Method for determining pathological tissue damage and diagnosing infectious disease in fish

专利摘要:
The present invention relates to methods for determining pathological tissue damage in fish. Also the present invention relates to methods for diagnosing infectious disease in fish. In particular, the present invention is based on the surprising finding that mixing fish serum with an aqueous protein precipitation solution according to the present invention will result in a protein precipitation reaction if the fish is suffering from pathological tissue damage. Moreover it was surprisingly observed that the degree of turbidity of the precipitate in the mix of sample and aqueous protein precipitation solution was dependent on the state of infectious disease in fish.
公开号:DK201770576A1
申请号:DKP201770576
申请日:2017-07-17
公开日:2017-07-24
发明作者:Peter David Eckersall；Mark James Thomas Braceland；David John Cockerill；John William Tinsley；Ralph Anthony Bickerdike
申请人:Biomar Group As；Marine Harvest (Scotland) Ltd；Univ Glasgow Court；
IPC主号:

专利说明:

Method for determining pathological tissue damage and diagnosing infectious disease in fish
Technical field of the invention
The present invention relates to methods for determining pathological tissue damage in fish. Also the present invention relates to methods for diagnosing infectious disease In fish. In particular, the present invention is based on the surprising finding that mixing serum from fish with an aqueous protein precipitation solution according to the invention will result in a protein precipitation reaction if the fish is suffering from pathological tissue damage. Moreover the degree of turbidity of the precipitate from diseased fish serum is indicative of the severity of the pathological tissue damage and serial samples could be used to determine the progression of disease and pathology in fish.
Background of the invention
Mortality and morbidity caused by infectious disease in e.g. Atlantic salmon,
Saimo safer, remains one of the most challenging issues to the productivity and growth of aquaculture of the species. Reducing the effects of a disease outbreak relies on efficient diagnosis of disease for the implementation of disease management strategies. However, tools for cost effective regular monitoring of fish health are severely lacking in industry.
Aquaculture is the world's fastest growing livestock producing industry with an average annual growth rate of 3,2 percent being observed from 1961 to 2009 (FAO, 2012) and an estimated 154 million tonnes offish being produced in 2011, overtaking production of wild catch fisheries. One of the most economically important ftnfish, in terms of North Western Europe in particular, is Atlantic salmon, Satmo safer. Aquaculture of this species alone has increased from 1990 to 2010 at an average annua! rate exceeding 9.2% (FAO, 2012) and is projected to continue to rise to meet demand with a continually growing giobal population.
However, the salmon farming industry is currently facing a number of sustainability issues which may in the foreseeable future limit growth. These issues include (but are not limited to) fish mea! and oil availability for feed (Tacon and Meitan, 2009) and suitable production sites (Asche et al 2013). In addition, in terms of Sts economic impact, one of the biggest challenges salmon aquaculture faces Is that of infectious disease (Kibenge et al 2012).
Diseases are abnormal conditions which affect the norma) function of cells and or organs within the body of an organism. They can be caused by a variety of factors such as bacterial, viral, parasitic, prions, radiation, toxins, inflammatory and autoimmune conditions and nutritional disorders. Physical trauma caused by an impact can cause pathological changes to tissues and organs as well.
Pancreas disease of Atlantic salmon (Satmo salar) which is caused by infection with salmonid alpha-virus (SAV) is associated with significant pathological damage to tissues, SAV infection leads to pancreatic pathology and marked myopathy affecting the heart and both red and white skeletal muscle. This is a major problem for the production of salmon by aquaculture,
Atlantic salmon aquacuiture relies on the production of an end product which is profitable. In fish culture the main aim, therefore, is to produce flesh, comprised of skeletal muscle and fat, which is desirable in the market place. Quality of fiesh is assessed in a number of ways (Larsson et al,, 2012) and is the result of a number of complex genetic and nutritional processes. Upon harvest of fish the quality of flesh is the major determinant of price and thus profitability, Whilst quality downgrading of fillet can be caused by nutritional factors, toxic myopathies, and muscle breakdown due to excessive exertion (Larsson et a!., 2012), one of the most important causes of skeletal muscle myopathy is infectious disease caused by viruses like (but not limited to) SAV,
Pancreas disease (PD) has been shown to have significant effects on fillet quality even after the fish is ctinicaiiy healthy following recovery from the disease. This is most probably due to chronic effects of pathological tissue damage to skeletal muscie tissue and to pancreas damage resulting in reduced ability to uptake nutrients (Lerfall et al., 2011; Larsson et al., 2012), Whilst there are a number of non-destructive tools to detect its aetiologicai agent, salmonid alpha-virus (SAV), such as identifying presence of genes of the virus by reverse transcriptase polymerase chain reaction or established serology methods to identify antibodies specific to the virus in blood samples, there are not any tissue specific biomarkers currently available to diagnose and quantify associated pathologies. Therefore, a method for diagnosing pathoiogicai tissue damage, such as (but not limited to) skeletal muscle myopathy would have important application In the field of aquacuiture, not only in diagnosing and quantifying the degree of PD-associated muscle myopathy but also pathological tissue damage caused by other diseases or stressors.
Despite various protein precipitation techniques being commonly used in the art in order to e.g, separate proteins from serum, the methods according to the present invention have hitherto not been disclosed in the art.
It is well-known that e,g. sodium acetate trihydrate is commonly used as a buffer in coiorimetric assays for example in an assay for the biood protein ceruloplasmin based on the oxidation of paraphenyienediamine (PPD) substrate which is added to the buffer resulting in colour change being proportional to the amount of ceruloplasmin in a given sample. This methodology has been used in many investigations of mammalian species using sodium acetate trihydrate as buffer; however with no precipitation being reported. Similar methods have also been used in finfish species.
For example, Haluzova et al. (2010) demonstrated that ceruloplasmin activities were increasing after treatment of carps with the pesticide Sparatakus; however, again, with no precipitation of proteins being reported despite the presence of pathological damage in a number of the tested tissues. A similar buffer system has aiso been used with an acetate buffer and sodium azide solution mixture (i.e. with PPD substrate added) in Rainbow trout; however, again, no precipitate formation was observed (Yada et al. 2004).
Within the patent literature the following prior art should be mentioned. WO2014/0411S9A1 discloses inter alia methods for treating, diagnosing, and tracking diseases associated with saimon alphavirus. W02014/0411S9A1 also discloses methods for determining and/or identifying and/or quantifying a PD virus in a sample (e.g., a biological sample such as serum) using reagents containing oligonucleotides encoding the SPDV, There is however no disclosure in W02Q14/Q411S9A1 of precipitation reactions of serum for use in a method for determining/diagnosing skeletal muscle damage caused by e.g. PD in saimon, EP 0 712 926 A2 discloses Infer alia that fish pancreatic disease virus (FPCV) causes PD in Atlantic salmon and furthermore discloses methods of isolating FPCV, e.g. through co-cultivation of infected tissues for the purpose of utilizing FPCV, or proteins or polypeptide derived therefrom as a PD-vaccine or in PD-diagnosis. Moreover, EP 0 712 926 A2 discloses salmon serum treated with e.g, phosphate buffers (e.g, PBS) for various types of antibody testing. There is however no disclosure in EP 0 712 926 A2 of precipitation reactions of serum for use in a method for determining/diagnosing skeletal muscle damage caused by e.g. PD in salmon. US 4,171.204 discloses inter alia a method for analysing blood serum or plasma wherein the blood serum or plasma Is treated with organic solvents such as acetonitrile, propionitriie, tetrahydrofuran or mixtures thereof in order to obtain a precipitate/supernatant of proteins which can be used in the examination for a component of interest. However, US 4.171,204 contains no disclosure offish, salmon, fish diseases such as PD or other muscle tissue related diseases In fish. Moreover, there is no disclosure of precipitation reaction capable of diagnosing skeletal muscle damage caused by e.g. PD in salmon.
Recently a multl-biomarker test of serum from human patients to assess rheumatoid arthritis has been proposed (Centoia et a!., 2013). In this study, however, no acetate buffer, let alone sodium acetate trihydrate buffer, was used to precipitate proteins. In addition, precipitation reactions have been used in the prior art to identify specific antibodies in a biological sample (so-called precipitin tests) which indicate infection of with a given pathogen (Nielsen, 2002).
The use of biological markers to assess fish health can be thought of in two categories. The first of these can be markers of infection by aetioiogicai agents of disease such as, acute phase proteins (Eckersall and Bell, 2010) where host Immune response components are used to give an indication of whether there is an infectious agent present at a given time. These can be extremely useful as non-destructive health monitoring tools and have advantages over specific tests of virus presence and antibody presence commonly used in salmon aquaculture (Adams and Thompson, 2011) by being raised to detectable levels in fish serum much earlier than antibody. Additionally, agents such as acute phase proteins can also be used as markers when the aetioiogicai agent is not known. However, it is widely established that aetiOlogica! agents can be present at a given site for a period of time before any clinical disease is observed or indeed any at ail (Kristofferson et a!., 2009; Jansen et al, 2010; Murray et al,, 2011). This, in terms of disease management strategies, creates a problem as one cannot be certain when a clinical outbreak occurs, using the current diagnostic armoury available, unless histopathoiogy assessment is routinely carried out which In turn reduces the output of fish to harvest and at significant sampling cost. As general biomarkers of pathological tissue damage, such as creatinine kinase, are being used ever more routinely in e.g, salmon aquaculture. Creatinine kinase assays have a number of limitations. For instance, the range of concentrations of creatine kinease in healthy sera due to a high and variabie activity is large. This creates a problem making it hard to identify subtie changes in concentrations on a site to base health management decisions. Thus, more sensitive and tissue specific markers are needed for accurate diagnosis of clinical disease.
Hence, hitherto, the most well-established and reliable way of assessing myopathy in fish has been by histopathology. However, histopathology, compared to other means of assessment, is time consuming, costly, and requires a high ievel of expertise from the practitioner. In fact, histological assessment taken over a time course providing a measure of whether fish are at severe disease state, or whether they are in recovery from diseases such as PD, can take several months to fuiiy resolve from initial infection.
However, despite these drawbacks, histopathology remains the gold standard for diagnosing myopathy in fish due to the limited number of reliable and sensitive alternative means of non-destructive health monitoring available. In light of this, there is a growing interest in identifying biomoiecules that are capable of detecting/determining pathological tissue damage or infection using nondestructive techniques based on e.g, blood derived serum or plasma.
Hence, there is a need in the art of a simple and reliable method of diagnosing whether a fish is infected with microorganisms causing pathological tissue damage and/or a simple and reliable method to determining the state and severity of infectious diseases in fish.
Summary of the invention
The present invention relates to methods for determining pathological tissue damage in fish as weii as methods for diagnosing infectious disease in fish. In particular, the present invention is based on the surprising finding that mixing fish serum with an aqueous protein precipitation solution according to the present invention will result in a protein precipitation reaction if one or more tissues of the fish is/are pathologically damaged. Moreover, method of the present invention if based on the surprising observation that the amount of precipitate formed, e.g. determined as the deg ree of turbidity of the precipitate, was dependent on the state and severity of the infectious disease in fish,
Thus, the serum precipitation reaction of the present invention is based on fish tissue protein released into the blood following infection or trauma of the fish.
In particular, the present invention relates to methods of diagnosing pathological tissue damage in fish, such as pancreatic, cardiac or skeletal muscle tissue damage caused by e.g. pancreas disease (PD), in e.g, Atlantic salmon, salmo salar.
Hence, the method of the present invention couid be used as a rapid and low cost test to indicate the presence and/or severity of infectious disease in fish, In addition, the method of the present invention couid complement existing histological assessment and couid be used as a way to quantify disease severity.
Since a common spectrophotometer and a low cost aqueous protein precipitation solution according to the present inventions can be used to measure the turbidity of the precipitate resulting from the method of the invention, the method could for example enable rapid analysis at fish farm locations.
Moreover, a rapid test kit could be easily and quickly developed by a pharmaceutical or diagnostics company to bring the invention to market. Thus, the method of the present invention could for example be used to provide documentation in the development or field validation of functional feeds or vaccines.
The precipitation reaction on which the present invention is based has e.g, been observed in serum of salmon with Pancreas Disease (PD), Heart and Skefetai Muscle Inflammation (HSMI) as wel! as Infectious Pancreatic Necrosis (IPN).
The method according to the invention relates to the hitherto unknown use of an aqueous protein precipitation solution for precipitating proteins from diseased fish serum in order to determine whether the fish is infected with pathogens that causes tissue damage. The inventors of the present invention have surprisingly found that the addition of serum from fish having pathological tissue damage, e.g, salmon infected with PD, results in a quantifiable precipitation of proteins which is differentlaliy dependant on disease state/severity and the extent of pathological damage, to a number of tissues, such as skeletal muscle.
Thus, a first aspect of the present invention concerns a qualitative test which involves simple mixing of fish serum and an aqueous protein precipitation solution according to the present invention followed by visually observing, e.g. with the naked eye, whether the mixture becomes turbid or remains clear. No visible turbidity occurs in samples from clinically healthy fish.
Hence, in its simplest form; of observable turbidity upon addition of sera from fish with a severe pathologicai state of infectious disease, such as PD positive salmon, to the aqueous protein precipitation solution according to the present invention has promise as a quick, onsite and inexpensive point of care qualitative and quantitative test.
Moreover, although a slight precipitate formation can occur when applying the method of the present invention to heaithy fish serum, it is not easily visible by the naked eye which, in turn, making the distinction between sera and tissue from healthy and diseased fish simple and effective.
Another aspect of the present invention relates to the quantitative monitoring of protein precipitation formation over a period of time, where differentia! precipitation over time has been shown to be an indicator not oniy of pathologicai damage to a given tissue but also to attain information on the extent/severlty of the tissue damage, i.e. an indication of the stage of the disease.
Yet another aspect of the present invention is to provide a precipitate from the mixture of fish serum and an aqueous protein precipitation solution according to the present invention which subsequently after separation from the serum proteins remaining in solution, can be reconstituted in water followed by a protein separation step, e.g. by using electrophoresis, in order to obtain protein band profiles which, in turn, are indicative of what tissues are pathologically damaged.
In addition, the method according to the present invention may be used as an economic and rapid test for assessing the efficacy of protective/functionai feeds, disease trial models and vaccines in fish health research.
More specifically, one aspect of the invention relates to a diagnostic method for determining whether a fish is infected with a pathogen capable of causing pathologicai tissue damage in a fish wherein the method comprises the consecutive steps of: S) mixing fish blood serum with an aqueous protein precipitation solution that causes increased protein precipitation when admixed with serum from tissue damaged fish compared to the corresponding protein precipitation observed in clinically healthy fish, ii) determining whether the fish is infected by establishing : iia) whether visible precipitate is formed, which in the affirmative is indicative of infection in the fish, or sib) whether the resulting mixture has no visible precipitate, which in the affirmative is indicative of clinically healthy fish.
