the process according to the invention, including many commercially available media.
- the medium contains one or more compounds selected from the group consisting of proteins, amino acids, carbohydrates, fatty acids, lipids and micronutrients.
- the medium is a medium for insect cells
- e er currently [most greferred embodiment the bacterja are ⁇ ultured in Sf-900_ II_SFM_ _ di e edie e l e er e medium as described by Godwin and Whitford, 1993, incorporated herein by e reference in its entirety. Alternatively the Sf-900 III medium may be used. As of i r e - deren e present, both media are commercially available from Invitrogen. i e e i
- the process comprises the steps of: ere e e e er ed
- the bacterial cells are harvested from the production culture after incubation for a period of 2-8 days, such as from 3-7 days or such as from 4-6 days, when an optical density (O.D.
- the invention provides a process, wherein ere r e e r dre id r d rdh *.r e de f ⁇ * r ere i a) said first pre-culture is inoculated with a volume of stock suspension corresponding to 0.1% or more (preferably 0.25 to 4%, more preferably 0.5 to 2%) of the volume of medium in said first pre-culture; and/or b) said second pre-culture is inoculated with a volume of first pre-culture corresponding to 0.1% or more (preferably 0.25 to 4%, more preferably 0.5 to 2%) of medium in said second pre-culture in order to obtain a production culture; and/or c) said production culture is inoculated with a volume of said second pre- culture corresponding to 0.1% or more (preferably 0.25 to 4%, more preferably 0.5 to 2%) of medium volume in said production culture.
- the process of the present invention may be performed by culturing the bacterium by batch fermentation, by fed-batch fermentation as well as by continuous fermentation.
- batch fermentation is preferred, also when using the process of the invention for culturing a bacterium of the Piscirickettsia genus at industrial scale.
- a batch volume of 100 - 5000 litres of medium is contemplated.
- Preferred batch sizes are from 200 - 4000 litres of medium, such as from 300 - 3000 litre of medium, or such as from 450 - 2000 litres of medium.
- the present invention provides a process for producing a vaccine comprising one or more steps of culturing a bacterium of the Piscirickettsia genus, wherein the bacterium is propagated or cultured in a substantially extracellular environment. It is to be understood that the process for producing a vaccine may be characterised by any of the features described above in relation to the process for culturing a bacterium belonging to the Piscirickettsia genus.
- the process for producing a vaccine may further comprise steps of harvesting the bacterium from the culture medium as well as steps of concentrating and/or disrupting, and inactivating the bacterium.
- inactivated bacteria can be attained in general by chemical or by physical means. Chemical inactivation can be carried out by treatment of the bacteria for example, but not limited to, with enzymes, with formaldehyde, beta-propiolactone, or ethyleneimine or derivative thereof, with organic solvents (e.g. Triton or Tween).
- Physiochemical inactivation can advantageously be carried out by subjecting the bacteria to energy-rich radiation, such as UV light, gamma irradiation or X-rays.
- the inactivation agent can be neutralized; for example formaldehyde-inactivated preparations can be neutralized with thiosulphate. If required, the pH is subsequently returned to about 7.
- Inactivation of bacteria by means of physical stress, using e.g. heat or a French Press provides a suitable starting material for the manufacturing of a vaccine.
- a bacterin need not necessarily be in the form of inactivated whole cells; the cells may be disrupted.
- the process may include a step of attenuating the bacterium.
- the bacteria have been attenuated.
- An attenuated strain of a bacteria belonging to the Piscirickettsia genus may be generated for instance by passing the bacteria through culture a number of times, or deleting or mutating a gene involved in a biosynthetic pathway.
- the step for concentrating the bacterium comprises the use of filtration techniques selected from the group consisting of: continuous cross flow filtration, diafiltration, centrifugation, or any combination hereof.
- a concentration factor is obtained that is larger than 0 and less than or equal to 12, such as from 1.2 to 11, from 1.4 to 10, from 1.6 to 9, from 1.8 to 8, from 2 to 7, from 2.25 to 6, from 2.5 to 5, or such as from 2.75 to 4.
- the concentration factor for a suspension of inactivated bacteria belonging to the Piscirickettsia genus is 2-6.
- the concentration factor is calculated from the total amount of antigen suspension fed to the system, divided on the product recovery including hold-up volume in the filtration unit.
- the "hold- up volume” is the volume of concentrate of bacteria belonging to the Piscirickettsia genus remaining in the filtration unit after collection of the concentrate.
