Patent Application: US-201113580015-A

Abstract:
gene expression technologies have the exciting potential of providing methods for monitoring long - term effects of contaminants and disease on free - ranging marine wildlife species . an added benefit is that these methods may elucidate the mechanisms by which these stressors can deleteriously affect an individual over a long period , and thereby aid in the design of therapeutic and preventative strategies to treat and protect susceptible individuals and populations at risk from oil exposure . our presentation will assess specific quantifiable genetic markers that can signify persistent pathological and physiological injury associated primarily with chronic hydrocarbon exposure . using empirical evidence from captive animals and recent captures , we will discuss how we are developing an understanding of gene expression as it relates to the immune system of the sea otter and other marine megafauna , and the potential effects of contaminants or disease .

Description:
to aid in understanding the invention , several terms are defined below . “ amplicon ” refers to a polynucleotide that is amplified from a bioagent in an amplification reaction . as used herein , a “ bioagent ” is any organism , cell , or virus , living or dead , or a nucleic acid derived from such an organism , cell or virus . examples of bioagents include , but are not limited , to cells , ( including but not limited to human clinical samples , bacterial cells and other pathogens ), viruses , fungi , protists , parasites , and pathogenicity markers ( including but not limited to : pathogenicity islands , antibiotic resistance genes , virulence factors , toxin genes and other bioregulating compounds ). samples may be alive or dead or in a vegetative state ( for example , vegetative bacteria or spores ) and may be encapsulated or bioengineered . as used herein , the terms “ complementary ” or “ complementarity ” are used in reference to polynucleotides ( i . e ., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid ) related by the base - pairing rules . for example , for the sequence “ 5 ′- a - g - t - 3 ′,” is complementary to the sequence “ 3 ′- t - c - a - 5 ′.” complementarity may be “ partial ,” in which only some of the nucleic acids &# 39 ; bases are matched according to the base pairing rules . or , there may be “ complete ” or “ total ” complementarity between the nucleic acids . the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands . this is of particular importance in amplification reactions , as well as detection methods that depend upon binding between nucleic acids . either term may also be used in reference to individual nucleotides , especially within the context of polynucleotides . for example , a particular nucleotide within an oligonucleotide may be noted for its complementarity , or lack thereof , to a nucleotide within another nucleic acid strand , in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand . the term “ complement of a nucleic acid sequence ” as used herein refers to an oligonucleotide which , when aligned with the nucleic acid sequence such that the 5 ′ end of one sequence is paired with the 3 ′ end of the other , is in “ antiparallel association .” certain bases not commonly found in natural nucleic acids may be included in the nucleic acids disclosed herein and include , for example , inosine and 7 - deazaguanine . complementarity need not be perfect ; stable duplexes may contain mismatched base pairs or unmatched bases . those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including , for example , the length of the oligonucleotide , base composition and sequence of the oligonucleotide , ionic strength and incidence of mismatched base pairs . where a first oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region ( or a portion of this region ) a “ region of overlap ” exists along the target nucleic acid . the degree of overlap will vary depending upon the extent of the complementarity . the term “ duplex ” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding the their complementary bases arrayed on a second strand . the condition of being in a duplex form reflects on the state of the bases of a nucleic acid . by virtue of base pairing , the strands of nucleic acid also generally assume the tertiary structure of a double helix , having a major and a minor groove . the assumption of the helical form is implicit in the act of becoming duplexed . the term “ gene ” refers to a dna sequence that comprises control and coding sequences necessary for the production of an rna having a non - coding function ( e . g ., a ribosomal or transfer rna ), a polypeptide or a precursor . the rna or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained . the terms “ homology ,” “ homologous ” and “ sequence identity ” refer to a degree of identity . there may be partial homology or complete homology . a partially homologous sequence is one that is less than 100 % identical to another sequence . determination of sequence identity is described in the following example : a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non - identical residues has 18 of 20 identical residues ( 18 / 20 = 0 . 