Another aspect of the present invention relates to a diagnostic method for determining the stage of a pathogen causing disease resulting in pathological tissue damage in fish, the method comprising the consecutive steps of: i) successively withdrawing biood samples from fish over a period of time (e.g. WO pc, Wipe, W2pc etc.) ii) mixing the fish blood serum from the blood samples obtained under i) with an aqueous protein precipitation solution that causes increased protein precipitation when admixed with serum from tissue damaged fish compared to the corresponding protein precipitation observed in clinically healthy fish,
Hi) determining the concentrations of the resulting protein precipitation samples from each of the mixtures obtained in step ii) iv) comparing the concentrations of each of the protein precipitation samples and thereby determining the stage of the disease by establishing: iva) whether the precipitation concentration of a sampie (e.g, W2pc) is higher than the concentration of the immediately preceding sampie (e.g. Wipe) which in the affirmative is indicative of progress of disease, or ivb) whether the precipitation concentration of a sample (e.g. W2pc) is essentially equal to the concentration of the immediately preceding sampie (e.g. Wipe) which in the affirmative is indicative of to steady state stage of the disease ivc) whether the precipitation concentration of a sample (e.g. W2pc) is Sower than the concentration of the immediately preceding sample (e.g. Wipe) which in the affirmative is indicative of recovery from the disease.
Yet another aspect of the present invention is to provide a diagnostic method for determining the stage of a pathogen causing disease resulting in pathological tissue damage in fish, the method comprising the consecutive steps of: i) successively withdrawing blood samples from fish over a period of time (e.g. WOpc, Wipe, W2pc etc.) ii) mixing the fish blood serum from each of the blood samples obtained under i) with an aqueous protein precipitation solution that causes increased protein precipitation when admixed with serum from tissue damaged fish compared to the corresponding protein precipitation observed in clinically healthy fish, iii) determining the optical density (level of turbidity) of the sample mixed with aqueous protein precipitation solution, iv) determining the optical density (ievei of turbidity) in a sample mixed with aqueous protein precipitation solution (e.g. W2pc) and in the immediately preceding sample (e.g. Wipe) v) optionally repeat step iv) with further successively obtained samples mixed with aqueous protein precipitation solution (e.g. W3pc, W4pc etc.), vi) determining the change in optical density of the samples mixed with aqueous protein precipitation solution determined in step iv) and optionally in step v) vil) thereby assessing the severity of the infectious disease as said severity is proportional with the degree of change in optical density (i.e. the higher the degree of change in optica! density the more severe is the infectious disease). A further aspect of the present invention is to provide a method for determining and/or screening and/or monitoring pathological tissue damage in a fish, the method comprises the steps of: i) obtaining blood from a fish, ii) mixing serum or plasma from the blood obtained in step (i), with an aqueous protein precipitation solution and obtaining a mixture, iii) visually determining the level of turbidity in the mixture obtained In step 00/ iv) visually comparing the ievei of turbidity in step (iii) with the ievei of turbidity in a reference solution, v) determining pathological tissue damage in said fish if the visual ievei of turbidity is higher in the mixture of step ii) compared to the level of turbidity in the reference solution and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
Another aspect of the present invention is to provide a method for determining and/or screening and/or monitoring pathological tissue damage in a fish, the method comprises the steps of: i) obtaining blood from a fish, it) mixing serum or piasma from the biood obtained in step i), with an aqueous protein precipitation solution, and obtaining a mixture iii) determining the ievei of turbidity in the mixture obtained in step ii) iv) comparing the determined level of turbidity in step iii) with a reference range, v) determining pathological tissue damage in said fish if the determined level turbidity is above the reference-range, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium suiphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium suiphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
Yet an aspect of the present invention is to provide a method for determining the stage and/or severity of pathological tissue damage in fish and/or for monitoring pathological tissue damage in a fish, the method comprising the steps of: i) successively withdrawing blood samples from a fish over a period of time, ii) mixing serum or plasma from the biood obtained in step i) with an aqueous protein precipitation solution, iii) determining the level of turbidity from each of the mixtures obtained in step ii) iv) comparing the ievei of turbidity of each of the mixtures and thereby determining: iva) whether the ievei of precipitation of the mixture is higher than the level of turbidity of the immediateiy preceding mixture which is indicative of progress of pathological tissue damage, or ivb) whether the level of precipitation of the mixture is essentially equal to the Ievei of turbidity of the immediately preceding mixture which is indicative of a steady state stage of the pathoiogical tissue damage, or ivc) whether the level of turbidity of the mixture is tower than the level of turbidity of the immediately preceding sample which is indicative of recovery from the pathological tissue damage, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium suiphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof, A further aspect of the present invention is to provide a kit for (i) determining and/or screening and/or monitoring pathological tissue damage or (ii) determining the stage and/or severity of pathologies! tissue damage in a fish and/or for monitoring pathoiogicai tissue damage in a fish, said kit comprises an aqueous protein precipitation solution, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium suiphate, magnesium suiphate, ammonium phosphate, sodium carbonate, sodium suiphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
Yet another aspect pertains to the use of the method according to the present invention, for developing and/or validating and/or monitoring efficacy of interventions to improve the immune response and/or host response in a fish including but not limited to vaccines, biosecurity practices, diet and combinations thereof.
Brief description of the figures
Figure 1A: Optimization of precipitate assay.(A) Illustrates the effect of pH of buffer of delta OD of muscle lysate over a one hour period at 32Qnm absorbance wavelength. 0,2M sodium acetate trihydrate buffer was used for pH range 3.7 to 5.6 while 0.2M sodium phosphate buffer was used for pH 5,8 - 8,0.
Figure lb: Optimization of precipitate assay, (B) Reference solution (blank) corrected optical density of resulting precipitation reaction over 60 minutes from the mixing of muscle lysate and buffer when using a range of different absorbance wavelength filters.
Figure lc: Optimization of precipitate assay. (C) The kinetics of muscle lysate precipitation reaction when OD is monitored at either 340nm or 550nm absorbance wavelength.
Figure Id: Optimization of precipitate assay. (D) Temperature effect on the formation of precipitate, monitored at 340nm absorbance wavelength using muscie lysate with OD recorded every second.
Figure le: Optimization of precipitate assay. (E) The effect of molarity on the delta OD at 340nm absorbance wavelength during a one hour time period when using W6pc sera and using sodium acetate trihydrate as protein precipitation solution.
Figure 2: Mean serum delta OD of precipitation reaction of serum from fish sampled at nine sample time points during a PD challenge. Bars represent mean f/~ error bars as SEM using microtitre plate assay at 340 nm absorbance wavelength. Pool A is serum sample from clinically health salmon.
Figure 3: Comparison of severity of tissue pathology (mean histopathoiogy score) in heart, read (slow) muscle pancreas or white (fast) muscle (as vertical histogram bars) with the mean deita change in turbidity in sera (straight line graph) according to week post challenge.
Figure 4: Coomassie blue stained 2DE separation of serum precipitate to compare protein profile at (A) W4pc serum (B) WO serum. Lane 1 contains molecular weight markers. Stained bands highlighted in circles represent individual proteins that were differentialiy expressed in W4pc serum compared to WO serum.
Figure 5: Protein precipitation from various organs of clinically healthy salmon tissue iysates. Bovine serum albumin (BSA) was used as a negative control Bars represent mean delta OD at 340nm absorbance wavelength using spectrophotometer method.
Figure 6. Time-course of pathology in pancreas, heart, red and white skeietai muscle in salmon with pancreas disease. Y-axis represents histopathoiogy lesion scores from 0 - 3 as degree of pathology severity for each tissue where 0 represents healthy tissue and 3 is severe histopathoiogy, according to a semi-quantitative scoring system (Table 1). X-axis is the time in weeks post chalienge whereby fish directly infected with SAV were introduced to the population by cohabitation. Histogram bars represent mean histopathoiogy score for pancreas, heart, red muscie then white muscle in the same sequence order for each time point post chalienge. Where no bar exists represents mean histopathoiogy lesion score of zero.
Figure 7: Monitoring of precipitate reaction using a range of aqueous buffers where salmon skeletal muscle lysate is mixed with the buffer/soiution. Y-axis is optical density at 34Gnm absorbance wavelength. X-axis is time in minutes. SAT ~ Sodium acetate trihydrate, SC = Sodium citrate, SR ~ Sodium phosphate, RA -Potassium acetate, SS = Sodium sulphate, AR = Ammonium phosphate, MS ~ Magnesium sulphate, Blank = Water.
Figure 8: Monitoring of precipitate reaction using different concentrations of organic solvent where salmon skeletal muscle lysate is mixed with 10%, 50%, 75%, or 100% ethanol compared to reference solution (blank) with no ethanol. Y-axis is optica! density at 340nm absorbance wavelength. X-axis is time In minutes.
Figure 9: Monitoring of precipitate reaction where salmon skeletal muscie iysate is mixed with aqueous ammonium sulphate (AS) at different ionic strengths; 0.2M, 0.5M, 0.75M, or 1M. Y-axis is optica! density at 340nm absorbance wavelength. X-axis is time in minutes.
Figure 10: Monitoring of precipitate reaction where salmon skeletal muscle lysate is mixed with polyethylene glycol (REG) at different molecular weights and concentration in Molarity. Y-axis is optical density at 340nm absorbance wavelength. X-axis is time in minutes. PEG1 represents l,0Q0Mw PEG and PEG2 represents an 8,000Mw agent, both at 0.01M or 0.05M concentration.
Figure 11: Differential precipitation in salmon serum of healthy (PLA) or with pancreas disease (W4) when using different aqueous buffers. The Y-axis is the difference in delta OD at 340nm absorbance wavelength for W4 compared to PLA sera for the same buffer. SAT = Sodium acetate trihydrate, SC - Sodium citrate, SP - Sodium phosphate, PA = Potassium acetate, SS == Sodium sulphate, Sea = Sodium Carbonate, AP = Ammonium phosphate, MS = Magnesium suiphate, AS 1M = Ammonium Suiphate 1M,
Figure 12: Difference in precipitation between salmon sera of heaithy (PLA) or with pancreas disease (W4) using three different buffers over 1 hour. The Y-axis is optica! density at 340nm absorbance wavelength. X-axis is time in minutes. Each fine represents a different combination of sera and buffer where SAT - Sodium acetate trihydrate, SC = Sodium Citrate, PA = Potassium acetate.
Figure 13: A box plot distribution of SPR OD in serum from healthy salmon (n~364) where the median is the centre line, 25-75th percentile are in the box, whiskers represent the l0-90th percentiles and dots are outliers .
Figure 14: Qualitative change in turbidity when sera from healthy (A) and diseased (B) A. salmon are added to 0.6M SAT buffer. Corresponding delta OD is also shown in figure 14C.
Figure 15: Boxplots of initial absorbance (A340) rSPR and the Δ340 for average for SPR in salmon following commercial vaccination.
Figure 16: Delta optical density of Atlantic salmon serum and plasma. Bars represent mean delta OD at 340nm (0.6M SAT buffer pH 5.6) 24 hours post separation. Error bars represent standard error of the mean.
Figure 17: Delta optical density of Atlantic salmon serum and piasma, Bars represent mean delta OD at 340nn (0.6M SAT buffer pH 5,6)48 hours post separation. Error bars represent standard error of the mean.
Figure 18 Delta optical density of Atlantic salmon serum after 72 hours (post separation) of being stored at 4«C or at room temperature. Bars represent mean delta OD at 340nm (0.6M SAT buffer pH 5.6). Error bars represent standard error of the mean.
Figure 19: Delta optical density of Atlantic salmon serum read at 96 hours post separation after storage at 4< or subjection to -80C and two freeze thaw cycles. Bars represent mean delta OD at 340nm (Q.6M SAT buffer pH 5.6) Error bars represent standard error of the mean.
Figure 20: Delta optical density of Atlantic salmon serum read at 96 hours post separation after storage at 4»C or subjection to ~8Q*C and four freeze thaw cycles. Bars represent mean delta optical density at 34Qnm (0.6M SAT buffer pH S.6).Error bars represent standard error of the mean.
Figure 21: Delta optical density of Atlantic salmon sera following challenge with Moritella Viscosa), Bars represent mean delta OD at 340nm pre~ challenge (n-30) or post-challenge (n=2G) (0.6M SAT buffer pH 5.6), Error bars represent standard error of the mean.
Figure 22: Delta optical density of Atlantic salmon serum during pancreas disease using 0.6M SAT buffer pH 5.6. Error bars represent standard error of the mean. Diet A (control) is indicated in black and diet B (functional feed) with grey line.
Figure 23: Delta optical density of sera from healthy and enteric red mouth (ERM) diseased rainbow trout. Bars represent mean delta OD at 34Gnm (0.6N SAT buffer pH 5.6). Error bars indicate standard error of the mean for these two groups using microtitre plate assay at 340 nm. * = significant difference to the control (t-test P *=< 0.0001).
Figure 24: Box plot distribution of SPR in serum from healthy sea bass (n=ll) and rainbow trout (n~26) where the median is the centre line, 25~75th percentile are in the box, whiskers represent the 10-90th percentiles and dots are outliers.
Figure 25: The delta optical density of control (NT CP), infected (NT IP), and moribund (NT MR) nile tllapia serum pools from a Streptococcus agalactiae challenge trial when introduced to 0.6M sodium acetate trihydrate buffer at a range of pH or a 2M sodium acetate trihydrate buffer at pH 4.8,
Figure 26: The resulting level of turbidity when pooled sera from control (NT CP), infected (NT IP) or moribund (NT MP) were added to G.6M sodium acetate trihydrate buffer at pH 3.8.
Figure 27: The delta optical density of control, infected,and moribund nile tilapia serum pools from a Streptococcus agalactiae challenge trial. Error bars represent standard error of the mean (SAT 0.6M pH 3,8).
Figure 28: Effect of altering serum volume on delta optical density at 340nm wavelength, where different volumes of serum from Atlantic salmon with pancreas disease were mixed with 230pl of 0.6M sodium acetate trihydrate pH 5.6.
The present invention will now be described in more detail in the following. Detailed description of the invention
The inventors of the present application surprisingly discovered that where serum of e.g, Atlantic saimon in a state of clinical Pancreas Disease (PD) was found to form a protein precipitate when mixed with sodium acetate trihydrate buffer, The resulting turbidity of the sampie can be measured and quantified by for example spectrophotometry or other quantitative measuring techniques known in the art, Also surprisingly, the inventors of the present application observed that the extent of the turbidity correlated with the severity of e,g. PD related histopathoiogy thereby providing an effective indicator of pathological tissue damage.
In addition/ it has been observed that the precipitation reaction is much more pronounced when the fish, e.g, a saimon, is infected by a pathogen causing pathological tissue damage, e.g. SAV the causative agent of PD.