- the vaccine may be based for instance on whole bacteria as described above it may according to some embodiments relating to the invention be preferred to develop a vaccine based on subunits of the bacterium of the Piscirickettsia genus. Therefore, in some embodiments, the process comprises one or more steps for isolation and/or purification of a subunit the bacteria.
- subunit refers to any component of the bacteria which alone or in combination with others is capable of eliciting an immune response.
- particularly suitable subunits are those that are capable of eliciting a protective immune response.
- Particular examples of subunits that may be suitable in the present context are lipopolysaccarides, proteins or peptides which are able to elicit an antibody response in fish, mice or rabbits.
- a "protective immune response” refers to an immune response which results in a relative percent survival of vaccinated fish of at least 20.
- the relative percent survival, ⁇ 1- (% mortalities in vaccinated group/% mortalities in control group) ⁇ *100, may be determined in experiments wherein a group of at least 20 fish of a relevant species is immunized intraperitoneally with 0.05 - 0.5 ml of an antigen preparation and compared to a group of fish which have received phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the fish are challenged intraperitoneally after 350-600 degree days with an appropriate amount of bacteria belonging to the Pisdrickettsia genus, such as 1,33 x 10 4 TCID 50 Pisdrickettsia salmonis isolate AL10007.
- the bacteria are titrated by end point dilution by the method of Karber (1931).
- the appropriate dose must be determined for each experiment by doing a pre challenge of the same stock of fish with dilutions of challenge material. The fish are followed for 30-40 days, and the number of mortalities in each group recorded.
- the subunit is isolated or purified in a process comprising one or more steps of chromatography, in particular one or more steps of affinity chromatography.
- publications in the field on how to develop and perform chromatographic purification of the relevant substances In particular the skilled person may consult publications such as "Protein purification methods; a practical approach" Eds.
- a third main aspect of the present invention pertains to a process for obtaining a bacterium belonging to the Pisdrickettsia genus.
- the process involves culturing a bacterium belonging to the Pisdrickettsia genus under conditions that favour bacteria which are capable of growing substantially in the absence of cells of non-bacterial origin as explained above.
- said process for obtaining a bacterium belonging to the Piscirickettsia genus comprises the steps of: a) providing a sample from a fish, which is infected with bacteria belonging to the Piscirickettsia genus; b) inoculating into an essentially cell free culture medium bacteria from said sample or dilution series hereof, or bacteria from a spread plate culture on a Cystein Heart Agar supplemented with 5% sheep blood, established from said sample of step (a) or dilution series hereof; and c) selecting a bacterium that propagate freely in the culture medium.
- the term "essentially cell free culture medium” refers to a culture medium comprising at the most 10 5 cells of non-bacterial origin/ml culture medium, such as at the most 2 x 10 5 , such as at the most 3 x 10 s , at the most 4 x 10 5 , at the most 5 x 10 s , at the most, 6 x 10 s , at the most 7 x 10 s , at the most 8 x 10 5 , at the most 9 x 10 s , or such as in the presence of at the most 1 x 10 6 cells of non-bacterial origin/ml culture medium.
- an "essentially cell free medium” will support growth of a bacterium belonging to the Piscirickettsia genus to the desired titres in a "substantially extracellular environment” and “substantially in the absence of cells of non-bacterial origin” as herein before defined. It is a further characteristic of the process that said sample may be taken from a body tissue, a body fluid, a secretion or from a tissue homogenate.
- a further aspect of the invention pertains to a bacterium of the Piscirickettsia genus, said bacterium having the ability to propagate in a substantially extracellular environment.
- the term "ability to propagate in a substantially extracellular environment” means that the bacteria will grow in a suitable culture medium - as for instance the Sf-900 II SFM medium with no supplements.
- the bacteria will grow to a titre of at least 1 x 10 4 , at least 5 x 10 4 , at least 1 x 10 5 , at leats 5 x 10 s , at least 1 x 10 6 , at least 5 x 10 6 , at least 1 x 10 7 , at least 5 x IfJ 7 , at least 1 x 10 8 , least 5 x 10 8 , least 1 x 10 9 , at least 2 x 10 9 , at least 3 x 10 9 , at least 4 x 10 9 , at least 5 x 10 9 , at least 6 x 10 9 , at least 7 x 10 9 , at least 8 x 10 9 , at least 9 x 10 9 , at least 1 x 10 10 in a culture without cells of non-bacterial origin, established by inoculating a volume of a stock solution corresponding preferably to 0.25 to 4%, more preferably 0.5 to 2%, most preferably 0.1%) of the of the final volume of medium
- the bacteria may be cultured for in ventilated spinner flasks at between 50 and 500 RPM and a temperature of 18.5 to 19.5 0 C for a period of from 1-10 days, such as from 1 to 8 days, such as from 2 to 5 days, such as from 2 to 4 days, such as from 2 to 3 days.