9 or 90 % sequence identity ). in another example , a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of a primer 20 nucleobases in length would have 15 / 20 = 0 . 75 or 75 % sequence identity with the 20 nucleobase primer . as used herein , sequence identity is meant to be properly determined when the query sequence and the subject sequence are both described and aligned in the 5 ′ to 3 ′ direction . sequence alignment algorithms such as blast , will return results in two different alignment orientations . in the plus / plus orientation , both the query sequence and the subject sequence are aligned in the 5 ′ to 3 ′ direction . on the other hand , in the plus / minus orientation , the query sequence is in the 5 ′ to 3 ′ direction while the subject sequence is in the 3 ′ to 5 ′ direction . it should be understood that with respect to the primers disclosed herein , sequence identity is properly determined when the alignment is designated as plus / plus . sequence identity may also encompass alternate or modified nucleobases that perform in a functionally similar manner to the regular nucleobases adenine , thymine , guanine and cytosine with respect to hybridization and primer extension in amplification reactions . in a non - limiting example , if the 5 - propynyl pyrimidines propyne c and / or propyne t replace one or more c or t residues in one primer which is otherwise identical to another primer in sequence and length , the two primers will have 100 % sequence identity with each other . in another non - limiting example , inosine ( i ) may be used as a replacement for g or t and effectively hybridize to c , a or u ( uracil ). thus , if inosine replaces one or more c , a or u residues in one primer which is otherwise identical to another primer in sequence and length , the two primers will have 100 % sequence identity with each other . other such modified or universal bases may exist which would perform in a functionally similar manner for hybridization and amplification reactions and will be understood to fall within this definition of sequence identity . the term “ highly conserved ,” it is meant that the sequence regions exhibit between about 80 - 100 %, or between about 90 - 100 %, or between about 95 - 100 % identity among all , or at least 70 %, at least 80 %, at least 90 %, at least 95 %, or at least 99 % of species or strains . as used herein , the term “ hybridization ” is used in reference to the pairing of complementary nucleic acids . hybridization and the strength of hybridization ( i . e ., the strength of the association between the nucleic acids ) is influenced by such factors as the degree of complementary between the nucleic acids , stringency of the conditions involved , and the tm of the formed hybrid . “ hybridization ” methods involve the annealing of one nucleic acid to another , complementary nucleic acid , i . e ., a nucleic acid having a complementary nucleotide sequence . the ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well - recognized phenomenon . the term “ oligonucleotide ” refers to a molecule comprised of two or usually more the dexoribonucleotides or ribonucleotides , such as primers , probes , nucleic acid fragments to be detected , and nucleic acid controls . the exact size of an oligonucleotide depends on many factors and the ultimate function or use of an oligonucleotide . oligonucleotides can be prepared by any suitable method , including , for example , cloning and restriction of appropriate sequences and direct chemical synthesis by method known in the art . the term “ primer ” refers to an oligonucleotide , whether natural or synthetic , capable of acting as a point of initiation of dna synthesis under conditions in which synthesis of a primer extension product , complementary to a nucleic acid strand is induced , i . e ., in the presence of four different deoxyribonucleic acid triphosphates and an agent for polymerization ( i . e ., dna polymerase or reverse transcriptase ) in an appropriate buffer at a suitable temperature . a primer is preferably a single stranded oligodeoxyribonucleotide . the appropriate length of the primer depends on the intended use of the primer but typically ranges from 5 to 25 nucleotides . a primer need not reflect exact sequence of the template that must be sufficiently complementary to hybridize with the template and search initiated dna synthesis . properties of the primers may include any number of properties related to structure including , but not limited to : nucleobase length which may be contiguous ( linked