The precipitation reaction on which the present invention is based has also been observed in serum of salmon with e.g. clinical Heart and Skeletal Muscie Inflammation (HSMI) as well as Infectious Pancreatic Necrosis (IPN), which is not associated with skeletal muscie pathology, so the method of the invention is also applicable as a general indicator of severe pathological state due to infectious disease and/or immune reaction during acute and chronic diseases caused by viruses.
Despite various protein precipitation techniques being commonly used in the art in order to e.g. separate proteins from serum or plasma, the method according to the present invention has hitherto not been disclosed in the art.
It is well-known that e.g. sodium acetate trihydrate is commonly used as a buffer in colorimetric assays for example in an assay for the blood protein ceruloplasmin based on the oxidation of paraphenyienediamine (PPD) substrate which is added to the buffer resulting in colour change being proportional to the amount of ceruloplasmin in a given sampie. This methodoiogy has been used in many investigations of mammalian species using sodium acetate trihydrate as buffer; however with no precipitation being reported (Maiac2ewska et a!. 2009; Ceron and Martinez-Subiela, 2004).
The serum or plasma precipitation reaction of the present invention is based on tissue proteins being released into the biood foliowing e.g. infection of the fish.
Hence, the methods of the present invention could be used as a rapid and low cost test to determine and/or screen and/or monitor pathologicai tissue damage in a fish or a population of fish. In addition, the methods of the present invention could complement existing histoiogical assessment and could be used as a way to quantify disease severity.
Since a common spectrophotometer and a low cost aqueous protein precipitation solution according to the present inventions can be used to measure the turbidity of the precipitate resulting from the method of the invention, the method couid for exampie enable rapid analysis at farm locations or at the veterinary clinic.
The precipitation reaction on which the present invention is based has e.g, been observed in serum or plasma from fish (please see the Examples).
The methods according to the invention relates to the hitherto unknown use of an aqueous protein precipitation solution for precipitating proteins present in serum or plasma from a diseased fish or a representative sample of a population of fish in order to determine whether the fish or a population of fish are e.g. infected with pathogens causing pathoiogicat tissue damage.
The inventors of the present invention have surprisingly found that the addition of an aqueous protein precipitation solution to serum or plasma from a fish having pathological tissue damage, results in a quantifiable precipitation of proteins which is differentially dependant on disease state/severity and the extent of pathological damage.
Hence, in its simplest form; of observable turbidity upon addition of serum or plasma from a fish with a severe pathological state of Infectious disease to the aqueous protein precipitation solution according to the present invention has promise as a quick, onsite and inexpensive point of care qualitative and quantitative test.
Another aspect of the present invention relates to the quantitative monitoring of protein precipitation formation over a period of time, where differentia! precipitation over time has been shown to be an indicator not oniy of pathologicai damage to a given tissue but also to attain information on the extent/severity of the tissue damage, i.e. an indication of the stage of the disease.
Yet another aspect of the present invention is to provide a precipitate from the mixture of fish serum or plasma and an aqueous protein precipitation solution according to the present invention which subsequently after separation from the serum proteins or plasma proteins remaining in solution, can be reconstituted in water followed by a protein separation step, e.g. by using electrophoresis, in order to obtain protein band profiles which, in turn, are indicative of what tissues are pathologically damaged.
The resulting precipitate (which provides turbidity of the mixture) can be measured and quantified by e.g. spectrophotometry or other quantitative measuring techniques known in the art. Turbidity is the result of precipitated proteins - thus, the ievel of turbidity correlates with the amount of precipitated proteins and increases if the amount of precipitated protein increases.
Also surprisingly, the inventors of the present invention observed that the extent of the turbidity correlated with the severity of e.g. infections related to histopathology, thereby providing an effective indicator of pathological tissue damage.
In addition, it has aiso been observed that the proteins (i) precipitate quickly and (ii) the precipitation reaction is more pronounced (increased turbidity) when pathological tissue damage is present in one or more intestinal organs of the fish.
In the following the terms "diagnosing pathological tissue damage" and the term "determining pathological tissue damage" are used herein interchangeably.
The methods of the present invention
The present invention relates to methods of determining pathologicai tissue damage in a fish or a population of fish.
As mentioned previously an aspect of the present invention pertains to the provision of a method for determining and/or screening and/or monitoring pathological tissue damage in a fish, the method comprises the steps of: i) obtaining blood from a fish, is) mixing serum or plasma from the blood obtained in step (i), with an aqueous protein precipitation solution and obtaining a mixture, iii) visually determining the level of turbidity in the mixture obtained in step (»)/ iv) visually comparing the level of turbidity in step (iii) with the level of turbidity in a reference solution, v) determining pathological tissue damage in said fish if the visual ievei of turbidity is higher in the mixture of step ii) compared to the level of turbidity in the reference solution and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium suiphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
The reference solution may be the aqueous protein precipitation solution or it may be e.g. water or any suitable buffer solution.
Compared to the methods known in the art the method of the present invention aliows for an on-site diagnosis of pathologicai tissue damage in a fish or a population offish. An on-site diagnosis saves time and allows the farmer or veterinarian to react quickly, either by initiating a treatment or by performing further tests (e.g. to identify the origin of the pathofogical tissue damage or to identify the tissue(s) affected). In an embodiment of the present invention, the precipitated proteins may be further anaiysed by methods known in the art (e.g. by electrophoresis or immunoassay).
If a visible precipitate is formed after mixing serum or piasma with an aqueous protein precipitation solution, this indicates that the fish suffers from pathologicai tissue damage.
If on the other hand, if no visible precipitate is formed after mixing serum or plasma and an aqueous protein precipitation solution, this affirm that the fish does not suffer from pathological tissue damage, Other test may be needed to confirm that the fish is in fact clinically healthy since not all diseases causes pathological tissue damage.
In one embodiment of the present invention the determination of visible precipitate is determined by the naked eye. Such a simple determination is indeed surprising and makes the method of the present invention highly commercially relevant, since it makes the farmer, veterinarian or health professional, by use of very simple equipment, capable of testing an individual fish or representative sample of a population on site and determining pathological tissue damage, in a quick but yet reliable way.
The ieve! of turbidity can be measured by use of e.g, a spectrophotometer at different wavelengths. In a preferred embodiment of the level of turbidity is measured as a change in optica! density at selected wavelengths and preferably at a wavelength of 340 nm (åOD340), A further aspect relates to a method for determining and/or screening and/or monitoring pathological tissue damage in a fish, the method comprises the steps of: i) obtaining blood from a fish, il) mixing serum or piasma from the blood obtained in step i), with an aqueous protein precipitation solution, and obtaining a mixture iii) determining the levei of turbidity in the mixture obtained in step ii) iv) comparing the determined ieve! of turbidity in step iii) with a reference range, v) determining pathological tissue damage in said fish if the determined ievei turbidity is above the reference-range, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium suiphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
The reference range may be established by the laboratory, the physician and on a species by species basis for each fish. A reference range may be calculated as plus or minus 2 standard deviations from the mean of a sample of a healthy population, or if a non-Gaussian distribution can be calculated as 2.5th to 97.5th percentlies. Either way It includes 95% of results from healthy individuals. In an embodiment the minimum number of individuals from a healthy population to establish a reference range will vary depending on the species and would be regarded as sufficient when a stable Sower (2.5%) and upper (97.5%) reference limits are obtained.
If the level of turbidity is measured using a spectrophotometer the following steps may be performed in addition to steps !)-v) above in order to obtain a reference range; i) measuring the optical density (OD) of the aqueous protein precipitation solution applied, it) mixing serum or piasma obtained from a population of healthy fish with the aqueous protein precipitation solution applied, iH) measuring the optical density {OD) of the mixture of step vi), iv) measuring the optical density (OD) of the mixtures in step vii) and calculating the mean optical density, v) subtracting the optical density measured in step viii) with the optical density measured in step ix) and obtaining a reference range.
Pathology is described as being a scientific discipline which is concerned with observing tissues in order to diagnose a disease, trauma or disorder. Pathology is routinely measured using histology. Formalin fixed tissues are rehydrated and embedded in paraffin wax blocks in order to generate a slide containing a tissue in a specific orientation. The blocks are cut on a microtome which produces fine sections of tissue which can be mounted onto a slide and stained to enhance their contract in a microscopic image. Stains such as but not limited to haematoxyiin and eosin are used routinely to highlight basophilic substances such as DNA/RNA and eosinophilic substances such as proteins. Histology scoring systems are based on the concept that the diagnosis of a specific disease or disorder is based on a collection of features rather than any individual feature which viewed using histology. Using a nominal category to describe a disease with histopathology does not give enough information to a clinician in order to make an accurate decision on prognosis and treatment. Therefore scoring and grading systems have been developed which provide additional information (Cross 199S), These systems are used in order to grade and assess tissues during health assessment. The criteria set out in a scoring system can be used to differentiate between diseases. Criteria are given to different tissue and cellular morphologies which are indicative of severity and used in order to create a histology scoring system. The criteria are analysed in a semi quantitative manner in order to stratify clinical samples. For example, Firshman et ai.(2006) developed a scoring system to differentiate between cases of polysaccharide storage myopathy in horses. For the various diagnostic criteria a severity score was assigned based on the frequency that feature occurred i.e. 0 - not present, l = present in .1 fibre in one 20x random microscopic field (rmf), 2 = present in >1 fibre in 2 rmf, and 3 == present in >1 fibre in > 3 rmf.
The present invention also relates to a method of determining the stage of pathological tissue damage and/or to a method of monitoring pathological tissue damage.
Another aspect relates to a method for determining the stage and/or severity of pathological tissue damage in fish and/or for monitoring pathological tissue damage in a fish, the method comprising the steps of: i) successively withdrawing biood samples from a fish over a period of time, si) mixing serum or plasma from the biood obtained in step i) with an aqueous protein precipitation solution, iii) determining the level of turbidity from each of the mixtures obtained in step ii) iv) comparing the level of turbidity of each of the mixtures and thereby determining: iva) whether the ievei of precipitation of the mixture is higher than the level of turbidity of the immediately preceding mixture which is indicative of progress of pathological tissue damage, or ivb) whether the level of precipitation of the mixture is essentially equal to the level of turbidity of the immediately preceding mixture which is indicative of a steady state stage of the pathological tissue damage, or ivc) whether the level of turbidity of the mixture is lower than the level of turbidity of the immediately preceding sample which is indicative of recovery from the pathological tissue damage, and wherein the aqueous protein precipitation solution comprises a salt seiected from the group consisting of ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
If the pathoiogical tissue damage is caused by a disease such as a viral, parasite or bacterial pathogen, the present inventions provides a method which makes it possible to determine not only the stage of the disease but also to continuously monitor the disease. Stages of disease are determined using histological scoring systems and elevated serum proteins such as creatine kinease, aspartate transaminase and iactate dehydrogenase (Castro &amp; Gourley 2011).
In the present context the term serum precipitation reaction (SPR) is used herein interchangeably with the term serum seiective precipitate reaction.
Intervals between withdrawal of blood sample
In certain embodiments of the method according to the invention, the withdrawal of blood samples form the fish are taken on an hourly basis, a daily basis, a weekly basis, a monthly basis or a yearly basis, most preferably on a weekly basis.
The aqueous protein precipitation solution
In the context of the present invention, "aqueous protein precipitation solution" is to be understood as an aqueous solution that causes protein precipitation when mixed with serum or piasma from a fish suffering from pathoiogical tissue damage. In an embodiment of the present invention the aqueous protein precipitation solution causes increased protein precipitation when mixed with serum or piasma from a fish suffering from pathological tissue damage when compared to the situation where the aqueous protein precipitation solution is mixed with serum or plasma from a fish not suffering from pathological tissue damage.
As evidenced in the examples of the present invention, the inventors have demonstrated that a protein precipitation reaction occurs when serum or plasma from fish suffering from pathological tissue damage is mixed with the aqueous protein precipitation solution of the present invention.
In the context of the present invention, "precipitation solution" means an aqueous solution that causes increased protein precipitation when admixed with serum from diseased fish compared to healthy fish.
It has been demonstrated that a protein precipitation reaction occurs when serum from e.g. diseased Atlantic saimon is mixed with an aqueous protein precipitation solution according to the present invention.
It has been further demonstrated that the proteins which are precipitated in serum when combined with the precipitation solution originate from tissues as such they leak into the circulatory biood system as a result of pathological tissue damage. Lysates obtained from tissues have been used to characterise the precipitate reaction and method optimisation which therefore apply to the precipitation reaction in serum.
In one embodiment the aqueous protein precipitation solution is a solution in water.
Thus, in certain embodiments of the method according to the invention, the aqueous protein precipitation solution is chosen from solutions in water of one or more acids, bases, salts, organic compounds, inorganic compounds, ionic buffers and mixtures thereof.
In certain embodiments of the method according to the invention, the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
In an embodiment sodium acetate may be sodium acetate trihydrate or anhydrous sodium acetate.
In certain embodiments of the method according to the invention, the aqueous protein precipitation solution contains an organic compound selected from the group consisting of methanoi, ethanol, acetone or mixtures thereof.
In certain embodiments of the method according to the invention, the aqueous protein precipitation solution contains polyethylenegiycol (PEG).
In a preferred embodiment the aqueous protein precipitation solution is selected from the group consisting of sodium acetate buffer, ammonium sulphate buffer, magnesium sulphate buffer, ammonium phosphate buffer, sodium carbonate buffer, sodium sulphate buffer, potassium acetate buffer, sodium citrate buffer, sodium phosphate buffer and mixtures thereof.
In an embodiment sodium acetate buffer may be sodium acetate trihydrate buffer or anhydrous sodium acetate buffer.
Sodium acetate is a buffer - thus, a commercially available sodium acetate buffer may be applied in the methods of the present invention.
Serum and plasma
Blood plasma is a pale yeiiow viscous liquid and makes up 55% of the blood's volume. The components of piasma are water 92%, dissolved protein 8%, metabolites and small molecules such as glucose, amino acids, vitamins, minerais, urea, uric acid, C02, hormones and antibodies. It also carries heat energy.
Blood plasma may be prepared by spinning a tube of fresh blood containing an anticoagulant in a centrifuge (e.g. a refrigerated centrifuge and e.g. for around 10 minutes at 1,000-2,000 x g) until the biood cells faii to the bottom of the tube. The blood piasma may then be poured or drawn off.
Blood serum on the other hand is blood piasma without clotting factors. Serum includes substantially all proteins not used in blood clotting (coagulation) and ail the electrolytes, antibodies, antigens, hormones, and any exogenous substances (e.g., drugs and microorganisms). Thus, in blood, the serum is the component that is neither a blood cell nor a clotting factor; it is essentially the blood plasma not Including the fibrinogens.
Serum may be prepared by collecting whoie biood and allowing the biood to clot by leaving it (preferably) undisturbed at room temperature for around 15-30 minutes or overnight at around 4°C. The clot may be removed by centrifuging at e.g. 1,000-2,000 x g for around 10 minutes in a refrigerated centrifuge. The resulting supernatant is designated serum.