- the bacterium is one that is obtainable by a process as described above.
- the bacterium is obtainable by a process comprising the steps of: a) providing a sample from a fish, which is infected with bacteria belonging to the Piscirickettsia genus; b) inoculating into a culture medium, which is preferably essentially cell free, bacteria from said sample or dilution series hereof, or bacteria from a spread plate culture established from said sample or dilution series hereof; and c) selecting bacteria that propagate freely in the medium.
- the bacterium is one which is in fact obtained by a process as described above.
- the bacterium is one which is capable of introducing the above-described symptoms and disease of piscirickettsiosis, and wherein said bacteria have genotypic and/or phenotypic characteristics related to those of the above mentioned deposited isolates.
- the bacterium belonging to the Piscirickettsia genus is characterised by having an internal transcribed spacer region which comprises a nucleic acid sequence which is more than 95% identical to the sequence of SEQ ID. NO. 1.
- said bacterium belonging to the Piscirickettsia genus is characterised by having an internal transcribed spacer region which comprises a nucleic acid sequence which is at least 95,0% identical to the sequence of SEQ ID. NO.
- said bacterium may be characterised by having an internal transcribed spacer region which comprises the sequence of SEQ ID NO: 1.
- said bacterium belonging to the Piscirickettsia genus is characterised by having a 16S rRNA gene which comprises a nucleic acid sequence which is at least 95,0% identical to the sequence of SEQ ID. NO. 2, such as at least 95,1%, at least 95,2%, at least 95,3%, at least 95,4%, at least 95,5%, at least 95,6%, such as at least 95,7%, at least 95,8%, at least 95,9%, at least 96%, at least 97%, at least 98%, at least 99%, or such as at least 99.5% identical to the sequence of SEQ ID. NO. 2.
- said bacterium may be characterised by having a Comment [MR5]: re er i 16S rRNA gene, which comprises the sequence of SEQ ID NO: 2.
- the e e er e e e i e e de der e re bacterium is any of the representative isolates that have been deposited under the Budapest Treaty with the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 OJG UK on the 9 June 2006 under the following accession numbers: 06050901 (isolate AL10005), 06050902 (isolate AL10007) and 06050903 (isolate AL10008), or the isolate which has
- Yet another main aspect of the invention pertains to a vaccine comprising a bacterium of the invention or a subunit of the bacterium.
- vaccines based on concentrated and/or disrupted and/or inactivated bacteria.
- the vaccine may be based on attenuated bacteria.
- the vaccine comprises an amount of antigen, which is higher than 0.05 mg/ml, such as from 0.05 to 5 mg/ml, from 0.1 to 4.5 mg/ml, from 0.2 to 4 mg/ml, from 0.3 to 3.5 mg/ml, from 0.4 to 3 mg/ml, from 0.4 to 3 mg/ml, from 0.5 to 2.5 mg/ml, from 0.6 to 2 mg/ml, from 0.7 to 1.5 mg/ml, or such as from 0.8 to 1 mg/ml.
- 0.05 mg/ml such as from 0.05 to 5 mg/ml, from 0.1 to 4.5 mg/ml, from 0.2 to 4 mg/ml, from 0.3 to 3.5 mg/ml, from 0.4 to 3 mg/ml, from 0.4 to 3 mg/ml, from 0.5 to 2.5 mg/ml, from 0.6 to 2 mg/ml, from 0.7 to 1.5 mg/ml, or such as from 0.8 to 1 mg/ml.
- the vaccine is in a formulation comprising an adjuvant.
- adjuvants frequently used in fish and shellfish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans, mineral oil and Carbopol ® .
- An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (1996), the content of which is incorporated herein by reference in its entirety.
- the vaccine of the invention may further comprise a suitable pharmaceutical carrier.
- the vaccine is formulated as an emulsion of water in oil.
- the vaccine may also comprise a so-called "vehicle".