together ) or non - contiguous ( for example , two or more contiguous segments which are joined by a linker or loop moiety ), modified or universal nucleobases ( used for specific purposes such as for example , increasing hybridization affinity , preventing non - templated adenylation and modifying molecular mass ) percent complementarity to a given target sequences . properties of the primers also include functional features including , but not limited to , orientation of hybridization ( forward or reverse ) relative to a nucleic acid template . the coding or sense strand is the strand to which the forward priming primer hybridizes ( forward priming orientation ) while the reverse priming primer hybridizes to the non - coding or antisense strand ( reverse priming orientation ). the functional properties of a given primer pair also include the generic template nucleic acid to which the primer pair hybridizes . for example , identification of bioagents can be accomplished at different levels using primers suited to resolution of each individual level of identification . broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division ( e . g ., an order , family , genus or other such grouping of bioagents above the species level of bioagents ). in some embodiments , broad range survey intelligent primers are capable of identification of bioagents at the species or sub - species level . other primers may have the functionality of producing bioagent identifying amplicons for members of a given taxonomic genus , cladespecies , sub - species or genotype ( including genetic variants which may include presence of virulence genes or antibiotic resistance genes or mutations ). additional functional properties of primer pairs include the functionality of performing amplification either singly ( single primer pair per amplification reaction vessel ) or in a multiplex fashion ( multiple primer pairs and multiple amplification reactions within a single reaction vessel ). as used herein , the terms “ pair of primers ,” or “ primer pair ” are synonymous . a primer pair is used for amplification of a nucleic acid sequence . a pair of primers comprises a forward primer and a reverse primer . the forward primer hybridizes to a sense strand of a target gene sequence to be amplified and primes synthesis of an antisense strand ( complementary to the sense strand ) using the target sequence as a template . a reverse primer hybridizes to the antisense strand of a target gene sequence to be amplified and primes synthesis of a sense strand ( complementary to the antisense strand ) using the target sequence as a template . the primers are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent , and which are amenable to molecular mass analysis . in some embodiments , the highly conserved sequence regions exhibit between about 80 - 100 %, or between about 90 - 100 %, or between about 95 - 100 % identity , or between about 99 - 100 % identity . the molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained , due to the variability of the variable region . thus design of the primers requires selection of a variable region with appropriate variability to resolve the identity of a given bioagent . bioagent identifying amplicons are ideally specific to the identity of the bioagent . in the disclosed embodiments of the invention , specific sequence primers and probes are provided . it will be apparent to those of skill in the art that provided with those embodiments , specific sequence primers and probes can be modified by , for example , the addition of nucleotides that either the 5 ′ or 3 ′ ends , which nucleotides are complementary to the target sequence or are uncomplimentary to the target sequence . so long as primer composition serves a point of initiation for extension on the target sequences , and the primers and probes comprise at least 5 consecutive nucleotides contained within those exemplified embodiments , such compositions are within the scope of the invention . the term “ primer ” may refer to more than one primer , particularly in the case when there is some ambiguity in the information regarding one or both ends of the target region to be amplified . if a “ conserved ” region shows significant levels of polymorphism in a population , mixtures of primers can be prepared that will amplifies such sequences , or the primers can be designed to amplify even mismatch sequences . a primer can be labeled , if desired , by incorporating a label detectable by spectroscopic , photochemical , biochemical , immunochemical , or chemical means . for example , useful labels include 32p , fluorescent dyes , electron - dense reagents , enzymes ( as is commonly used in elisas ), biotin , or haptens and proteins for which antisera or monoclonal antibodies are available . a label can also be used to “ capture ” the primer , so as to facilitate immobilization