The serum or plasma proteins which precipitates when mixed with the aqueous protein precipitation solution of the present invention, originate from the affected tissue since they leak into the circulatory blood system as a result of pathological tissue damage.
Aliquots of whole blood may be in volumes ranging from X0pL-4000 μΙ, such as but not limited to 50pL, 100 μί, 200 pi, 300 μ!, 400 μΙ, 500 μΐ, 600 μΙ, 700 μ!, 800 μί, 900μΙ, 1000 μΙ, 1100 μΙ, 1200 μ!, 1300 μΙ, 1400 μί, 1500 μΙ, 1600 μ!, 1700 μΙ, 1800 μΙ, 1900μ1, 2000μΙ, 2100 μί, 2200 μ!, 2300 pi, 2400 μί, 2500 μί, 2600 μί, 2700 μΙ, 2800 μί, 2900μΙ or 3000μΙ before separating the blood into serum or plasma.
Since proteins are specific to the tissue from which they are released, it may be possible to identify which tissue(s) are affected (Le. by pathological tissue damage) by analysing the precipitated proteins creating turbidity (by applying methods known in the art - such as but not limited to 2 dimension electrophoresis (DE) gel electrophoresis, ELISA, western blot, and mass spectrometry.. 20E is a commonly used method of gel electrophoresis used to separating proteins based on their isoelectric point and mass, 2DE may begin with 1~D electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first. The two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, and protein mass. To separate the proteins by isoelectric point is called isoelectric focusing (IEF). Thereby, a gradient of pH is applied to a gei and an electric potential is applied across the gei, making one end more positive than the other. At aii pH values other than their isoelectric point, proteins wii! be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulied to the more positive end of the gel. The proteins applied in the first dimension wsli move along the gel and will accumulate at their isoelectric point. The result of this is a gei with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used stains are silver and Coomassie Brilliant Slue staining. ELISA is a immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein in a solution.
The western blot (sometimes caiied the protein immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.
Mass spectrometry is an analytical technique which separates components of a sample by their mass. A sample is vaporised into a gas and ionized. The resulting ions are accelerated and focussed into a beam. The ion beam passes through a magnetic field which bends the charged tons. Lighter components or tons with more ionic charge will deflect in the field more than heavier or iess charged components, A detector counts the number of different deflections and the data can be plotted s a spectrum of different masses. Different proteins have different masses which can be compared against a database to identify the constituents of a sampie. Mass spectrometry can be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples.
By identifying the tissue(s) affected it may limit the number of possible disease candidates since some of these diseases are very tissue specific - accordingly, by applying this concept, time may be saved and also the process economy may be affected in a positive manner since the number of possible tests to be run may be reduced.
Determining the concentration of precipitated proteins
The concentration of precipitated proteins may be measured applying any of the methods known in the art.
The level of turbidity is indicative of the amount of precipitated proteins in a sample. Thus, the level of turbidity resembles the concentration of precipitated proteins.
As mentioned previously the leve! of turbidity may be determined by measuring the change in optica! density at seiected wavelengths and preferable at a wavelength of 340nm (aoD-mo).
Thus, in order to determine the concentration of precipitated proteins, the sample (comprising serum or plasma and an aqueous protein precipitation solution) is centrifuged and the pellet washed in water or any suitable buffer solutions. The washed pellet is resuspended in water and the concentration of precipitated proteins may e.g. be determined by Bradford assay, using Bradford Reagent. The Bradford protein assay can be used to measure the total protein concentration of an aqueous sample of unknown protein content. The theory behind the method is that proteins bind to a Coomassie dye under acid conditions which resuits in a colorimetric change from brown to blue which can be measured quantitatively by spectrophotometry. The intensity of the blue colorimetric change is dependent on protein concentration and can be compared to a standard curve of known protein concentration, such as with Bovine Serum Albumin (BSA) as standard.
Equally applicable are the optical identity which may be monitored by applying e.g, a plate reader (measuring absorbance (Ab) or e.g. a spectrophotometer (measuring optical density (OD)) at seiected wavelengths and preferably at 340nm. In the present context it is to be understood that the absorbance and optica! density are equivalents. In certain embodiments of the present invention, the optical density may be determined by spectrometry using a wavelength between preferably 160-595nm and most preferably 340nm,
Selective precipitation, from diseased and heaithy animals, requires buffer conditions to be optimised for each individual species. This requires optimisation of a buffer which minimises destabilisation of normal circulatory proteins in biood serum or plasma whiie causing precipitation of proteins which are abnormaiiy increased in abundance in serum or piasma during disease. By following this optimisation methodology the difference between healthy and diseased both qualitatively and quantifiabiy is maximised. The foiiowing methodology describes can be optimised in other species of interest
In certain embodiments of the method according to the invention, the determination of visible precipitate is detected by the naked eye.
Typically the naked eye will respond to wavelengths of 390~700nm. The absorbance of a solution is dependent upon the colour. It has been stated low concentrations of protein invisible to the naked eye protein (<0.012 mg/mL) (Ngo et al. 2014).
In certain embodiments of the method according to the invention, the optical density of precipitates formed on mixing sample with aqueous protein precipitation solution is determined by spectrometry using a wavelength between preferably 160-595nm and most preferably 340nm.
Sample Volume
The volume of serum or piasma to use in the test has been established as 15pi (Figure 28). Optimising sample volume Is important for determining the amount of blood required from each individual animal and to minimise the amount of serum required to obtain a detectable reaction. Figure 28 demonstrates the effect of using decreasing volumes of a sample on detectable precipitation levels. From 40 to 60μΙ there was an extremely high delta change in OD though the little difference between the three volumes indicates a saturation effect. This delta OD falls dramatically when using 30pi, Sample volume of 15pl was determined based on the detectable change in turbidity while minimizing sample volume.
Buffer Volume
The optimal volume of buffer for the reaction that has been identified was approximately 245pl when Ι5μί of serum or plasma is used. This buffer volume has been determined as too small a volume leads to little precipitation while too high a volume leads to a dilution of observable change in turbidity when sera or plasma from a diseased animal is added. Another consideration is the type of equipment being used in the test 245μΙ is standard for when using 300pi capacity welled microtitre plates, but when a single channel traditional spectrophotometer is used volume must be increased to a point where Sight is passed through the mixture e.g. 1ml. However, such am increase in buffer volume requires a relative increase in sample used also which may be limited.
Temperature
Temperature is a major catalyst for the selective precipitation reaction (SPR), It has been shown the rate of precipitation occurs much more quickly at 45*C compared to 2G«C (Figure ID). While SPR still takes place at lower temperatures (e.g. room temperature) this may not be idea! where high throughput is required. Many automated readers have standard temperatures such as 37°C. This temperature is preferred for the reaction as it gave sensitive, accurate, and repeatable results for samples over a 60 minute monitoring period. Figure ID shows the effect of temperature on the spectrophotometer assay. Increasing the temperature from 20ο€ to 37°C caused a steady increase in the absorbance at 340nm for the precipitation reaction. At 45°C there was a rapid increase followed by a fall in absorbance due to a sedimentation effect with precipitate settling out of the solution which is not desirable.
In a preferred embodiment the temperature of the mixture of serum or plasma and the aqueous protein precipitation solution Is in the range from 20-45 °C, such as 21-44 °C , e.g. 22-43 °C, such as 23-42 °C, e.g. 24-41 °C, such as 25-40 °C, e.g. 26-39 °C, such as 27-38 °C, e.g. 28-37 °C, such as 29-36 °C, e.g. 30-35 °C, such as 34-36 °C, e.g. 35-36 °C and even more preferably in the range from 33-37°C.
Wavelength
The wavelength at which changing optical density is measured is pivotal for quantification of delta change (Δ). By testing different wavelengths it has been shown that the optimal wavelength for identifying changing OD caused by turbidity/ precipitation is 340 nanometres (nm). The optimal wavelength for detecting changing turbidity by precipitation was examined by assessing a range of fixed wavelength detection filters to establish the optimal wavelength for observing precipitate formation by ELISA plate reader. For example, the 340 nm filter yielded the greatest CD reading (Figure IB), confirming that a wavelength of 340nm was optimal.
Steps for Optimisation in a Given Species 1. Create a stock solution of different molarities of sodium acetate or (other precipitation solutions). This stock solution is used to investigate at which ionic strength it causes precipitation of proteins in healthy or diseased sera. Pools of healthy and disease serum or plasma are to be used in this initial phase of optimisation due to large values required. Using the optimised buffer volume, wavelength and temperature values as carried out previously, measure to delta of individual serum samples. A suitable molarity is identified when a visual and quantifiable change in turbidity is observed. The differential between healthy and diseased may at this point not be very high but this may be due to the pH not being optimal or too high a molarity. However, the difference should be observed. If solution remains clear for both health states molarity must be increased. Though if both become extremely turbid due to a mass (unseiective) precipitation then molarity should be lowered 2. The pH of the precipitate buffer is optimised in order to dictate the amount of proteins within a serum or plasma sample to precipitate. Adjusting the pH of the precipitation buffer is achieved by adding an acid or base, e.g. hydrochloric acid or sodium hydroxide respectively. The optimal buffer should be at a pH where normal circulatory proteins are kept in a soluble state and those which are markers of disease are precipitated. For example Figure 1A shows the relationship of changing pH and delta OD in Atlantic saimon serum. Changes in pH result in the reaction becoming less applicable as it destabilises the majority of proteins as shown by the delta OD. 3, Molarity of the buffer is optimised to reduce normal circulatory proteins precipitating from serum which reduces the tests sensitivity and specificity. This effect of molarity on the magnitude of difference in Atlantic saimon can be seen in Figure IE. It has been found that for Atlantic saimon a Q.6M buffer is optimal and causes a higher precipitate reaction whilst normal circulatory proteins remain soluble. The optimal buffer conditions in Atlantic saimon for the SPR was found to be 0.6MM SAT at a pH of 5.6.
If suitable conditions for SA buffer cannot be found using the outlined methodology the process can be repeated using different salt based buffers and precipitation agents. Such salt based buffers include sodium citrate, potassium acetate, sodium carbonate or sodium phosphate.
Ratios o f serum and aqueous protein precipitation solution
In certain embodiments of the method according to the invention, the ratios between serum and aqueous protein precipitation solution is: 1: 10-20, 1: 11-19, 1:12-18, 1: 13-17, 1:14, 1:15, 1:16
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on sodium acetate, the ratio between serum and aqueous protein precipitation solution is: 1: 10-20, 1: 11-19, 1:12-18, 1: 13-17, 1:14, 1:15, 1:16 and most preferably 1:16 1/3 or 16 2/3,
Heating of serum and aqueous protein precipitation solution before mixing In certain embodiments of the method according to the invention, the serum is heated prior to the mixing with aqueous protein precipitation soiution to: preferably, 20-40°C, more preferably 32-39°C, even more preferably 34-3S°C and most preferably to 37°C.
In certain embodiments of the method according to the invention, the aqueous protein precipitation solution is heated prior to the mixing with serum to: 20-40°C, more preferably 32-39°C, even more preferably 34-38°C and most preferably to 37°C.
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on sodium acetate, the aqueous protein precipitation solution is heated prior to the mixing with serum to: preferably 20-4Q°C, more preferably 32-39°C, even more preferably 34-38°C and most preferably to 37°C.
Reaction time
If a fish suffers from pathological tissue damage, proteins precipitate when applying the method of the present invention ~ i.e. when serum or piasma are mixed with an aqueous protein precipitation solution. Depending e.g, on the amount of proteins and e.g. the origin of the protein precipitation of proteins may take time. Thus, in a preferred embodiment the mixture of serum or piasma and the aqueous protein precipitation solution may react for at least 30 seconds, e.g, at least 1 minute, such as at least 5 minutes, e.g. at least 10 minute, such as at least 15 minutes, e.g. at least 20 minute, such as at least 25 minutes, e.g, at least 30 minute, such as at least 35 minutes, e.g. at least 40 minute, such as at least 45 minutes, e.g. at least 50 minute, such as at least 55 minutes, e.g, at least 60 minute, such as at least 70 minutes, e,g, at least 80 minute, such as at feast 90 minutes, e.g. at least 2 hours, such as 3 hours, e.g. 4 hours, such as 5 hours, in a preferred embodiment 60 minutes,
The inventors surprisingly discovered that if the pathological tissue damage originates from intestinal tissue the proteins precipitate very quickly and at a high turbidity, Thus, in particular the method of the present invention may be applied to determine pathological tissue damage originating from the intestines if proteins are precipitated (preferably visually) no more than 10 minutes, such as no more than 9 minutes, e.g, no more than 8 minutes, such as no more than 7 minutes, e.g. no more than 6 minutes, such as no more than 5 minutes, e.g. no more than 4 minutes, such as no more than 3 minutes, e.g. no more than 2 minutes, such as no more than i minute following mixing of the serum or piasma with the aqueous protein precipitation solution.
Molarity/ionic strength/strength of buffer in the aqueous protein precipitation solution
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on sodium acetate, the molarity of sodium acetate buffer is between Q.OSM -iM, more preferably between 0,114 -114, more preferably between 0.2M ~ IM, more preferably between G.3M - 114, more preferably between 0,414 - IM, more preferably between 0,514 - IM, more preferably between 0.5(4 - 0.9M, more preferably between 0.5M - 0.8M, more preferably between 0.514 - 0.7M and most preferably 0.6 M,
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on ammonium sulphate (AS), the ionic strengths of ammonium sulphate (AS) is between 0.214 ~ 114, most preferably 0.2M,
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on polyethylene glycol (PEG) where PEG1 represents l,0G0Mw PEG, the ionic strengths of PEG! is between 0.01M -0.0514, most preferably O.OSM.
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on polyethylene glycol (PEG) where PEG2 represents 8,00GMw PEG, the ionic strengths of PEG2 is between 0.0114 -O.OSM, most preferably 0.05M.
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on ethanol, the strengths of ethanol is between 10% -100%, most preferably 10%, pH of the aqueous protein precipitation solution
In certain embodiments of the method according to the invention, the pH of the aqueous protein precipitation is preferably between 3.7 ~ 8,0, more preferably between 5,2 ~ 6,0, more preferably between 5,4 - 5,8, and most preferably 5,6,
In certain embodiments of the method according to the invention, when the aqueous protein precipitation solution is based on sodium acetate, the pH of the solution is preferably between 3,7 - 5,6, more preferably 5, more preferably 5.2, more preferably 5.4, most preferably 5.6,
In certain embodiments of the method according to the invention, when solution is based on sod ium phosphate, the pH of the aqueous protein precipitation is preferably between 5.6 ~ 8,0, more preferably 6,0, more preferably 5,8 and most preferably 5,6.
