Patent Application: US-201414193380-A

Abstract:
the present invention provides a method of diagnosing a cyathostomin infection , said method comprising the step of identifying a level of anti - cyathostomin larval antigen antibodies in a sample , wherein a level of anti - cyathostomin larval antigen antibodies is indicative of a cyathostomin infection .

Description:
the present invention will now be described in detail and with reference to the following figures which show : fig1 . clustalw alignment of cy - gala - 1 with its orthologues in other nematode species . cyathostomin ( cy ) gala - 1 is compared to n . brasiliensis keratin - like protein ( nb - klp ) ( accession number : bab68205 ); t . circumcincta ( tc ) ( aam45145 ); o . ostertagi ( oo ) ( cad22110 ); c . elegans ( ce ) klp - 1 ( np 502026 ) and ce - klp - 2 ( np 501448 ). the signal peptide for each sequence is underlined and the domain of unknown function ( duf148 ) is boxed . the histidine - rich region is highlighted in grey and the glycine - rich regions of the c . elegans sequences are shown in bold . fig2 : development transcription pattern of cy - gala - 1 . rt - pcr was performed using gene - specific primers for cy - gala - 1 and the housekeeping gene cytochrome oxidase c subunit i ( coxi ), from mixed - species pools of el3 ( lane 1 ), dl ( lane 2 ) and lp ( lane 3 ) cdna . for each reaction no - template controls were performed ( n ). sizes in base pairs ( bp ) are labelled on the left - hand side . fig3 a and 3b . immunoreactivity of rcy - gala - 1 . igg ( t ) reactivity to rcy - gala - 1 in horses infected with cyathostomins or other helminths as assessed by ( a ) immunoblot and ( b ) elisa . fig3 a . lane 1 : coomassie blue . lanes 2 - 11 : igg ( t ) reactivity of specific equine sera : hf ( 2 ); ci ( 3 ); a pool of sera from cyathostomin - free horses ( n = 5 ) from an abattoir ( 4 ); a pool of sera from cyathostomin - infected horses which harboured total mucosal larval burdens of & gt ; 100 , 000 ( n = 6 ) from an abattoir ( 5 ); horses mono - specifically infected with p . equorum ( 6 ), s . edentatus ( 7 ), s . westeri ( 8 ) or s . vulgaris ( 9 ). also shown is igg reactivity in sera from a rabbit before ( lane 10 ) and after two immunisations ( lane 11 ) with a 20 kda complex purified from el3 / dl somatic extracts [ 11 ]. fig3 b . elisa indicating igg ( t ) reactivity to rcy - gala - 1 antigen in equine sera over an experimental infection [ 29 ]. responses in the ci group are depicted by the solid lines and black shapes and in the hf group by dashed lines and white shapes . fig4 : reactivity of anti - rcy - gala - 1 antiserum to cyathostomins and other equine helminths . igg ( t ) responses were assessed by ( a ) elisa and ( b ) immunoblot . elisa results depict binding of anti - rcy - klp - 1 anti - sera ( black ) and pre - immunisation serum ( white ). for both assays , the antigens were as follows : 1 = rcy - gala - 1 ; 2 = cyathostomin il3 ; 3 = cyathostomin el3 ; 4 = cyathostomin dl ; 5 = cyathostomin lp ; 6 = adult a . perfoliata ; 7 = adult p . equorum ; 8 = adult s . edentatus ; 9 = adult s . vulgaris ; 10 = adult s . equinus . fig5 : immunolocalisation of cy - gala . transverse sections of dl cyathostomins were probed with anti - rcy - gala - 1 antiserum ( a ) and pre - immunization serum ( b ). specific binding of antiserum in the parasite gut is indicated by the black arrows . the vertical bar represents 40 μm . fig6 : schematic representation of cy - gala - 1 and the 220 bp fragment of the gene amplified from 10 cyathostomin species . cy - gala - 1 cdna sequence is represented by black boxes ( a ). the 220 bp region pcr amplified from genomic dna samples from 10 cyathostomin species is represented by the white box . the latter is expanded to indicate the position of the intron ( hatched box ). the range in interspecies variation for the whole gene fragment ( and also without the intron sequence ) are