of either the primer or a primer extension product , such as amplified dna , on a solid support . as used herein , the terms “ purified ” or “ substantially purified ” refer to molecules , either nucleic or amino acid sequences , that are removed from their natural environment , isolated or separated , and are at least 60 % free , preferably 75 % free , and most preferably 90 % free from other components with which they are naturally associated . an “ isolated polynucleotide ” or “ isolated oligonucleotide ” is therefore a substantially purified polynucleotide . the term “ reverse transcriptase ” refers to an enzyme that catalyzes the polymerization of nucleoside triphosphates to form primer extension products that are complementary to a ribonucleic acid template . the term “ sample ” in the present specification and claims is used in its broadest sense . on the one hand it is meant to include a specimen or culture ( e . g ., microbiological cultures ). on the other hand , it is meant to include both biological and environmental samples . a sample may include a specimen of synthetic origin . biological samples may be animal , including human , fluid , solid ( e . g ., stool ) or tissue , as well as liquid and solid food and feed products and ingredients such as dairy items , vegetables , meat and meat by - products , and waste . biological samples may be obtained from all of the various families of domestic animals , as well as feral or wild animals , including , but not limited to , such animals as ungulates , bear , fish , lagamorphs , rodents , otters , minks , sea lions , etc . environmental samples include environmental material such as surface matter , soil , water , air and industrial samples , as well as samples obtained from food and dairy processing instruments , apparatus , equipment , utensils , disposable and nondisposable items . these examples are not to be construed as limiting the sample types applicable to the methods disclosed herein . the term “ source of target nucleic acid ” refers to any sample that contains nucleic acids ( rna or dna ). particularly preferred sources of target nucleic acids are biological samples including , but not limited to blood , saliva , cerebral spinal fluid , pleural fluid , milk , lymph , sputum and semen . as used herein , the term “ sample template ” refers to nucleic acid originating from a sample that is analyzed for the presence of “ target ” ( defined below ). in contrast , “ background template ” is used in reference to nucleic acid other than sample template that may or may not be present in a sample . background template is often a contaminant . it may be the result of carryover , or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample . for example , nucleic acids from organisms other than those to be detected may be present as background in a test sample . a “ segment ” is defined herein as a region of nucleic acid within a target sequence . a “ target ” dna is used in this application also includes nucleic acids which are added to a test specimen to provide positive controls and the assets . a “ pcr reagent ” refers to any of the reagents considered essential to pcr , namely primers for the target nucleic acid , a thermostable dna polymerase , a dna polymerase cofactor , and one or more deoxyribonucleoside - 5 ′- triphosphates . other optional reagents and materials used in pcr may be used and are described below . the term “ thermostable polymerase enzyme ” refers to an enzyme that is relatively stable to heat and catalyzes polymerization of nucleoside triphosphates to form primer extension products that are complementary to one of the nucleic acid strands of the target sequence . the present invention may employ conventional molecular biology , microbiology and recombinant dna techniques within the skill of the art . some of these techniques are set forth in the literature — i . e ., “ qiaex ii ® handbook — for dna extraction from agarose and polyacrylamide gels and for desalting and concentrating dna from solutions ,” october ( 2008 ); “ invitrogen ™— topo ta cloning ® kit for sequencing — five - minute cloning of taq polymerase - amplified pcr products for sequencing ,” user manual , version o ( invitrogen 2006 ); “ paxgene ® blood rna kit handbook ,” version 2 ( paxgene 2009 ); sambook , molecular cloning : a laboratory manual , third edition ( sambook 2001 ). applicants selected fifteen ( 15 ) sea otter genes in six different systems as genes of interest . these are set forth in table 1 . these were selected based on applicants &# 39 ; knowledge of the effects of oil exposure on sea otters . the six different systems from which these genes were selected are ( 1 ) immune defense , ( 2 ) signal transduction , ( 3 ) tumorigenesis , ( 4 ) cellular injury , ( 5 ) xenobiotic metabolism , and ( 6 ) reproduction . specific primer sets for these genes were designed , and are set forth in table 2 . the primer sets were designed using the general laboratory methodology , and set forth in greater detail below . each primer set comprises a forward primer and corresponding reverse primer . these novel primer sets are a result of extensive trial - and - error experimentation . although applicants employed conventional , industry - developed techniques in designing these novel primer sets , the specific methodology employed in making these primer sets are a result of considerable experimentation , and required performance of very specific steps under very specific conditions . the methods of the present invention employ these novel primers for use in identifying and measuring numerous , pathophysiological changes in sea otters due to their exposure to toxins — i . e ., oil . these primers may be used to analyze genetic information contained in the blood , or other bodily fluids collected from sea otters so as to assess and monitor the effect of exposure to toxins on the health and wellbeing of the animal . the primers identified herein are “ function specific ” primers . they amplify genes having specific functions . note that table 1 sets forth the function of each gene of interest . the present invention further comprises a diagnostic or monitoring tool for prognosing or predicting disease or other health issues or , in general , assessing the health of sea otters by detecting the quantitative expression levels of specific genes affected by exposure or possible exposure to toxins , such as petroleum - based compounds . these expression levels are compared to the expressed levels of the same genes obtained from control sea otters that were maintained in clean aquarium environments and routinely monitored for health . six systems of the sea otter upon which crude oil or components of crude oil were found to have an effect were selected . these six systems include the ( 1 ) immune defense , ( 2 ) signal transduction , ( 3 ) tumorigenesis , ( 4 ) cellular injury , ( 5 ) xenobiotic metabolism , and ( 6 ) reproduction . fifteen ( 15 ) genes within these systems were identified , and are set forth in table 1 . degenerate primers for these genes were designed based upon multi - species alignments ( genbank ). once designed , all degenerate or nonspecific primers were ordered from eurofins mwg operon ( huntsville , ala .). complementary dna ( cdna ) was generated from sea otter blood samples obtained from dr . dave jessup , a california fish and game veterinarian permitted by the state and federal government to maintain captive sea otters . the degenerate primers were utilized on the cdna generated from the bloods of three sea otters in his facility . polymerase chain reaction ( pcr ) amplifications using these degenerate primers were performed on 20 ng of each cdna sample in 50 μl volumes containing 20 - 60 pmol of each degenerate primer , 4 . 0 mm tris - koh ( ph 8 . 3 ), 15 mm koac , 3 . 5 mm mg ( oac ) 2 , 3 . 75 μg / ml bovine serum albumin ( bsa ), 0 . 005 % tween - 20 , 0 . 005 % nonidet - p40 , 200 μm each dntp , and 5 u of advantage ™ 2 taq polymerase ( clontech , palo alto , calif .). the pcr was performed on an mj research ptc - 200 thermal cycler ( mj research , watertown , mass . ), and consists of 1 cycle at 94 ° c . for 3 minutes , 40 cycles at 94 ° c . for 30 seconds , 60 ° c . for 30 seconds , and 72 ° c . for 2 minutes , with a final extension step of 72 ° c . for 10 minutes . the products of these reactions were electrophoresed on 1 . 5 % agarose gels and visualized by ethidium bromide staining . bands representing pcr products of the predicted size were excised from the gel , and extracted and purified using a commercially available nucleic acid - binding resin ( qiaex ii ™ gel extraction kit , ( qiagen , valencia , calif .). isolated fragments were ligated into a t / a type cloning vector ( pgem - t easy ™ vector systems ; promega , madison , wis .). following transformation , growth , and blue - white selection in competent cells ( se dh5α competent cells , life technologies inc ., rockville , md . ), the dna from positive clones was isolated . nucleotide sequences of both strands were determined by dideoxy nucleotide methodology using an automated sequencer ( model 373 ; applied biosystems , foster city , calif .). nucleotide sequences of the pcr products were analyzed using align ™ and contig ™ sequence alignment software programs ( vector nti ™; informax inc ., north bethesda , md .) and compared to known sequences using the ncbi blast program , and the imgt / hla database . sea otter - specific quantitative pcr primers within the scope of the present invention were subsequently designed using primer express ( primer design software , applied biosystems , foster city , calif .). sea otters were captured within each of two geographically distinct populations ( california