The fish
In certain embodiments of the method according to the invention, the fish which is subjected to the diagnostic method(s) of the invention is of the family Saimomdae or may be of the subfamily Saimonidae. In a preferred embodiment, the fish may be of a genus selected from the group consisting of Saimo, Oncorhynchus and Saiveiinus.
In certain embodiments of the method according to the invention, if the fish is of the genus Saimo the fish may be selected from the group consisting of Atlantic salmon (Saimo salar L), Adriatic trout (Saimo obtusirostris), Fiathead trout (Saimo piatycephafus), Marble trout (Saimo marmoratus), Ohrid trout (Saimo ietnica), Sevan trout (Saimo ischchan) and Brown trout (Saimo trutta),
In certain embodiments of the method according to the invention, if the fish is of the genus Oncorhynchus, the fish may be selected from the group consisting of rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch) and Chinook salmon (Oncorhynchus tshawytscha).
In certain embodiments of the method according to the invention, if the fish is of the genus Saiveiinus, the fish may be selected from the group consisting of Arctic char (Salvelinus alplnus).
In certain embodiments of the method according to the invention, the fish is selected from the group consisting of fry, fingerlings, parr, smoits and post-smoits.
In certain embodiments of the method according to the invention, if the fish is of the genus Sparus, the fish may be selected from the group consisting of gilt-bead (sea) bream (Sparus aurata).
In certain embodiments of the method according to the invention, if the fish is of the family Serranidae, the fish may be selected from the genus Dicentrarchus of the group consisting of European seabass (Dicentrarchus lahrax), and from the genus Dicentrarchus of the group consisting of Japanese seabass (Lateoiabrax japonicas), and from the genus Epinephelus consisting of the Arabian grouper (Epinephelus tauvina) .
In certain embodiments of the method according to the invention, if the fish is of the genus Oreochromis, the fish may be selected from the group consisting of Nile tilapia, (Oreochromis niloticus).
In certain embodiments of the method according to the invention, if the fish is of the genus Pangasius, the fish may be selected from the group consisting of Pangas catfish, (Pangasius pangasius), and basa fish, (Pangasius bocourti),
In certain embodiments of the method according to the invention, if the fish is of the genus Seriola, the fish may be selected from the group consisting of Japanese amberjack or yeiiowtaii, (Seriola quinqueradiata).
In certain embodiments of the method according to the invention, if the fish is of the family Cyprinidae, the fish may be selected from the genus Ctenopharyngodon of the group consisting of grass carp (Ctenopharyngodon idella), the genus Hypophthaimichthys of the group consisting of silver carp (Hypophthaimichthys molitrix) and blghead carp {Hypophthaimichthys nobtits), the genus Cyprinus of the group consisting of common carp {Cyprinus carpio), the genus Carassius of the group consisting of crucian carp (Carassius carassius) and gibel carp (Carrassius gibelio), the genus Catla of the group consisting of major (Indian) carp (Catla catla or Gibelion catla), and the genus Labeo of the group consisting of Rohu or roho labeo {Labeo rohita),
In certain embodiments of the method according to the invention, if the fish is of the family Sciaenidae, the fish may be selected from the genus Larimichthys of the group consisting Yellow croaker (Larimichthys crocea).
In certain embodiments of the method according to the invention, if the fish is of the famiiy Latidae, the fish may be selected from the genus Lates of the group consisting of Barramundi (Lates caicarifer)
The pathogens
In an embodiment of the present invention the pathological tissue damage may be caused by a pathogen selected from the group consisting of virus, bacteria, parasites and combinations thereof.
In certain embodiments of the method according to the invention, the pathogen causing tissue damage in the fish is chosen from the group of viruses consisting of but not limited to Salmon α-virus, saimonid alphavirus, Salmon Pancreas Disease Virus (SPDV), saimonid afphavirus (SAV), Piscine Myocarditis Virus, Piscine reovirus (PRV), Atlantic Salmon Caicivirus (ASCV), Aquabirnaviruses, Infectious pancreatic necrosis virus (IPNv), ISA virus or virai hemorrhagic septicemia virus (VHSV).
In certain embodiments of the method according to the invention, the pathogen causing tissue damage in the fish is chosen from the group of bacteria consisting of but not limited to Aeromonas salmoniddia subsp salmonicidia, Vibrio salmoniddia l Vibrio anguiliarum, Moriteiia vsscosus, Pisdrickettsia salmonis, Yersinia ruckeri or Flavobacterium psychrophiium,
In certain embodiments of the method according to the invention, the pathogen causing tissue damage in the fish is chosen from the group of fungi consisting of but not limited to Saproiegnia spp.
In certain embodiments of the method according to the invention, the pathogen causing tissue damage in the fish is chosen from the group of parasites consisting of but not limited to Neoparameabe peruans, ichthyophthirius mufbWiis,
The diseases in certain embodiments of the method according to the invention, the disease that causes the pathological tissue damage in the fish is caused by one or more of the following virus diseases: Pancreas Disease (PD), Saimon Pancreas Disease (SPD), Heart and Skeletal Muscle Inflammation (HSMI), infectious pancreatic necrosis (I PN), Card ίο Myopathy Syndrome (CMS), Infectious salmon anaemia (ISA), viral hemorrhagic septicemia (VHS), acute myopathy and immune reactions during chronic diseases caused by viruses,
In certain embodiments of the method according to the invention, the pathological tissue damage is caused by one or more of the following bacteria diseases: furunculosis, coid water vibriosis, vibriosis, winter uScer disease, piscirickettsiosis, salmonid rickettsial septicemia (SRS), enteric redmouth (ERM), rainbow trout fry syndrome (RTFS), or bacterial kidney disease (8KD),
In certain embodiments of the method according to the invention, the pathological tissue damage is caused by one or more other acute or chronic stressors including fungi disease; Saproiegniasis, parasite diseases; Ameobie gill disease (AGP) or freshwater white spot disease.
Kit
Another aspect of the present invention pertains to the provision of a kit for (i) determining and/or screening and/or monitoring pathologicai tissue damage or (ii) determining the stage and/or severity of pathological tissue damage in a fish and/or for monitoring pathological tissue damage in a fish, said kit comprises an aqueous protein precipitation solution, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
Thus, the present invention further contemplates a kit for assessing pathological tissue damage in a fish. The kit is conveniently in eompartmenta! form with one or more compartments adapted to receive a sample from a fish such as serum or plasma.
Generally, the kit is in a form which is packaged for saie with a set of instructions. The instructions would generally be in the form of a method for determining pathologicai tissue damage in a fish, said method comprising coiiecting a serum or plasma sample from said fish, incubating said sample with an aqueous protein precipitation solution supplied with kit and then measuring the concentration of precipitated proteins, wherein the presence or level of said precipitated proteins is indicative of pathological tissue damage.
In one embodiment the kit contains blood collection tubes, graduated plastic pipettes for solution transfer and a aqueous protein precipitation solution &amp; porta b le s pectroph otometer.
The contemplated kit of the present invention may be in a multicomponent form wherein a first component comprises serum or plasma collection tube, a second complement comprising a graduated pipette for solution transfer, a third component comprises an aqueous protein precipitation solution and a third component comprises a set of instructions which instructions comprise the following: (I) collect serum or plasma in the collection tube; (ii) mix serum or plasma with the aqueous protein precipitation solution; (iii) incubate the mixture; and (v) determine the concentration of protein precipitation. As mentioned previously the level of turbidity is indicative of the amount of precipitated proteins.
If blood is added to the collection tube containing a gei based substance, serum will automatically separate from the whole blood. If the blood container does not contain a gei based substance then serum would have to be collected via centrifugation. Whole blood is preferably centrifuged for 10 min at 1,000 x rpm. Serum can be stored at ~2G°C or analysed immediately.
The Isolated serum may be analysed using the spectrophotometer method with 1m! of sodium acetate (SA buffer) dispensed into a cuvette and incubate at 37°C. 180μΙ of 2% (w/v) bovine serum albumin (BSA) was mixed with 60pl of serum in an micro centrifuge tube and then mixed with the SA buffer. Mixture is incubated for 1 hr and read in a spectrophotometer at 340 nm wavelength and compared to a standard reference sample.
In a preferred embodiment the kit is adapted for using the methods of the present invention. Thus, in use the kit may perform the methods of the present invention.
Use
The present invention also pertain to the use of the above mentions for developing and/or validating and/or monitoring efficacy of interventions to improve the immune response and/or host response in a fish including but not limited to vaccines, biosecurity practices, diet and combinations thereof.
Evaluating the efficacy of a health intervention or biosecurity practice can be either carried out on an individual or representative number of indiuviduals for a population across a farming location or in comparison between farming locations.
Determining whether an intervention has caused a change in health status is carried out by evaluating the change in delta optical density using the serum precipitation reaction pre and post intervention or over a time course following the intervention. An effective health intervention is determined by a reduction in delta opticai density for serum or plasma samples taken after the intervention to the reference range or where the delta opticai density analysed repeatidiy over a time course and calculated as area under the curve (AUC) is statistically significantly lower compared to that for an individual or a representative number of individuals of the population, calculated at each time point as the mean of the subset of the population, for which no intervention took place. A failed treatment or intervention is determined when the delta optical density for serum or plasma samples taken after the intervention does not change or increases above the reference range post intervention or where the delta optical density analysed repeatidiy over a time course and calculated as area under the curve (AUC) is not statistically significantly different or statistically significantly higher compared to that for an individual or a representative number of individuals of the population, calculated at each time point as the mean of the subset of the population, for which no intervention took place.
Population
Monitoring the efficacy of interventions or health status of a fish population is carried out by sampling a sub group of individuals from a representative number of cages, tanks, ponds across a farm site. The data gained from this sampling is extrapolated to determine the health status of the site. Individuals should be selected in a way to reduce a sampling bias effect i.e. not selecting oniy diseased individuals.
Definitions
Prior to discussing the present invention in further details, the following terms and conventions wiil first be defined: Δ340; In the context of present application, any references to Δ340 refers to the change in optical density (Δ340) calculated by subtracting the first reading from the final for a specified time period. Said optical density refers to the reconstituted serum/buffer sample measured as the absorbance (Ab) measured at a wavelength 340nm by spectrophotometry in e.g. a Mictotitre piate reader or single cuvette instrument. 2DE: 2 dimensional electrophoresis.
Ab: absorbance as equivalent to optica! density.
Buffer: A buffer is a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH.
Clinically healthy salmon: in the context of the present application, clinically healthy salmon refers to clinical parameters are within a normal range and absence of infectious agent.
Reference solution: in the context of the present application, reference solution refers to the aqueous protein precipitation solution or it may be e.g. water or any suitable buffer solution or an admixture of a serum or plasma sample with the aqueous protein precipitation solution from a healthy fish of the sample species and may contain BSA to maintain precipitation In suspension. CMS: Cardiomyopathy syndrome, CV: coefficient of variation (CV) is defined as the ratio of the standard deviation v to the mean μ. It shows the extent of variability in relation to mean of the population,
Differentia! precipitation: the precipitation of one sample being not the same as another sample
Microtiter plate reader: In the present applications, any references to "ELISA piate reader" refers to laboratory instrument designed to detect biological, chemical or physical events of samples in small volumes in a microtiter plate, typically with a spectrophotometer.
Fish serum: In the context of present application, any references to "fish serum" refers to plasma component of blood with clotting factors removed.
Fish tissue lysate: In the context of present application, any references to "fish tissue fysate" refers to a fluid containing the contents of lysed cells from fish tissues. HSMI; Heart and Skeletal Muscle Inflammation IPN: Infectious Pancreatic Necrosis MS/MS: a "tandem mass spectrometry" method for structure determination and analysis of molecules, Typically, MS/MS uses two mass spectrometers in tandem, wherein the two analyzers are separated by a collision gas ceil.
Myopathy; Myopathy basically means muscle disease. A myopathy is a muscular disease in which the muscle fibers do not function for any one of many reasons, resulting in e.g, muscular weakness. In fish, skeletal muscie myopathies - causing muscle breakdown which in turn reduces the quality of the fish meat - are some of the most common symptoms of infectious diseases such as PD, QD: optical density
Pathogen: in the context of the present application, pathogen refers to anything that can produce disease in a host. Pathogen may refer to an infectious agent such as a microorganism, virus, bacterium, prion, fungus or protozoan, that causes disease ih its host.
Pathological damage: Predicted or actual progression of a particular disease in an organ, tissue or cell, PD: Pancreas Disease.
Serum precipitation reaction (SPR): in the context of the present application, SPR is measured by change in absorbance over 60 minutes at 340nm
Pyloric caeca: in many fish, processed in finger-shaped pouches called pyloric caeca, which secrete digestive enzymes and absorb nutrients,
Salmon id: is a family of ray-finned fish, which includes salmon, trout, chars, freshwater whitefishes and graylings, SAV: salmoinid alpha-virus, SPR: serum precipitate reaction.
Turbidity: In the context of the present application, turbidity refers to the measure of relative clarity of a liquid solution.
Wpc: week post challenge with infectious microorganism (for example: WOpc refers to non-challenged fish - W4pc refers to fish, which have been challenged (infected for 4 weeks).
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non-limiting examples.
Examples
Example 1 (obtaining serum samples)
Serum was collected from fish during the pathogenesis of PD from an experimental trial study described in BraceSand efc al (2013; 2014).
The feeding trial consisted of an acclimatisation phase and challenge phase which was carried out at Veso Vikan (Namsos, Norway). 360 Atlantic salmon parr of 30g with no previous contact with SAV were randomly distributed into duplicate lm3 tanks and acclimated. Following acclimation, 120 fish were transferred from the acclimation tanks into triplicate 0,6 m3 tanks to be used for the disease challenge.
Naive fish to be used as infective viral shedders (Trojans) were marked by dipping their adipose fin and injected with SAV 3 infected CHSE cell culture supernatant at ca. 10s TOD/fish into their intraperifconeal cavity. Thirty inoculated Trojans were added to each of the challenge tanks 6 days after their assembly. Cohabitant fish serum was sampled at 0, 2, 3, 4, 5, 6, 8, 10 and 12 weeks post challenge. At each time point 9 fish per tank were killed by lethal overdose of anaesthetic benzocaine and biood collected in non-heparinised sampie tubes from the caudai vein,
From 6 of the sampled fish, pyloric caecae and pancreas (hereafter referred to as pancreas), heart and skeletal muscle tissue were processed from standardised locations for histology, Tissues were routinely processed and stained with hematoxylin and eosin. Tissue samples were examined os a blind study by a pathologist and the scoring system used to semi-quantify the distribution and severity of the tissue lesions is described by McLoughlin et al. (2006) (Table 1).
Tabie 1. Semi-quantitative lesion score system used to PD compare lesion severity in Atlantic salmon, (a) Pancreatic lesion classification, (b) Heart iesion classification, (c) Red and white skeietai muscie lesion classification.