depicted . a representative pcr product of cy - gala - 1 is shown for each species ( b ): c . catinatum ( 1 ); c . nassatus ( 2 ); c . goldi ( 3 ); c . longibursatus ( 4 ); c . coronatum ( 5 ); c . pateratum ( 6 ); c . ashworthi ( 7 ); c . leptosomum ( 8 ); c . minutus ( 9 ) and c . labiatus ( 10 ). fig7 a : optimisation of antigen cocktails . the antibody response of encysted cyathostomin infected ( positive ) and non - infected ( negative ) animals is shown for varying concentrations of antigen and two different cocktails of antigen ( ct1 and ct2 ). ct1 contains gala 1 , gala 2 , gala 3 . ct2 contains gala 1 , gala 2 , gala 3 and cid 1 . individual antigen concentration is shown on the x axis and optical density ( o . d ) on the y axis . 7b : ratio of signal for encysted cyathostomin infected ( positive ) to uninfected ( negative ) animals in an elisa . individual antigen concentration is on the x axis and ratio of positive to negative optical density on the y axis . c : shows mean serum antibody response to cocktail 1 ( ct1 ) in groups of horses with varying infection levels . ct1 contains gala 1 , gala 2 , gala 3 . horses were grouped as follows according to total mucosal parasite burden ( tmb ). neg ; uninfected horses tmb = 0 ( n = 5 ), low ; tmb = 0 - 20000 , ( n = 8 ), medium ; tmb = 20000 - 100000 , ( n = 7 ), high ; tmb =& gt ; 100000 ( n = 26 ). error bars show +/− standard error of the mean . o . d = optical density . d : shows mean serum antibody response to cocktail 2 ( ct2 ) in groups of horses with varying infection levels . ct2 contains gala 1 , gala 2 , gala 3 and cid 1 . horses were grouped as follows according to total mucosal parasite burden ( tmb ). negative ; uninfected horses tmb = 0 ( n = 5 ), low ; tmb = 0 - 20000 , ( n = 8 ), medium ; tmb = 20000 - 100000 , ( n = 7 ), high ; tmb =& gt ; 100000 ( n = 26 ). error bars show +/− standard error of the mean . o . d = optical density . fig8 a - e : roc analysis of elisa data derived from cocktail ( ct ) 1 ( which includes gala - 1 , 2 and - 3 ) and ct2 ( which includes gala - 1 ,- 2 , - 3 and cid - 1 ). the areas under the curve ( auc ) are shown on each graph for each ct at the specified cyathostomin burden cut - off value indicated on each set of charts . the results indicate that ct1 and ct2 allow clear discrimination at different levels of cyathostomin mucusal burden , especially developing larval ( dl ) burdens above 120 , 000 ; however , it is likely that the auc values could be improved by developing the assay to take into account cyathostomin species complexity and by including proteins that specifically relate to el3 . these additional proteins have been identified and will be added systematically to the cocktails to test their effect on auc in the roc analysis .′ cyathostomins were collected from equine large intestinal tissue as described previously [ 9 ]. briefly , caecum and ventral colon samples were removed at an abattoir and luminal parasites ( lp ), consisting of fifth stage larvae and adults , were collected from intestinal washings using sieves . mucosal larval stages were recovered by pepsin - hcl digestion [ 9 ]. the mucosal parasites were separated into two populations based on size following previous recommendations [ 13 ]: ( i ) el3 and ( ii ) late third stage ( ll3 )/ developing fourth stage ( dl4 ), collectively termed developing larvae ( dl ). nematode samples for rna extraction were placed into rnalater ( ambion ) at 4 ° c ., while those for protein extraction and genomic dna isolation were snap frozen in liquid nitrogen and stored at − 80 ° c . for immunolocalisation experiments , dl were fixed in 10 % formal saline . infective third - stage larvae ( il3 ) were collected from horse feces as described previously [ 8 ]. individual adult cyathostomins were identified to species according to published recommendations [ 16 ]. adult stage large strongyles , anoplocephala perfoliata and parascaris equorum , were