and southeast alaska ) using tangle nets , hand - held dip nets , or under - water diver - held traps . captured animals were anesthetized , weighed , measured , and blood samples obtained . following sampling , anesthesia was reversed and the animal was released near the capture location . all captures were conducted under existing federal oma permits issued to mr . j . bodkin and dr . m . t . tinker , united states department of the interior , united states geological survey ( usgs ), and procedures approved by the usgs animal care and animal use committee according to federal guidelines . blood was immediately injected into paxgene blood rna collection tubes ( preanalytix , switzerland ), refrigerated in the field , and then frozen at the usgs western ecological research center facilities until processing . rapid rna degradation and induced expression of certain genes after blood draws has led to the development of methodologies for preserving the rna expression profile immediately after blood is drawn . paxgene tubes contain a blend of rna stabilizing reagents that protect rna molecules from degradation by rnases and prevent induction of gene expression . without this stabilization , copy numbers of individual mrna species in whole blood can change more than 1 , 000 - fold during storage and transport . rna from blood in the paxgene tubes was then isolated according to manufacturer &# 39 ; s standard protocols ( silica - based microspin technology ). the extracted rna was treated with 10 u / μl of rnase free dnase i ( dnase , amersham pharmacia biotech inc ., piscataway , n . j .) to remove contaminating gdna at 37 ° c . for 20 minutes followed by heat inactivation at 95 ° c . for 5 minutes and chilling on ice . extracted rna was stored at − 80 ° c . until processing and analysis . a standard cdna synthesis was performed on 2 μg of rna template from each animal . reaction conditions included 4 units reverse transcriptase ( omniscript ™, qiagen , valencia , calif . ), 1 μm random hexamers , 0 . 5 mm each dntp , and 10 units rnase inhibitor , in rt buffer ( quiagen , valencia , calif .). reactions were incubated for 60 minutes at 37 ° c ., followed by an enzyme inactivation step of 5 minutes at 93 ° c . and stored at − 20 ° c . until further analysis . quantitate real - time pcr ( q - rtpcr ) systems for sea otter s9 gene ( 18s ribosomal subunit / housekeeping gene — reference gene that does not change as a result of exposure to toxin ) and the genes of interest were run in separate wells using the cdna synthesized and the primer sets identified in table 2 . there were fifteen separate wells , one for each gene of interest , run in duplicate . complimentary dna was examined using an intercalating fluorescent dye pcr . each reaction contained 500 ng dna in 25 μl volumes with 20 pmol ssp , tris - cl , kcl , ( nh 4 ) 2 so 4 , 2 . 5 mm mgcl 2 ( ph 8 . 7 ), dntps , hotstar ™ taq dna polymerase ( quantitect sybr green pcr master mix , qiagen , valencia , calif . ), and 0 . 5 units uracil - n - glycosylase ( rocke , indianapolis , ind .). amplifications were performed in an abi 7300 ( applied biosystems , california ) under the following conditions : two minutes at 50 ° c ., followed by 15 minutes at 95 ° c ., and 40 cycles of 94 ° c . for 30 seconds , 58 ° c . for 30 seconds , and 72 ° c . for 30 seconds . reaction specificity was monitored by melting curve analysis using a final data acquisition phase of 60 cycles at 65 ° c . for 30 seconds and verified by direct sequencing of randomly selected amplicons . gene expression is measured as ct values — threshold crossing values . the lower the ct value , the more the gene is expressed . gene expression data obtained from these samples were then compared to similar data generated from samples obtained from control animals in known and documented good health . these control animal samples were obtained from captive sea otters residing at the vancouver aquarium , vancouver , british columbia , canada and shedd aquarium , chicago , ill . as we increase our sample size from known healthy captive sea otters , we will be able to establish diagnostic ranges of expression that can be used as a standard for field investigations and veterinary use . these ranges would be able to be determined by one having ordinary skill in the art having knowledge of the present invention . the experimental procedure described above was used in the following examples ( case studies ), the results of which are graphically set forth in fig2 - 20 . this sea otter suffered exposure to oil from a natural seep , and was captured by california fish and game . blood was drawn at the time of capture and periodically during its rehabilitation at the monterey bay aquarium . using the novel primers as described herein , an increase in aryl hydrocarbon receptor ( ahr ) expression was observed , followed by a decrease in