Example 2 (obtaining ceil lysates of tissue samples)
Tissue samples were collected from clinically healthy fish from a commercial production fish farm site with no previous history of disease. The tissue lysates were e.g. compared to see which proteins from different organs were similar to those observed in the serum protein precipitation reaction of infected fish.
Tissue samples were immediately stored on dry ice and subsequently frozen at ~ 80°C until processing. Lysates were created using mortar and pestle technique known in the art, where tissue pieces were ground while kept frozen by periodic addition of liquid nitrogen until a fine powder formed. This was then transferred to another mortar and iOml of ceil lysis buffer (20mM Tris~HCL, pH 7.5) per Ig of tissue was added prior to further grinding and mixing for 5 minutes. The grinded tissue was then centrifuged at 13,000rpm at 4°C for ten minutes whereafter the removed supernatant was transferred into another tube and the centrifugation step was repeated. Solid residue was discarded and the supernatant was passed through a 0,45pm filter twice before storage at -80°C.
Example 3 (the Serum Precipitation Reaction (SPR) assay)
Plate reader
For use on a microtitre plate; 15μΙ of serum is dispensed to a 96 weil plate and placed inside a microtitre plate reader (Fiuorostar Optima BMG Labtech) equilibrated to 37°C, Sodium acetate trihydrate buffer (0.6 M at a pH of 5,6) was also heated to 37°C before addition of 245pl to each weil. Absorbance (Ab) was measured with the plate reader at 34Gnm over a 60-minute duration with readings being taken every 30 seconds for each well to give optical density values at each time point. The change in optica! density (Am) in each sampie was calculated by subtracting the first reading from the final for each.
The results are shown in figure 2.
Spetrophotometer
The precipitate assay was performed on a spectrophotometer (Jencons 7315} as follows. An aliquot of 180μΙ 2% (w/v) bovine serum albumin (BSA) was added to 60μΙ of serum sample before mixing in a cuvette. 1 ml of sodium acetate trihydrate buffer (0,5 M at a pH 5.6 was added to the cuvette. The BSA was shown not to have any effect on the reaction kinetics or precipitation reaction but reduced the sedimentation of precipitate allowing for more reiiable OD to be monitored in the spectrophotometer test as by adding viscosity the sedimentation process takes longer, thus keeping precipitate suspended in the solution during the test allowing for more accurate readings. Monitoring of the OD was carried out at 34Gnm with readings taken every second for 60 min. Results are shown in Figures 1C, ID, and IE.
Example 4 (SPR to determine changes in precipitation over time in fish infected with PD)
The SPR method according to the present invention was applied on individual samples to investigate the changes in precipitation over time and the relationship between degree of precipitation and pathological tissue damage caused by PD. A pool of sera from clinically healthy fish (pool A) and mean serum precipitation reaction response of fish at week 4 post challenge (W4pc) in a severe pathological state were used to calculate interassay %CV, which were 10.6% and 9,7% respectively (n = 25 plates), average intra assay %CV was 7.2%,
Figure 2 shows SPR measured by the mean Δ340 of serum from fish according to week post challenge (Wpc).
Thus, by using the method of the present invention the precipitation potentiai of individual samples was assessed with a;mo from 0 to 60 minutes. As is evident from figure 2, there were significant differences in SPR during the triai as the disease progresses with a significant rise in precipitation in sera at W4pc, before its peak at W6pc and returning to homeostasis near levels at W12pc. Also observed was a significant correlation between the pathological tissue damage and SPR (figure 3) which makes the SPR assay of the present invention applicable as a diagnostic test in the field as the test requires little specialised reagent and with a visible end-point couid be easily performed at fish production sites. Figure 3 shows the four tissues affected by pancreas disease and the mean lesion scores during the study compared to the mean precipitate/ delta OD change during the progression of pancreas disease pathology.
These surprising findings makes the method a simple and effective tool for determining the severity of pathological tissue damage in fish over time, which can be of importance when monitoring of the health status of fish in aquaculture.
The invention has potential to improve fish health management decisions when a disease outbreak occurs in a fish farming facility, which in turn will lead to lower health related losses of the production unit. Specifically this invention will provide information on the timing of critical period of severe pathology and the degree of severity.
This knowledge can be used to implement mitigation strategies to minimise mortality and decrease the negative impact on farmed animal growth and feed utilisation caused by the disease pathology, thereby reducing the economic impact of the disease, These mitigation strategies may include reducing feeding rate, increased biosecurity measures and avoiding handling or movement offish during the critical pathology period. Decreasing the number of destructive samples that are required will reduce fish health management costs and reduce the number of farmed animais to be culied thereby improving fish welfare standards of the farming industry,
This invention can also be used as a method for developing and assessing the efficacy of functional feeds, immune modulators or stimulants, medicines, disease trial models and vaccines by providing information and documentation on the severity of pathology in both the laboratory development of the products and for in the field validation and product support. The invention may reduce the number of animais required to be culled for biological tissue samples in these studies. Greater knowledge of disease processes within populations may also be obtained as results of the ability to sample from Individual fish for epidemiology studies.
Example 5 (SPR to determine pathological tissue damage in different fish tissues}
The inventors of the present application have thus observed that pathological damage to the tissues depicted in figures 3 and 6 would, as in the case of PD, cause a change in serum proteome constitution, that would increase the SPR and thus be applied as a general health test as a non-destructive tool for diagnosing clinical disease. Moreover, the ability for precipitate to be reconstituted in water and electrophoresis separation of proteins contained means there is the potential for identifying specific band profiles depending on disease/ pathology.
This observation was further validated by the significant correlation of Δ340 to histopathology of ail tested tissues by using Pearson correlation coefficient (as previously pubiished in Braceiand et ai 2013) thus indicating the precipitate potential of a given serum sampie may be influenced by multiple pathologies which could be valuable as a biomarker of damage to tissue In genera!,
Also, the precipitation reaction based on the Δ340 using the Microtitre plate assay was found to be significantly correlated with histopathoiogicai data as described in Braceland et ai. 2013 by Pearson coefficient analysis, with δ,μο correlating positively with severity of histopathoiogy in heart (p = 0.001), pancreas (p = <0.001), red muscle (p = <0,001), and white muscle (p = <0,001), thus indicating that pathological damage to all of the examined tissues had a significant effect on the precipitate potential of serum.
Example β (optimization of the precipitation assay)
The quantifiable difference of turbidity depending on health status of individual fish providing the sera has ied to the development of a SPR based method of the present invention based upon A340.
Through development and optimization it was found that this turbidity phenomenon is effected by a number of parameters, such as; temperature, and the molarity pH of the buffer used. For instance, precipitation formation is much faster at higher temperatures (figure Id) and aiso more precipitate is formed over a given time when using a buffer of higher molarity (figure le) or pH of buffer (figure la). In addition, forming precipitate which manifests as turbidity is detected differently dependent on the wavelengths used (figure lb and lc).
Thus, a range of buffers with the desired pH were prepared by titrating sodium acetate (Q.2M) with acetic add (0.2M) and di-sodium hydrogen phosphate (0.2M) with sodium dihydrogen phosphate-2-hydrate to achieve a range from pH 3.7 to pH 8.0, Results indicated that a sodium acetate buffer with pH 5,6 was optima! for maximizing &amp;wo (figure la).
The optima! wavelength for determining changing turbidity caused by precipitation was examined by assessing a range of fixed wavelength detection filters to establish the optima! wavelength for observing precipitate formation by Microtitre plate reader with 340 nm yielding the greatest OD reading (figure lb). This wavelength also gave higher absorbance using a spectrophotometer when compared to SSOnm (figure lc), confirming that a wavelength of 34Gnm was optima!.
Figure Id shows the effect of temperature on the spectrophotometer assay. Increasing the temperature from 20°C to 37C,C caused a steady increase in the absorbance at 340nm for the precipitation reaction. At 45°C there was a rapid increase followed by a fall in absorbance due to a sedimentation effect with precipitate settling out of the solution.
The effect of buffer moiarity on precipitation carried out using a plate reader with 60μ! muscle lysate being added to 200μ! buffer. Figure ie iliustrates the wide range of moiarity where precipitate is formed, although a significant drop in Δ340 being observed when sodium acetate buffers of <0,2 M were used. From these results, the optimal assay conditions for the serum precipitate reaction (SPR) were determined to be: 0.6M sodium acetate trihydrate buffer at pH 5,6, and reaction temperature at 37°C,
Other factors, such as, buffer to sample ratio were also optimized and was found to be 15pl sample with 245μ! buffer for analysis on Microtitre plate reader.
The optima! buffer to sampie ratio for the spectrophotometer assay was found to be 1ml of buffer being added to 240μl of sampie mix (the sampie mix consisting of 60μΙ sample and 180μ! 2% BSA) with changing absorbance being monitored over a 60 minute period (Δ340).
Example 7 (identification of the precipitated proteins) 2 Dimensional Electrophoresis (2DE) of precipitate
In order to investigate the cause of the serum precipitation reaction (SPR), WOpc and W4pc sera was mixed with sodium acetate buffer and after extensive washing was examined after reconstitution in water by using 2 dimensionai electrophoresis (2DE) examination.
More specifically, to investigate the composition of the proteins forming the precipitate, separate WOpc and W4pc pools were created for examination with equal volumes from each time point. Samples of the serum poois (300pi) were added to 0.6M sodium acetate trihydrate buffer (iml) and allowed to incubate for one hour in a water bath at 37°C> The solution was then centrifuged at 3000rpm for 10 minutes and supernatant collected. Precipitate was washed by resuspending in 1ml of SAT buffer and mixed prior to spinning at 3000rpm. This was repeated twice and each time supernatants removed, the final time the precipitate was re-suspended in 200μ1 of distilled water and mixed until proteins had gone back into solution.
One microiitre of each serum sampie collected from each fish sampled at each time point was pooled according to week to create pooled samples for the analysis of changing protein composition throughout the time course. The protein concentration of the pooied samples was determined by Bradford assay, using Bradford Reagent.
The Bradford protein assay is used to measure the totai protein concentration of an aqueous sample of unknown protein content. The theory behind the method is that proteins bind to a Coomassie dye under acid conditions which results in a colorimetric change from brown to blue which can be measured quantitatively by spectrophotometry. The intensity of the blue colorimetric change Is dependent on protein concentration and can be compared to a standard curve of known protein concentration, such as with BSA as standard. An equal protein loading for 2DE protein separation by isoelectric focusing based on isoelectric point (pi) and sodium dodecy! sulphate poly- acrylamide gel electrophoresis (SDS-PAGE) based on molecular weight. Three replicate 2DE gels were run of the pool of samples from each time point. Separation by pi was carried out using 11 cm immobilized pH Gradient (IPG) strip with a pH range of 3 tolO (BioRad, Hemel Hempstead, UK). After protein loading of the IPG strips with serum diluted In a rehydration buffer (8 M Urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte®} (BioRad, Hemel Hempstead, UK) and covered in 500 pi of mineral oil, a combined rehydration and focusing step was carried out over 17 h with a total of 35,000 V-h. The IPG strips were removed, oil drained and then treated with two equilibration buffers both made from a stock solution comprised of 6 M urea, 0.375 M Tris-HCI, pH 8.8, 2% (w/v) SDS, 20% (v/v) glycerol, the first of these containing 2% (w/v) dithiothreitol (Sigma-Aidrich, Poole, UK) to reduce the proteins and subsequently the alkylating agent iodoacetamide at 2.5% (w/v) (Sigma-Aldrich, Poole, UK). IPG strips were then placed onto Criterion SDS-PAGE gels and submerged in XT Mops running buffer and subjected to electrophoresis at 200 V for one hour (Bio-Rad, Hemel Hempstead, UK). Subsequently gels were stained in Coomassie brilliant blue G-25Q dye 0.1% (w/v) in de-stain solution for 1 h and then de-stained using a solution of methanol:water:acetic acid, (4:5:1) overnight, scanned and saved in 16-bit grey TIFF format images for gel image analysis.
As can be seen from figure 4, the 2DE gel profile of precipitate formed from PD diseased (W4pc) serum is much more complex and diverse than that of the WOpc serum. The main difference in proteome that causes this differential precipitation is an increase in proteins (spot 6-22, 27-31, 35} that are usually intraceiiuiar being passively increased in the serum due to pathologica! tissue damage during PD.
For example, the detection of multiple isoforms of enzymes, such as CK (CKl, CK2, and CK3) indicates that the diagnostic method according to the present invention is not restricted to identifying pathologies in one specific tissue.
Example 8 (SPR in muscle iysate)
The use of tissue lysates in this experiment was to determine whether proteins found in the serum precipitation reaction were tissue specific.
Moreover, through the introduction of muscle lysate to a number of different buffer types it has been shown that precipitation reaction is not limited to sodium acetate trihydrate (Figure 7),
All of these buffers were used at an ionic strength of 0,6M which was found when using sodium acetate trihydrate buffer (Figure 7) to cause a high level of precipitation. While ali of these buffers seemed to also cause a precipitation reaction sodium acetate trihydrate (SAT) or Potassium acetate (PA) had the greatest effect. However, each buffer may have a greater precipitation effect at different molarities.
This has also been extended to a number of well-recognised general precipitation agents including ethanol, ammonium sulphate, and polyethylene glycol (PEG) (Figures 8, 9 and 10).
Example 9 (further identification of precipitated protein)
Using the afore mentioned buffers it was shown (Figures 11 and 12) that precipitation using these is differentia! when using equai amounts of sera from fish depending on disease state with sodium acetate, sodium citrate and potassium acetate appearing to result in the highest degree of precipitation.
Mass spectroscopy of proteins
In total 36 protein spots of the W4pc pooi from the resulting 2DE gel were excised and subjected to trypsin digestion before protein identification via eiectrospray ionisation (ESI) mass spectrometry on an Amazon ion trap MS/MS (Bruker Daltonics) as described in Braceiand et al. (2013),
Chosen protein spots were excised manually by scalpel and placed In Individual vials to be subjected to in~ge! digestion for protein extraction prior to identification via mass spectrometry analysis. Gel pieces were washed with 10G mM NH4HC03 for 30 min and then for a further hour with 100 mM NH4HC03 in 50%(v/v) acetonitrile.
After each wash all solvent was discarded. Gel plugs were then dehydrated with 100% acetonitrile for 10 min prior to solvent being removed and dried completely by vacuum centrifugation. Dry gei pieces were then rehydrated with 10 pi trypsin at a concentration of 20 ng/plin 25 mM NH4HC03 (Cat No. V5li 1, Promega, Madison, wi, USA) and proteins allowed to digest overnight at 37 °C. This liquid was transferred to a fresh tube, and gel pieces washed for 10 min with 10 pi of 50% acetonitrile. This wash was pooled with the first extract, and the tryptic peptides dried to completion. Tryptic peptides were solubilized in 0.5% (v/v) formic acid and fractionated on a nanoflow uHPLC system (Thermo RSLCnano) before analysis by electrospray ionisation (ESI) mass spectrometry on an Amazon ion trap MS/MS (Bruker Daltonics).