- a vehicle is a device to which the antigen adheres, without being covalently bound to it.
- Such vehicles are i.a. biodegradable nano/micro-particles or -capsules of PLGA (poly-lactide-co-glycolic acid), alginate or chitosan, liposomes, niosomes, micelles, multiple emulsions and macrosols, all known in the art.
- ISCOM European patents EP 109.942, EP 180.564 and EP 242.380, the content of which is incorporated herein by reference in its entirety).
- the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Cremophore, Tween® and Span®. Also adjuvants such as interleukin, CpG and glycoproteins may be used. It is to be understood that the vaccine may further be in a formulation comprising an antigen from a bacterial source other than a bacterium of the Piscirickettsia genus, an antigenic material obtained from a viral source, an antigenic material obtained from a parasitical source, and/or an antigenic material obtained from a fungal source.
- suitable surface-active compounds or emulsifiers e.g. Cremophore, Tween® and Span®.
- adjuvants such as interleukin, CpG and glycoproteins may be used. It is to be understood that the vaccine may further be in a formulation comprising an antigen from a bacterial source other than a bacterium of the Piscirickettsia genus, an
- said antigen from a bacterial source other than bacterium of the Piscirickettsias genus is selected from the group consisting of live, attenuated or killed bacteria of the species, but not limiting to, Aeromonas sp, Vibrio sp. Listonella sp., Moritella viscosa, Photobacterium damsela, Flavobacterium sp., Yersinia sp. Renibacterium sp.
- Streptococcus sp. Lactococcus sp., Leuconostoc sp., Bifidobacterium sp., Pediococcus sp., Brevibacterium sp., Edwarsiella sp. Francisella sp. Pseudomonas sp., Cytophaga sp., Nocardia sp., Mycobacerium sp., subunits of these bacteria, and any combination hereof.
- VHSV Viral Hemorrhagic Septicemia Virus
- VHSV nucleoprotein of Viral Hemorrhagic Septicemia Virus
- IHNV Infectious Hematopoietic Necrosis virus
- IPNV inactivated Pancreatc Necrosis Virus
- SVC Spring Viremia of Carp
- CMV Channel Catfish Virus
- viral source is selected from the group consisting of pancreatic disease virus (SPDV), Iridovirus, Infectious Salmon Anaemia virus (ISAV), and heart and skeletal muscle inflammation virus.
- said parasitic source is selected from the group consisting of Lepeophtheirus Sp., Caligus Sp., and Ichthyophthirius Sp.
- the vaccine comprises an antigenic material obtained from a fungal source wherein said fungal source is selected from the group consisting of Saprolegnia Sp., Branchiomyces sanguinis, Branchiomyces demigrans and Icthyophonus hoferi.
- the vaccine comprises antigenic amterial from Vibrio ordalii and Infectious Pancreatic Necrosis virus.
- the vaccine according to the invention may in particular be formulated for administration to a fin fish. More specifically the vaccine may be (formulated) for administration to a telostei.
- the teleostei include, but are not limited to salmonids, basses, breams, cods, snappers, flatfish, catfish, yellowtails and tilapias.
- the vaccine is formulated for administration by a route selected from the group consisting of: Bath, immersion, intraperitoneal injection, intramuscular injection and oral administration.
- the vaccine would be administered to young fish in the fresh-water stage..
- the vaccine is one that is obtainable or one that is actually obtained by a process as described above.
- the present invention provides a feed comprising the vaccine according to the invention, said feed may for example be pelleted or extruded feed.
- a bacterium according to the invention or a subunit hereof for use in veterinary medicine.
- Other aspects pertain to a bacterium according to the invention or a subunit of said bacteria in the manufacture of a medicament for the prevention of infections with bacteria of the Piscirickettsia genus.
- a medicament comprising a bacterium according to the invention or a subunit hereof is relevant for the prevention of infections with Piscirickettsia salmonis or other species of the Piscirickettsia genus, hereunder for the prevention of piscirickettsiosis.
- aspects of the invention pertain to a method for prevention of infections in fish with bacteria of the Piscirickettsia genus, said method comprising administering to the fish a vaccine according to the invention.
- Figure 1 Data from experiment conducted to reveal the ability of P. salmonis to grow on traditional bacterial media.
- Figure 2 Pulsed Field Electrophoresis gel showing a band pattern of the AL 10005 isolate of P. salmonis.