expression during rehabilitation . fig2 provides a graphical representation of this with reference to the average ahr expression of two healthy sea otters from the vancouver aquarium . in addition , an increase in mx - 1 expression was observed after oiling indicating viral infection possibly exacerbated by exposure that decreased with rehabilitative care — see fig3 . blood samples were obtained from three shedd aquarium sea otters ( chicago , ill .) before being transported to a temporary facility in minnesota . the three sea otters are identified as kiana , mari and yaku . using the novel primers as described herein , a high expression of ahr and hsp70 ( heat shock protein 70 ) was observed . this was probably indicative of less than ideal conditions at the chicago facility that was in need of and scheduled for renovation . expression of these genes decreased after they were removed from the shedd aquarium — see fig4 . note that the normalized c t values are provided in the following order , from left to right — kiana aug . 19 , 2008 , kiana mar . 25 , 2009 , mari aug . 19 , 2008 , mari mar . 24 , 2009 , yaku aug . 19 , 2008 and yaku mar . 24 , 2009 , respectively first for expression of ahr , and then for expression of hsp70 . note that fig4 illustrates this with reference to the average corresponding gene expression for two healthy sea otter . blood samples were obtained from twenty - one ( 21 ) sea otters from katmai , ak . using the novel primers as described herein , the average levels of expression in ten ( 10 ) genes was determined , and compared with the average values from two healthy sea otters — see fig5 . the katmai sea otter population was assumed to be relatively healthy due to its isolation from anthropogenic discharges but the findings of increased expression of the drb and complement cyt inhibitor genes indicated that this group may have elevated incidence of bacterial infection relative to two known healthy sea otters . blood sample was obtained from an adult male sea otter from the katmai population that was diagnosed with hyperthermia by a veterinarian assisting with captures . using the novel primers as described herein , we reaffirmed field diagnosis of hyperthermia compared to average values from two healthy sea otters ( expression of hsp70 gene ). we also identified the adult male sea otter &# 39 ; s possible inability to combat viral disease ( extreme lack of expression in mx - 1 gene ). see fig6 . blood sample was obtained from an adult female sea otter from katmai population that was diagnosed with enlarged lymph nodes by a veterinarian assisting with captures . using the novel primers as described herein , we diagnosed highly elevated expression of drb suggesting bacterial infection that reaffirms the field diagnosis — see fig7 . blood samples were drawn from thirty - one ( 31 ) sea otters from monterey , calif . and thirty - nine ( 39 ) sea otters from big sur , calif . using the novel primers as described herein , the levels of expression in a suite of genes for each was detected — suite of genes set forth in fig8 . the average levels of expression in the suite of genes for all of the monterey sea otters , and the average levels of expression in the suite of genes for all of the big sur sea otters were determined . these average levels of expression in the suite of genes relative to the average expression values from two healthy sea otters is set forth in fig8 . a scatter plot showing the results of a discriminant function analysis comparing the levels of gene expression between two populations of sea otters ( monterey vs . big sur ) is shown at fig9 . blood samples were drawn from twenty ( 20 ) sea otters from knight island , forty - one ( 41 ) sea otters from montague island , and twenty - one ( 21 ) sea otters from katmai , ak . using the novel primers as described herein , the levels of expression in a suite of genes for each was detected — suite of genes set forth in fig1 . the average levels of expression in the suite of genes for all of the sea otters from each given location were determined . these average levels of expression in the suite of genes relative to the average expression values from two healthy sea otters is set forth in fig1 . the knight and montague island sea otters captured in 2006 - 2008 were from prince william sound , alaska , the area impacted by the exxon valdez oil spill in 1989 . a scatter plot showing the results of a discriminant function analysis comparing the levels of gene expression between three populations of sea otters ( knight vs . montague vs . katmai ) is shown at fig1 . at the time of this filing , all field data are presented relative to two clinically diagnosed , healthy captive zoo animals that are provided for example and do not constitute a reference for free - ranging or wild sea animals . further planned sampling from such animals will be necessary