also obtained and stored at − 80 ° c . cyathostomin rna was extracted from dl populations by homogenisation in a mortar and pestle under liquid nitrogen , then using trizol ( invitrogen ) according to the manufacturer &# 39 ; s instructions . integrity of rna samples was assessed using a 2100 bioanalyser ( agilent technologies ) and rna stored in rnase - free water at − 80 ° c . a mixed - species dl cdna library was constructed using a smart cdna library construction kit ( clontech laboratories , inc ) using long distance pcr according to manufacturer &# 39 ; s instructions . briefly , the cdna was synthesised by reverse transcriptase ( rt )- pcr using 1 μg total rna pooled from 11 separate dl rna samples collected over a 6 - month period from a range of intestinal sites . this was done to maximise cyathostomin species representation within the cdna library . after ligation into the λtriplex2 vector , the cdna was packaged into gigapack gold iii packaging extract ( stratagene ) and amplified in escherichia coli xl1 - blue strain , ( stratagene ). library quality was assessed by analysing insert size in 40 plaques chosen at random . length and identity of the inserts were determined by pcr and sequencing ; the majority of plaques contained an insert with an average size of 500 base pairs ( bp ). an el3 cdna library was constructed using the same method as for the construction of the dl cdna library with the exception being the use of a sl1 primer to amplify nematode specific dna prior to ligation into the triplex2 vector . ( martin , et al , 1995 ). briefly , the cdna was synthesised by reverse transcriptase ( rt )- pcr using 1 μg total rna pooled from el rna samples from el3 larvae collected from a range of intestinal sites from 6 individual horses . this cdna was then used in a pcr with sl1 forward primer sequence : ggtttaattacccaagtttgag and reverse primer sequences : attctagaggccgaggc and ttctagaggccgaggcg . products of this pcr were then used for packaging into the triplex2 vector as described for generation of the dl cdna library . immunoscreening was performed according to the manufacturer &# 39 ; s protocol . for immunoscreening , two types of sera were used : cyathostomin - infected ( ci ) and helminth - free ( hf ) sera [ 29 ]. ponies in the ci group ( n = 3 ) had been trickle infected with a total of 3 . 9 million cyathostomin il3 over a period of 9 weeks , while the hf control group ( n = 3 ) were maintained helminth - free . serum was obtained weekly from both groups . for immunoscreening , a pool of ci sera was prepared by combining samples obtained from the three ponies at 12 , 13 , 14 and 16 weeks pi . the pool of hf sera was made by combining samples obtained from the three ponies at 2 , 3 , 4 and 6 weeks before the start of the infection period . to reduce background reactivity , both pools of sera were pre - absorbed with e . coli lysate by incubating equal volumes of each and rocking for 4 h at room temperature [ 37 ]. after centrifugation at 18 , 000 × g for 10 min , the supernatant was retained for probing library filter lifts . the primary immunoscreen consisted of approximately 108 , 000 cdna clones in e . coli xl1 - blue strain . plaque lifts were made onto nitrocellulose filters ( hybond - c extra , ge healthcare ). the membranes were washed [ five × 10 min in tris - buffered saline ( 10 mm tris , 150 mm nacl , ph 7 . 4 ) containing 0 . 