- Figure 4 Cumulative % mortality of fish injected either with PBS or with a vaccine containing inactivated Piscirickettsia salmonis cultured by the method according to the invention.
- Example 1 Isolation of P. salmonis from fish without the use of host cells.
- Liver samples from moribund Atlantic salmon diagnosed with P. salmonis were aseptically removed and homogenised.
- the homogenate was diluted in SF900II cell culturing medium and centrifuged at 2000 x g to remove liver tissue.
- the clarified homogenate was inoculated in a dilution series in SF900II medium and incubated as static cultures at 18 degrees C. Bacterial growth could be observed within a few days.
- the bacterial suspension was identified to be P. salmonis by immunoflourescens microscopy using monoclonal antibodies as described in example 2.
- the bacterial isolate was verified to be P. salmonis using a commercial kit "SRS Fluorotest Directo" from Bios Chile, Chile.
- the kit is based on FITC-conjugated monoclonal antibodies against P salmonis.
- Positive controls were slides with P. salmonis, fixed in methanol/acetone and frozen at -20 0 C.
- Negative controls were slides with Aeromonas salmonicida, fixed in methanol/acetone and frozen at - 2O 0 C. Positive samples were observed as containing spherical fluorescent bacteria when viewed in a fluorescence microscope set at the appropriate wavelength.
- Example 3 Propagation of P. salmonis without host cells.
- Example 3 illustrates multiple passages of P. salmonis in spinner flasks, without the presence of host cells initiated from a frozen stock.
- All the bacterial cultures were grown in SF900II media from Invitrogen with no supplements.
- the cultures were cultivated in ventilated spinner flasks at 75 rpm and 20 degrees C.
- the growth was monitored by OD 600 nm measurement and titration according to Karber, 1931 of the cultures.
- Table 1 clearly demonstrate the ability of P. salmonis to replicate without the presence of host cells. After approximately 6 days post resuscitation the bacteria reaches a typical growth pattern, and was sub cultured every 3-5th day. Maximum OD 600 values of more than 7 were reached within 4-5 days. Table 1. Passage of P. sal in spinner flasks without Sf-9 cells in SF-900 II medium
- Example 5 16S rRNA and ITS gene analysis, Pulsed field Gel Electrophoresis (PFGE) analysis of Piscirickettsia salmonis
- PFGE Pulsed field Gel Electrophoresis
- the PCR products were sent to GATC Biotech for sequencing.
- the sequences were analysed using programme DNASIS Max and the comparison of the sequences were done at http://www.paraliqn.org/This service is based on the two comparison algorithms called Smith-Waterman (SW) and ParAlign.
- SW Smith-Waterman
- ParAlign is a heuristic method for sequence alignment; details on the method is published in Rognes (2001). Default settings for score matrix and Gap penalties as well as E-values were used.
- PFGE pulse field gel electrophoresis
- the plugs were washed two times with purified water for 10 minutes at 50 0 C and two times with TE buffer for 15 minutes at 50 0 C.
- Bam I, Xho I, Xba I, Ecori I, Age I, MIu I, Rsr II, Sma I, NcO I, Sac II, Kpn I, AfI III were used for restriction endonuclease digestion in accordance with the instructions of the manufacturers.
- the fragments were resolved by PFGE in SeaKem Gold agarose (1%) by using the CHEF Mapper XA system (Bio-Rad). The following parameters were used: running time 24 h; temperature, 14 0 C; Voltage gradient, 6.0 V/cm, included angle, 120°; an initial pulse time of 0.1 s and a final pulse time of 15 s.
- the gel was stained with ethidium bromide (O.l ⁇ l/ml) for 30 min, distained in distilled water for 3x 30 minutes and photographed under UV light.
- a photo of the PFGE gel is shown in figure 2.
- Lane 8 and 9 where DNA from AL 10007 is treated with enzymes Rsr I or Sma I displays how a PFGE band pattern from Piscirickettsia sp able to grow without the support of eukaryotic cells may appear.
- the pH was maintained using automatically addition of Sodium Hydroxide or Phosphoric acid.
- Example 7 Detailed description of each step in the production process for Piscirickettsia salmonis without the support of eukaryotic cells.
- a flask containing 50-125 ml medium is inoculated with Working Seed suspension at a ratio of 0.5-2% of medium volume.
- the flask is incubated at a temperature of 19 ⁇ 2 C C for 4-7 days or longer.