Peptide separation was performed on a Pepmap CIS reversed phase column (LC Packings), using a 5-85% v/v acetonitrile gradient (in 0,5% v/v formic acid) run over 45 min, at a flow rate of 0.2pS/min. Mass speetrometric (MS) analysis was performed using a continuous duty cycle of survey MS scan followed by up to five MS/MS analyses of the most abundant peptides, choosing the most intense multiply-changed ions with dynamic exclusion for 120 seconds. MS data were processed using Data Analysis software (Bruker) and the automated Matrix Science Mascot Daemon server (v2.i,06). Protein identifications were assigned using the Mascot search engine to interrogate protein sequences in the NCBI databases restricting the search to teieostei, allowing a mass tolerance of 0.4 Da for both MS and MS/MS analyses. In addition, the search consisted of a carbamidomethyi fixed modification and a variable oxidation.
Mass spectroscopy is a tool currently used to study proteins. Proteins are initially separated using gel electrophoresis as described above. Proteins of interest are excised and subjected to in~gei digestion for extraction prior to identification via mass spectrometry analysis.
Mass spectrometry is an analytical technique which separates components of a sample by their mass. A sample is vaporised into a gas and ionized. The resulting ions are accelerated and focussed into a beam. The ion beam passes through a magnetic field which bends the charged ions. Lighter components or ions with more ionic charge will deflect in the field more than heavier or less charged components. A detector counts the number of different deflections and the data can be plotted s a spectrum of different masses. Different proteins have different masses which can be compared against a database to identify the constituents of a sampie. Mass spectrometry can be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples.
Thirty-six spots were identified for mass spectrometry analysis from W4pc sera (Table 2). Analysis of these spots identified a number of tissue derived enzymes which due to the pathological tissue damage associated with PD are increased in sera (Table 2).
Table 2 shows the protein spots of interest at W4pc. Spots excised and their corresponding identities found via mass spectrometry, score, peptide matches and percent (%) sequence coverage. Score is related to the confidence of the database to identify a given protein. Identification of protein identity is carried out using an algorithm or score where confidence is given through matching peptides in a sample which make up part of a whole protein (= matches) the % sequence coverage is an indication of how identified peptides equate to the full length of protein. In general a score of over 70 indicates a high degree of confidence in the assumed protein identification.
Matches is concerned with the number of peptides in the protein matched with the number of known proteins available on the database and the % sequence coverage is the alignment of the identified peptides with amino acid sequences.
As can be seen from table 2 below, a number of intracellular enzymes were identified such as; three isoforms of creatine kinase, enolase, aidoiase, glyeeraldehyde 3-phosphate dehydrogenase, and pyruvate kinase. There is also an increase in apoiipoprotein AX (spot 23-25, 33, 34) complement C9 (spots 2-4) serotra nsferri n {spot 5, 25) cathepsin (spot 8) and haemoglobin (36), The smaii precipitate formed with WOpc pooled sera comprises mainly apolipoprotein and haemoglobin.
Indeed many of these enzymes, such as; creatine kinase (CK), enolase, aidoiase, and giyceraldehyde-3-phosphate dehydrogenase (GAPDH) have previously been shown in BraceSand et ai, (2013) to be increased in serum concentration associated with pathological damage e.g. muscle tissues.
Table 2, Table 2 shows the protein spots of interest at W4pc, Spots excised and their corresponding identities found via mass spectrometry, score, peptide matches and percent (%) sequence coverage, A clear difference in the precipitate was observed between W4pc and WOpc. The majority of spots identified that were present in W4pc precipitate but absent in WOpc. Creatine kinease, enolase, aldolase and giyceraldehydrate-3-phosphate dehydrogenase were found to precipitate in W4pc serum when combined with sodium acetate trihydrate buffer due to their increasing abundance associated with PD related pathological damage to tissues.
The development of lesions over time is iiiustrated in Figure 6, which shows mean lesion scores for heart, pancreas, white muscle and red muscle at each sampling point. Lesions developed in the pancreas from week 2 and were the slowest to recover, with a minority of tissue samples still not showing fuii recovery by week 12, The heart demonstrated a quick recovery, with a peak in lesion severity in fish sampled in week 4 and then a rapid recovery. The histopathoiogical damage to red and white muscle was delayed with the peak damage occurring at 6 and 8 weeks respectively.
Example 10 (Optimisation of the SPR reaction and application of SPR in cultured fish species)
Tabfe 3. Table 3 shows a summary of the optimisation performed Optimisation of the assay conditions.
The SPR can successfully differentiate between clinically healthy Rainbow trout and those individuals infected with bacteria pathogen Yersinia ruckeri, Application in identifying infection by this specific bacteria! pathogen and for systemic infection by other bacterial species.
Optimisation of buffer conditions for investigating disease In tilapia found 0.6M pH 3,8 SAT be the preferred buffer. Significant Increase in deita OD and visual turbidity during infection with bacterial pathogen Streptococcus agalactiae infection and resulting disease.
The SPR was optimised with selected buffers and salt solutions. Sait solutions were kept at 0.6M in order to keep continuity with the preferred SPR buffer sodium acetate trihydrate. Salt buffers evaluated were ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate trihydrate. Magnesium sulphate, sodium carbonate or ammonium sulphate all produced little or no difference in the Δ340 between diseased and healthy sera potentially making these buffers not applicable for the test at G.6M (Table 3),
Table 3: Change in optical density (Δ340) on addition of either 30μΙ of control serum from a healthy site (Pool A) or 30pl of serum from diseased A. salmon (week 4 post cohabitation infection with SAV where clinical pancreas disease was diagnosed) to wells on a microtitre plate containing 230pi of 0.6M buffers and monitored over a 60 minute period,
Atlantic Salmon Reference Range
In order to determine a reference range the SPR was evaluated from 364 healthy serum samples. From these results, which had a classic bell shaped, gaussian distribution (Figure 13) the reference range for SPR in healthy salmon serum was calculated to be In the range of 0.06 to 0.18 Δ340 AU, The upper limit Is the cutoff point used to differentiate healthy serum from that from fish with diseases. This healthy range serves as an indicator of health status in that any result which is above this threshold would be deemed to be from a diseased individual(s). Moreover, this can be visualized In the SPR qualitative form as seen in Figure 14, This clearly shows the differences observed between diseased and healthy sera are observable without the use of a spectrophotometer,
Atlantic salmon response to vaccination.
The SPR's potential to investigate mechanical damage was carried out on serum samples from Atlantic saimon which had been mock vaccinated with P8S, adjuvant (ADJ), and a commercial vaccination. Serum samples were collected at 70, 250, 450, 600, 750, and 850 days post injection. Results from this study showed that while there appeared to be a slight effect on the precipitate reaction in terms of Δ 340 there was an extremely high initial absorbance reading for SPR at 250 and 450 degree days (Figure 15) resulting from the instant precipitation of SPR, The rapid development of the precipitation may provide an indication to which organs or tissues are affected. These results suggest that the SPR is in response to mechanical damage / physical trauma due to the vaccination procedure. The SPR therefore has application in monitoring of vaccination in fish to identify mechanical damage and this principal wouid apply to monitor health status following invasive surgical procedures by testing serum immediately after the procedure and for a time course to identify when the physiological damage had resolved to identify recovery without the need for further invasive procedures.
Sample Treatment Effects on the SPR in Atlantic salmon (Salmo saiar)
The way in which sampling Is carried out and then sample treatment has been shown numerous times to affect the results of a number of biochemical and immunological assays. Therefore, a number of investigations were carried out to examine any effects on the serum precipitate reaction (SPR),
Unless otherwise stated all assays in Example 10 were carried out using the standard methodology below.
Methodology
Blood was taken from the caudal vein of clinically healthy Atlantic salmon from commercial aquaculture production sites and transferred from syringe into lithium heparin vacutainers. Plasma was then separated from cells by centrifugation at 10,000 g for 2.5 minutes and then placed into eppendorf tubes and stored at 4*C until analysis. Differences between groups were statistically assessed by student T-tests.
Samples were processed on a microtire plate. 15pi of sample (in duplicate) was dispensed to a 96 well piate and placed inside the plate reader (Fluorostar Optima BMG Labtech) at 37°C. Sodium acetate trihydrate buffer (0,6 M at a pH of 5.6) was heated to 37°C before addition of 245pi to each well, Absorbance (Ab) was measured at 340nm over a 60 minute duration with readings being taken every 30 seconds. The change in optical density (Δ 340) was calculated by subtracting the first reading from the final,
Investigation 1: Effect of Collection Tube Type
The effect of collection tube type was assessed through the use of splitting 1.5ml (total) of blood equally Into three different tubes. Tube type 1 and 2 were both lithium heparin vacutainers for plasma collection and standard non heparinised tubes for serum collection,
Separated samples were stored at 4°C and SPR assay carried out on all samples at 24 hours and 48 hours post separation. Mean delta OD results can be seen in Figure 16 and 17 for 24 and 48 hours post separation assays respectively. There was no significant tube type effect indicating that both serum and plasma yields similar results.
Investigation 2: Sample Storage 4«C vs Room Temperature (RT)
The effect of storage temperature of samples was investigated by taking 3ml of blood from fish and separating this equally into two 1.5ml capacity lithium heparin vacutainers. Plasma was separated from cells 3 hours post collection of blood from fish (n = 40) and either stored at 4C or room temperature (RT) for 72 hours before being assayed.
This investigation found that there was a significant decrease in mean SPR activity of samples when stored at room temperature when compared to those stored at 4°C (P ~ 0.005) (Figure 18). Therefore serum samples should be kept at cool temperature e.g. 4C until analysis.
Investigation 3: Storage at 4°C vs Freeze Thaw Cycles To investigate the effects of freeze thaw cycles plasma was collected by centrifugation three hours post blood sampling. In total 3mi was taken from each fish and split equally into two iithium-heperinised vacutainers and plasma separated. Plasma from 20 fish (40 tubes) was then split into aliquots with one going into storage at 4«C until analysis and additional replicated serum samples from the same corresponding 20 fish subjected to two freeze thaw cycles using -8Q»C temperature (Figure 19). In addition, a separate set of plasma from 20 other fish were split and held at either 4«C or subjected to four freeze thaw cycles (Figure 20). There was no significant effect on SPR results as a consequence of either 2 or 4 freeze thaw cycles. These assays were carried out 96 hours post separation and the SPR results from those held at 4«C are comparable to results from assays carried out at times doser to sampling. Therefore storage for up to one week at 4< does not result in significant breakdown of protein and thus does not affect the SPR. The assay can therefore be applied in serum samples taken up to 1 week previously without compromising the analysis resuit which would be beneficial for application in remote geographical areas, Furthermore, as the assay is not affected by 4 freeze thaw cycles indicates that the recommended directions for transport of samples should be to freeze samples before despatch to the place of analysis to keep samples as cool as possible while in transit.
Key Findings
Both Plasma and Serum can be used for the SPR and results from the same fish are almost identical when serum and plasma from it are compared.
Storing at room temperature reduces the SPR delta OD after 72 hours crucially this lowering of SPR activity may lead to wrong judgement of health status. Storage at freezing temperatures is preferable, though at 4*C the SPR remains stable at ieast for 96 hours and transport on ice packs is sufficient. SPR in Atlantic Salmon Challenged With bacterial pathogen Moritelia Viscosa
To investigate the use of the SPR in diagnosing, monitoring, and establishing the severity of Moriteila vtscosa 30 pre-chaiienge sera samples were subjected to the assay (0.6M SAT at pH 5,6) aiong with 20 post-chaiienge samples , Table 4 shows the mean delta OD of these two groups along with the standard deviation and error, this is represented as a histogram in Figure 21,
Table 4: The average delta OD at 340nm when sera from pre challenge (n=30) and post Moritefia viscosa challenge
Key Findings SPR may be used as an effective tool in diagnosing, monitoring, and establishing the severity of moritella viscosa outbreaks in Atlantic salmon.
Application of SPR in Atlantic salmon (Salmo salar) during pancreas disease challenge.
The ability of functional feeds to reduce tissue pathology in Atlantic salmon was assessed using the SPR on serum samples from a pancreas disease laboratory challenge. 27 serum samples per diet were analysed at nine time points throughout a disease challenge. Figure 22 shows the separation of results by diet through sampling time course from WOpc to W12pc. This analysis showed that from W4pc to W8pc that mean Δ340 was lower in fish fed diet B (functional feed) than those fed diet A (control) indicating a lowering of severity of pathological damage.
Key findings
Serum SPR was reduced in the experimental group of fish fed diet B (functional feed)
Rainbow Trout,
Sera from twenty seven healthy and twenty five rainbow trout infected with Yersinia ruckeri (the aetological agent of red mouth disease) were examined using the SPR, Figure 23 shows the mean delta OD from these samples when results were grouped as healthy and diseased (RM), A statistically significant difference (P =< 0.0001) in delta OD change was observed in the diseased group using a t test compared to the healthy group. While gross pathology or visual inspection of mouth and skin of the fish is a reliable tool in diagnosing the condition the SPR may be useful in non-destructively diagnosing the severity of haemorrhaging and pathology to internal organs.
Establishing Reference Ranges in Trout and Sea Bream .
In order to determine a reference range the SPR was evaluated from 11 healthy sea bream and 26 rainbow trout. The preferred reference ranges of these two species was found to be 0.09 to 0.25 in sea bream and <0.026 to 0.21 in trout. Figure 24 shows median results for these two species using healthy sera with 25* and 75th percentiles in box plot and 10-9Qth percentile in whiskers. SPR in Nile Tiiapia {Oreochromis nitoticus)
Serum samples from Nile tiiapia {Oreochromis niioticus) infected with Streptococcus agalactiae were assayed for SPR to investigate the potential for the test to be used in this specific infection and as a health monitoring tool in Nile tiiapia aquaculture production, initial Investigations using 0.6M pH 5.6 sodium acetate trihydrate (SAT) buffer found no clear difference between control and diseased samples. Therefore, the effect of pH was investigated in this species. Figure 25 shows the delta OD of control (NT CP), infected (NT IP) and moribund (NT MP) sera pools when introduced to 0.6M SAT at differing pH and a 2M SAT at pH 4,8.
This showed that at a more acidic pH it was possible to optimise the SPR in diseased individuals while minimizing the precipitation levels in healthy control samples. Not only was this difference pronounced at a quantitative level but is easily visualised in the assays qualitative format as seen in Figure 26.
The SPR can be applied in tiiapia in identifying clinical disease of strep, (species) but also in other bacterial diseases also.
Figure 27 shows the deita OD of control, infected and moribund Nile tiiapia sera pools following Streptococcus agalactiae challenge (0.6M SAT at pH 4.8).