to establish ranges of values for healthy animals that can be used for comparisons in field studies . however , the findings demonstrate diagnostic utility in individual captive animals , as well as differences among populations of wild or free - ranging sea otters that are scientifically defensible , i . e ., ( 1 ) relatively low expression in the assumed pristine katmai , ak . sea otter population ; ( 2 ) increased gene expression , particularly the hdcmb21p gene implicated in tumorigenesis in sea otters from the area of prince william sound , alaska , which suggests evidence of carcinogenesis possibly influenced by a major oil spill 20 years ago in the area where the sea otters were sampled ; ( 3 ) increased expression in sea otters from the more urbanized monterey bay coastline than the more rural big sur coastline . one having ordinary skill in the art with knowledge of the present invention would readily be able to establish ranges of values for healthy animals that can be used for comparisons in field studies and veterinary use . it is applicants &# 39 ; intention that the present invention not be limited to ranges of values for healthy animals described and set forth herein . the novel primer sets herein may be synthesized using conventional oligonucleotide synthesis methodologies . the present invention demonstrates that a small sample of blood from a sea otter may be used to quantitatively assess expression of various genes , which levels of expression may be used as indicators of pathophysiological changes in sea otters , which may be used to assess and monitor sea otter health due to its exposure to fuels / oils or other toxins . although each novel primer set herein may be run separately for the purpose described herein , one having ordinary skill in the art with knowledge of the present invention may wish to run more than one or , perhaps , all of the primer sets to best render a diagnostic assessment on the health of a sea otter . although the present invention is primarily described with reference to oil - exposed sea otters , the invention is not intended to be so limited . applicants have identified a suite of genes that react to exposure to various toxins . applicants submit that the present invention may be used to evaluate pathophysiological changes in sea otters that may have been exposed to toxins such as viruses , bacteria , or xenobiotics . applicants submit that one skilled in the art with knowledge of the present invention would recognize that the present invention encompasses the use of the present invention to assess pathophysiological changes in sea otters that may have been exposed to any variety of environmental contaminants / toxicants . moreover , applicants submit that the present invention may be used , in general , to monitor the health of sea otters . applicants submit that the present invention has ecological and veterinary applications for monitoring the health of captive as well as free - ranging sea otters . in addition , although the present invention is described with reference to sea otters , applicants submit that the invention is not intended to be so limited . applicants submit that the present invention may be suitably employed to evaluate pathophysiological changes in mustelidae family in general . applicants further submit that the present invention may be used by veterinarians in their treatment of otters and other species belonging to the mustelidae family . while particular embodiments of the present invention have been shown and described , it will be obvious to those skilled in the art that changes and modifications may be made without departing from this invention . therefore , it is intended that the claims herein are to include all such obvious changes and modifications as fall within the true spirit and scope of this invention . the teachings of the references cited herein are incorporated herein in their entirety : ( 2008 ). qiaex ii ® handbook — for dna extraction from agarose and polyacrylamide gels and for desalting and concentrating dna from solutions . altschul , s . f ., w . gish , et al . ( 1990 ). “ basic local alignment search tool .” j mol biol 215 ( 3 ): 403 - 410 . bartosiewicz , m ., s . penn , et al . ( 2001 ). “ applications of gene arrays in environmental toxicology : fingerprints of gene regulation associated with cadmium chloride , benzo ( a ) pyrene , and trichloroethylene .” environ health perspect 109 ( 1 ): 71 - 74 . bowen , l . e . a . ( 2006 ). “ differential expression of immune response genes in steller sea lions : an indicator of ecosystem health ?” ecohealth 3 : 109 - 113 . bowen , l . e . a . ( 2007 ). “ differential gene expression induced by exposure of captive mink to fuel oil : a model for the sea otter .” ecohealth 4 : 298 - 309 . burczynski , m . e ., m . mcmillian , et al . 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