- the Optical Density (OD 6 oonm) of the culture at the moment of harvest will be at least 1.
- the culture is transferred aseptically to the seed fermenter.
- a fermenter containing 7-9 litres of medium is inoculated with a suspension of pre-culture no. 1 at a ratio of 0.5-2% of medium volume.
- the culture is incubated at a temperature of 19 ⁇ 2 "C for a period of 2-4 days or longer until the culture has an Optical Density (OD 6 oonm) of at leastl.
- Copolymer solution is added automatically to control foaming.
- the culture is transferred aseptically to the main fermenter.
- a fermenter containing 450 - 2000 litres of medium is inoculated with a suspension of pre-culture no. 2 at a ratio of 0.5-2% of medium volume.
- the culture is incubated at a temperature of 19 +2 0 C.
- samples are taken to ensure that the culture is harvested in the logarithmic phase of the growth curve.
- the Optical Density (OD 60 onm ) of the culture at the moment of harvest will be between 6-14. This value is normally obtained after incubation for 3-6 days.
- P. sal, -04 Inactivation Formaldehyde solution (35 per cent) is added to the culture to a final concentration of 0.2% formaldehyde.
- the inactivant is thoroughly mixed with the bacterial suspension, which is then immediately transferred aseptically to the sterile inactivation tank.
- the culture is kept at a temperature of 19 ⁇ 2 0 C during a period of 90 minutes under continuous agitation.
- the inactivated bacterial suspension is transferred aseptically to the holding tank where the suspension is cooled.
- P.sal.-05 Concentration The inactivated P. salmonis suspension is concentrated by continuous cross flow filtration followed by diafiltration, using microfilters with a pore size of ⁇ 0.2 ⁇ m.
- Example 8 Immunisation of fish, and challenge with Piscirickettsia salmonis.
- Vaccines Piscirickettsia salmonis, isolate AL 10015 was grown in Sf900 II medium to an OD of 12.7 and inactivated with formalin. After inactivation, the antigen was diafiltrated and concentrated against PBS, resulting in an antigen solution with a protein content of 17.9 mg/ml. The antigen was formulated in mineral oil together with inactivated Vibrio ordalii and Infectious Pancreatic Necrosis virus. The Piscirickettsia salmonis antigen dose was 2.2 mg/ml vaccine.
- Groups of 50 fish with an average weight of 34.6 grams were immunized intraperitoneal ⁇ with a vaccine containing inactivated Piscirickettsia salmonis, Vibrio ordalii and Infectious Pancreatic Necrosis virus or phosphate buffered saline (PBS).
- the fish were kept at 12 0 C and challenged intraperitoneally after 45 days with 1,33 x 104 TCID 50 Piscirickettsia salmonis, isolate AL10007. The fish were followed for 42 days, and the number of mortalities in each group recorded.
- Figure 4 shows the cumulative % mortality of fish injected either with PBS or with a vaccine containing inactivated Piscirickettsia salmonis grown as described.
- the antigen was concentrated In the control group, 77.1% of the fish died, while 27.6 % of the fish died in the group vaccinated with a vaccine containing inactivated Piscirickettsia salmonis, Vibrio ordalii and Infectious Pancreatic Necrosis virus.
- This experiment clearly shows that a vaccine produced with Piscirickettsia salmonis produced as described in this patent, mixed with other antigens reduced mortality against and elicits protection against piscirickettsiosis.
- Example 9 Growth of Piscirickettsia salmonis on cystein heart agar supplemented with 5% sheep blood.
- AL 10005 was inoculated onto cystein heart agar supplemented with 5% sheep blood. After one week incubation on 2O 0 C, bacterial growth was observed as whitish lines on the plates.