Items 1, Diagnostic method for determining whether a fish is infected with a pathogen capabie of causing pathologies! tissue damage in a fish wherein the method comprises the consecutive steps of; i) mixing fish biood serum with an aqueous protein precipitation solution that causes increased protein precipitation when admixed with serum from tissue damaged fish compared to the corresponding protein precipitation observed in cilnicafiy healthy fish, ii) determining whether the fish is infected by establishing: iia) whether visible precipitate is formed, which in the affirmative is indicative of infection in the fish, or iib) whether the resulting mixture has no visible precipitate, which in the affirmative is indicative of clinically healthy fish, 2, Diagnostic method for determining the stage of a pathogen causing disease resulting in pathological tissue damage in fish, the method comprising the consecutive steps of: i) successively withdrawing blood samples from fish over a period of time (e.g. WOpc, Wipe, W2pc etc.) ii) mixing the fish blood serum from the blood samples obtained under i) with an aqueous protein precipitation solution that causes increased protein precipitation when admixed with serum from tissue damaged fish compared to the corresponding protein precipitation observed in clinically healthy fish, iii) determining the concentrations of the resulting protein precipitation samples from each of the mixtures obtained in step ii) iv) comparing the concentrations of each of the protein precipitation samples and thereby determining the stage of the disease by establishing: iva) whether the precipitation concentration of a sample {e.g. W2pc) is higher than the concentration of the immediately preceding sample (e.g. Wipe) which in the affirmative Is indicative of progress of disease, or ivb) whether the precipitation concentration of a sample (e.g . W2pc) is essentially equal to the concentration of the immediately preceding sample (e.g. Wipe) which in the affirmative is indicative of to steady state stage of the disease ivc) whether the precipitation concentration of a sample (e.g. W2pc) is Sower than the concentration of the immediately preceding sample (e.g. Wipe) which in the affirmative is indicative of recovery from the disease. 3 Diagnostic method for determining the stage of a pathogen causing disease resulting in pathological tissue damage in fish, the method comprising the consecutive steps of: i) successively withdrawing biood sampies from fish over a period of time (e.g. wopc, wipe, W2pc etc.) ii) mixing the fish blood serum from each of the biood samples obtained under i) with an aqueous protein precipitation solution that causes increased protein precipitation when admixed with serum from tissue damaged fish compared to the corresponding protein precipitation observed in clinically healthy fish, iii) determining the optical density (level of turbidity) of the sample mixed with aqueous protein precipitation solution, iv) determining the optical density (level of turbidity) in a sample mixed with aqueous protein precipitation solution (e.g. W2pc) and in the immediately preceding sample (e.g. Wipe) v) optionally repeat step iv) with further successively obtained sampies mixed with aqueous protein precipitation solution (e.g. W3pc, W4pcetc.), vi) determining the change in optical density of the samples mixed with aqueous protein precipitation solution determined in step iv) and optionally in step v) vii) thereby assessing the severity of the infectious disease as said severity is proportional with the degree of change in optical density (i.e. the higher the degree of change in optical density the more severe is the infectious disease). 4. Method according to any of claims 1-3, wherein the aqueous protein precipitation solution is chosen from soiutions in water of one or more acids, bases, salts, organic compounds, inorganic compounds, ionic buffers or mixtures thereof. 5. Method according to ciaim 4, wherein the salt is chosen from ammonium sulphate, magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate trihydrate or mixtures thereof. 6. Method according to ciaim 4, wherein the organic compound is chosen from methanol, ethanol, acetone or mixtures thereof. 7. Method according to ciaim 4, wherein the inorganic compound is polyethyieneglycol (PEG). 8. Method according to any of claims 1-7, wherein the fish is of the family Salmonidae such as Atlantic salmon (Saimo salar L.), Adriatic trout (Salmo obtmlrostris), Flathead trout (Salmo platycephalus), Marble trout (Salmo marmoratus), Ohrid trout (Salmo letnica), Sevan trout (Salmo ischchan), Brown trout {Salmo trutta), also including fish from the genus Saiveiinus, such as Arctic char (Saiveiinus alpinus), also including fish from the genus Oncorhynchus, such as rainbow trout (Oncorhynchus my kiss), coho salmon (Oncorhynchus kisutch) and Chinook salmon (Oncorhynchus tsbawytscba)« 9. Method according to any of claims 1-8, wherein the pathogen is chosen from the group consisting of Salmon o-virus, salmonid alphavirus, Salmon Pancreas Disease Virus (SPDV), salmonid alphavirus (SAV), Piscine Myocarditis Virus, Piscine reovirus (PRV), Atlantic Salmon Calclvirus (ASCV), Aquabirnaviruses, Infectious pancreatic necrosis virus (IPNv), ISA virus or viral hemorrhagic septicemia virus (VHSV). 10. Method according to any of claims 1-8, wherein the pathogen is chosen from the group consisting of Aeramonas saimonlcidia subsp saimoniddia, Vibrio saimoniddia, Vibrio angutiiarum, Moritelia viscosus, Pisdrickettsia saimonis, Yersinia ruckeri or Flavobacterium psychrophilum. 11. Method according to any of claims 1-8, wherein the pathogen is Saprolegnia SPP· 12. Method according to any of claims 1-8, wherein the pathogen is chosen from the group consisting of Neoparameabe peruans or Ichthyophtbirius muftifiliis, 13. Method according to any of claims 1-8, wherein the pathological tissue damage is caused by one or more of the following diseases: Pancreas Disease (PD), Salmon Pancreas Disease (SPD), Heart and Skeletal Muscle Inflammation (HSMI), infectious pancreatic necrosis (IPN), Myopathy Syndrome (CMS), Infectious salmon anaemia (ISA), viral hemorrhagic septicemia (VHS), acute myopathy, immune reactions during chronic diseases caused by viruses, furunculosis, cold water vibriosis, vibriosis, winter ulcer disease, pisdrickettsiosis, salmonid rickettsial septicemia (SRS), enteric redmouth (ERM), rainbow trout fry syndrome (RTFS), bacterial kidney disease (BKD), Saprolegniasis, Ameobic giil disease (AGD) or freshwater white spot disease. 14. Method according any of claims 1-13, wherein the determination of visible precipitate is detected by the naked eye.
References
Adams A &amp; Thompson KD (2011) Development of Diagnostics for Aquaculture -Challenges and Opportunities, Aquaculture Research, 42, 93-102,
Asche Fr., Guttormsen A.G., Nielsen R. (2013) Future challenges for the maturing Norwegian salmon aquaculture industry: An analysis of total factor productivity change from 1996 to 2008. Aquaculture, 396-399, 43-50. M. Braceland, R. Bickerdike, 3, Tinsley, D. CockeriSS, M.F, Mcloughlin, D.A, Graham,, RJ. Burchmore, W. Weir, C, Wallace, P.D. Eckersall (2013) The serum proteome of Atlantic salmon, Salmo salar, during pancreas disease (PD) following infection with saSmonid alphavirus subtype 3 (SAV3)J Proteomics, 94, 423-436.
Braceland M. M.F. McLoughlsn, 1 Tinsley, C. Wallace, D. Cockerill, M. McLaughlin, P.D. Eckersall (2014) Serum enolase: a non-destructive biomarker of white skeletal myopathy during pancreas disease (PD) in Atlantic salmon Salmo salar L Journal of fish diseases 38 (9), 821-831
Centoia et a!., 2013: Centoia, M<, Cavet, G., Shen, Y,, Ramanujan, S,, Knowlton, N. , Swan, K. A, Curtis, 1. R. (2013). Development of a multi-biomarker disease activity test for rheumatoid arthritis. PioS One, 8(4), 1-6.
Eckersall PD, Bell R (2010). Acute phase proteins: Biomarkers of infection and inflammation in veterinary medicine. Veterinary Journal. 185. 23-27.
Haluzova et al. ¢2010): Haluzova L, Modra H., Blahova., Marsalek P., Siroka Z., Groch., Svobodova. (2010) Effects of subchronic exposure to Spartakus (prochloraz) on common carp Cyprinus carpio. Neruro endocrino lett. 2, 105-113,
Jansen M.D., Wasmuth M.A., Olsen A.B., Gjerset B., Modahl L, Breck 0., Haldorsen R.N., Hjelmeland R, Taksdal T. (2010) Pancreas disease (PD) in sea-reared Atlantic salmon, Salmo salar L, in Norway; a prospective, longitudinal study of disease development and agreement between diagnostic test results. Journal of Fish Diseases 33, 723-736.
Kibenge FSB, Godoy MG, Fast M, Workenhe S, Kibenge PUT, (2012), Countermeasures against viral diseases of farmed fish. Antiviral Research.
Larsson et al,, 2012: Larsson T,, Mørkøre T.,. Koistad K, Østbye, S, Afanasyev T.-K,, Krasnov A. (2012) Gene expression profiling of soft and firm Atlantic salmon fillet. PLOS ONE, 7, p. e39219.
LerfiaS! et a!,, 2011: Lerfal! X, Larsson 1., Birkeland S., Taksdal T., Daigaard P., Afanasyev S,, Bjerke M.T., Mørkøre 1(2011), Effect of pancreas disease (PD) on quality attributes of raw and smoked fillets of Atlantic salmon (Salmo salar L) Aquaculture, 324-325, 209-217
Nielsen, 2002: Nielsen K., (2002), Diagnosis of brucellosis by serology, Veterinary Microbiology, 90, 447- 459.
McLoughiin M.F., Graham D.A., Norris A., Matthews D.,Foyle L, Rowley H.M., jewhurst H., MacPhee J. &amp; Todd D. (2006) Viroiogical, serological and histopathologicaS evaluation of fish strain susceptibility to experimental infection with salmonld alphavlrus. Diseases of Aquatic Organisms 72, 125-133.
Tacon A.G.T, Metian M. (2008) Globa! overview on the use offish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects 285, 146-158.
Yada et al. 2004:Yada T,, Muto K., Azuma T,, Ikuta K. (2004) Effects of prolactin and growth hormone on plasma levels of lysozyme and ceruloplasmin in rainbow trout, Comparative Biochemistry and Physiology Part C: Toxicology &amp; Pharmacology. 139, 57-63.

权利要求:
Claims (14)
[1] 1. A method for determining and/or screening and/or monitoring pathological tissue damage in a fish, the method comprises the steps of: i) obtaining blood from a fish, ii) mixing serum or plasma from the blood obtained in step (i), with an aqueous protein precipitation solution and obtaining a mixture, iii) visually determining the level of turbidity in the mixture obtained in step (ii), iv) visually comparing the level of turbidity in step (iii) with the level of turbidity in a reference solution, v) determining pathological tissue damage in said fish if the visual level of turbidity is higher in the mixture of step ii) compared to the level of turbidity in the reference solution and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
[2] 2. A method for determining and/or screening and/or monitoring pathological tissue damage in a fish, the method comprises the steps of: i) obtaining blood from a fish, ii) mixing serum or plasma from the blood obtained in step i), with an aqueous protein precipitation solution, and obtaining a mixture iii) determining the level of turbidity in the mixture obtained in step ii) iv) comparing the determined level of turbidity in step iii) with a reference range, v) determining pathological tissue damage in said fish if the determined level turbidity is above the reference-range, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
[3] 3. A method for determining the stage and/or severity of pathological tissue damage in fish and/or for monitoring pathological tissue damage in a fish, the method comprising the steps of: i) successively withdrawing blood samples from a fish over a period of time, ii) mixing serum or plasma from the blood obtained in step i) with an aqueous protein precipitation solution, iii) determining the level of turbidity from each of the mixtures obtained in step ii) iv) comparing the level of turbidity of each of the mixtures and thereby determining: iva) whether the level of precipitation of the mixture is higher than the level of turbidity of the immediately preceding mixture which is indicative of progress of pathoogical tissue damage, or ivb) whether the level of precipitation of the mixture is essentially equal to the level of turbidity of the immediately preceding mixture which is indicative of a steady state stage of the pathological tissue damage, or ivc) whether the level of turbidity of the mixture is lower than the level of turbidity of the immediately preceding sample which is indicative of recovery from the pathological tissue damage, and wherein the aqueous protein precipitation solution comprises a salt selected from the group consisting of magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
[4] 4. Method according to any one of claim 1 or 2, wherein the aqueous protein precipitation solution is a solution in water.
[5] 5. Method according to any of claims 1-3, wherein the aqueous protein precipitation solution comprises one or more compounds selected from the group consisting of acids, bases, salts, organic compounds, inorganic compounds, ionic buffers and mixtures thereof.
[6] 6. Method according to claim 4, wherein the organic compound is selected from the group consisting of methanol, ethanol, acetone and mixtures thereof.
[7] 7. Method according to claim 4, wherein the inorganic compound is polyethyleneglycol (PEG).
[8] 8. Method according to any one of the proceeding claims, wherein the aqueous protein precipitation solution is selected from the group consisting of sodium acetate buffer, magnesium sulphate buffer, ammonium phosphate buffer, sodium carbonate buffer, sodium sulphate buffer, potassium acetate buffer, sodium citrate buffer, sodium phosphate buffer and mixtures thereof.
[9] 9. Method according to any of the preceding claims, wherein the fish is selected from the group consisting of the family Salmonidae, the family Serranidae, the family Cyprinidae, the family Sciaenidae and the family Latidae
[10] 10. Method according to any of the preceding claims, wherein the fish is selected from the group consisting of the genus Salvelinus, the genus Oncorhynchus, the genus Sparus, the genus Dicentrarchus, the genus Dicentrarchus, the genus Epinephelus, the genus Oreochromis, the genus Pangasius, the genus Seriola, the genus Ctenopharyngodon, the genus Hypophthalmichthys, the genus Cyprinus, the genus Carassius, the genus Catla, the genus Labeo, the genus Larimichthys and the genus Lates.
[11] 11. A method according to any one of the preceding claims, wherein the pathological tissue damage is caused by a pathogen selected from the group consisting of virus, bacteria, parasites and combinations thereof.
[12] 12. Method according any of the preceding claims, wherein the determination of visible precipitate is detected by the naked eye.
[13] 13. Use of a kit for performing the method according to claims 1-12, said kit comprises an aqueous protein precipitation solution comprising a salt selected from the group consisting of magnesium sulphate, ammonium phosphate, sodium carbonate, sodium sulphate, potassium acetate, sodium citrate, sodium phosphate, sodium acetate and mixtures thereof.
[14] 14. Use of the method according to claims 1-12, for developing and/or validating and/or monitoring efficacy of interventions to improve the immune response and/or host response in fish including but not limited to vaccines, biosecurity practices, diet and combinations thereof.

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法律状态:
2018-09-28| PHB| Application deemed withdrawn due to non-payment or other reasons|Effective date: 20180722 |

优先权:

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EP14199554.8A|EP3037823A1|2014-12-22|2014-12-22|Method for determining pathological tissue damage and diagnosing infectious disease in fish|

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