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CA2656032A CA2656032C (en) | 2006-06-29 | 2007-06-27 | Process for culturing bacteria of the piscirickettsia genus |
GB0823188A GB2452892B (en) | 2006-06-29 | 2007-06-27 | Process for culturing bacteria of the piscirickettsia genus |
NO20090442A NO347865B1 (en) | 2006-06-29 | 2009-01-29 | Method for cultivating bacteria of the Piscirickettsia genus |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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GB2457158A (en) * | 2008-02-08 | 2009-08-12 | Pharmaq As | Polyvalent fish vaccines |
WO2014198913A2 (en) * | 2013-06-14 | 2014-12-18 | Cermaq As | Novel piscirickettsia salmonis isolate |
WO2016082050A1 (en) * | 2014-11-24 | 2016-06-02 | Pontificia Universidad Católica Del Valparaíso | Culture medium allowing the growth of the bacterium piscirickettsia salmonis |
EP3241909A4 (en) * | 2014-12-30 | 2018-06-20 | Universidad De Chile | Pcr-rflp-based method for identifying, and determining the purity of piscirickettsia salmonis |
WO2024003035A1 (en) | 2022-06-27 | 2024-01-04 | Vaxxinova Norway As | Vaccines for piscirickettsiosis (salmonid rickettsial septicaemia) |
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US9993541B2 (en) | 2013-11-13 | 2018-06-12 | University Of Oslo | Outer membrane vesicles and uses thereof |
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Non-Patent Citations (5)
Title |
---|
BIRKBECK T HARRY ET AL: "Growth of Piscirickettsia salmonis to high Titers in insect tissue culture cells" INFECTION AND IMMUNITY, vol. 72, no. 6, June 2004 (2004-06), pages 3693-3694, XP002459547 ISSN: 0019-9567 * |
CVITANICH J D ET AL: "THE ISOLATION OF A RICKETTSIA-LIKE ORGANISM CAUSING DISEASE AND MORTALITY INCHILEAN SALMONIDS AND ITS CONFIRMATION BY KOCH'S POSTULATE" JOURNAL OF FISH DISEASES, OXFORD, GB, vol. 14, no. 2, 1991, pages 121-146, XP009092260 ISSN: 0140-7775 * |
FRYER J L ET AL: "ISOLATION OF A RICKETTSIALES-LIKE ORGANISM FOR DISEASED COHO SALMON (ONCORHYNCHUS KISUTCH) IN CHILE" FISH PATHOLOGY, XX, JP, vol. 25, no. 2, 1990, pages 107-114, XP001206465 * |
FRYER J L ET AL: "PISCIRICKETTSIA SALMONIS GEN. NOV., SP. NOV., THE CAUSATIVE AGENT OF AN EPIZOOTIC DISEASE IN SALMONID FISHES" INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 42, no. 1, January 1992 (1992-01), pages 120-126, XP001029383 ISSN: 0020-7713 * |
FRYER J L ET AL: "Piscirickettsia salmonis: A Gram-negative intracellular bacterial pathogen of fish" JOURNAL OF FISH DISEASES, OXFORD, GB, vol. 26, no. 5, 20 May 2003 (2003-05-20), pages 251-262, XP002253317 ISSN: 0140-7775 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2457158A (en) * | 2008-02-08 | 2009-08-12 | Pharmaq As | Polyvalent fish vaccines |
GB2457158B (en) * | 2008-02-08 | 2013-04-03 | Pharmaq As | Polyvalent vaccine against salmonid alphavirus infections |
WO2014198913A2 (en) * | 2013-06-14 | 2014-12-18 | Cermaq As | Novel piscirickettsia salmonis isolate |
WO2014198913A3 (en) * | 2013-06-14 | 2015-02-26 | Cermaq As | Novel piscirickettsia salmonis isolate |
GB2530210A (en) * | 2013-06-14 | 2016-03-16 | Cermaq As | Novel piscirickettsia salmonis isolate |
GB2530210B (en) * | 2013-06-14 | 2017-12-27 | Cermaq Group As | Novel piscirickettsia salmonis isolate |
WO2016082050A1 (en) * | 2014-11-24 | 2016-06-02 | Pontificia Universidad Católica Del Valparaíso | Culture medium allowing the growth of the bacterium piscirickettsia salmonis |
EP3241909A4 (en) * | 2014-12-30 | 2018-06-20 | Universidad De Chile | Pcr-rflp-based method for identifying, and determining the purity of piscirickettsia salmonis |
WO2024003035A1 (en) | 2022-06-27 | 2024-01-04 | Vaxxinova Norway As | Vaccines for piscirickettsiosis (salmonid rickettsial septicaemia) |
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CA2656032A1 (en) | 2008-01-03 |
CL2007001893A1 (en) | 2008-01-04 |
GB2452892A (en) | 2009-03-18 |
GB0823188D0 (en) | 2009-01-28 |
WO2008002152A3 (en) | 2008-03-06 |
GB2452892B (en) | 2011-10-12 |
CA2656032C (en) | 2017-06-13 |
WO2008002152A8 (en) | 2009-07-23 |
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