05 % tween - 20 ( tbst )], then blocked for 1 h with 1 % gelatin / tbst . in the first screen , the serum pool from the ci ponies was used at 1 : 200 in tbst and incubated with the membranes overnight at 4 ° c . the secondary antibody ( goat anti - equine igg ( t ), serotec ) and tertiary antibody ( rabbit anti - goat [ igg ]: hrp , sigma ), were incubated at 1 : 200 and 1 : 500 respectively , for 1 h each , with washing ( as above ) between steps . filters were developed using sigmafast dab with metal enhancer ( sigma ). positive clones were isolated by taking agar plugs from the corresponding plate . plaques that reacted non - specifically with equine sera ( false positives ) were identified by performing a second screen . here , clones selected in the first round were screened as described above , except that filters were cut in half and one half probed with the ci serum pool and the other with the hf serum pool . only plaques that reacted with the ci serum pool and not the hf serum pool were selected for sequence analysis . vector - specific primers were used to amplify selected phage inserts and the pcr products purified using a qiaquick pcr purification kit ( qiagen ). each purified pcr product was sequenced using a commercial service ( mwg biotech ). the resultant sequences were translated and searched against the genbank ‘ non - redundant protein ’ database using blastp , and then against the ‘ non - human , non - mouse ’ est database using tblastn , from the national centre for biotechnology information [ 3 ]. sequence alignments were performed using clustalw2 [ 21 ] from the european bioinformatics institute ( http :// www . ebi . ac . uk / tools / clustalw2 /) and analysis for signal peptides performed using signalp 3 . 0 [ 5 ]. sequence identities were calculated using megalign 8 . 0 . 2 ( dnastar ) based on clustalw alignments . molecular mass estimations were made using an online tool from the sequence manipulation suite ( http :// www . bioinformatics . org / sms2 / protein_mw . html ) and glycosylation sites identified using expasy pro site ( http :// ca . expasy . org / prosite /). rt - pcr to determine temporal transcription pattern of the mrna encoding the cyathostomin gut - associated larval antigen - 1 ( cy - gala - 1 ) stage - specific cdna was synthesised from 1 μg each of el3 , dl and lp total rna using a smart cdna library construction kit ( clontech laboratories , inc .). briefly , first - strand cdna was synthesised and amplified using the long - distance pcr method ( 22 cycles ). double - stranded cdna was purified using a qiaquick pcr purification kit ( qiagen ), eluted in 50 μl dh 2 o and stored at − 20 ° c . until required . integrity and loading of each cdna population was assessed by amplifying a portion of the cytochrome oxidase c subunit i ( coxi ) gene using primers designed to conserved sequences among cyathostomins ( sense : 5 ′- aaaaaggaggtgtttggttc - 3 ′ ( seq id no : 62 ); antisense : 5 ′- cttgaatttgataaaactacacc - 3 ′ ( seq id no : 63 )). pcr conditions were as follows : 0 . 3 μm primers , 0 . 25 μm dntps and 1 . 5 mm mgcl 2 with the following cycling : 94 ° c . for 5 min , 40 cycles at 94 ° c . for 30 sec , 60 ° c . for 30 sec and 72 ° c . for 30 sec , with a final extension at 72 ° c . for 7 min . pcr was performed using platinum taq ( invitrogen ) with 1 μl cdna from each developmental stage . primers were designed for the most abundant immunoreactive clone identified in section 2 . 2 and designated cyathostomin gut - associated larval antigen - 1 ( cy - gala - 1 ). the primer sequences were as follows : sense , 5 ′- aattgtggggaacaggag - 3 ′ ( seq id no : 64 ); antisense , 5 ′- aatgaaaatcagactcctagg - 3 ′ ( seq id no : 65 ). pcr conditions were as above , but using 35 cycles . this experiment was repeated twice and the pcr products were analysed on 2 % w / v agarose gels using tracklt 100 bp dna ladder ( invitrogen ) for size determination . the gels were stained with 1 × gelred ( biotium ). the cy - gala - 1 clone from the library immunoscreen that contained the largest insert was chosen for expression of recombinant protein . this clone incorporated the full - length coding sequence of cy - gala - 1 including the putative initiating methionine , signal peptide and poly - a tail . primers were designed to amplify the coding sequence of cy - gala - 1 ( minus the sequence that encoded the signal peptide ) for sub - cloning into pet - 22b (+) vector ( novagen ). appropriate sequences encoding flanking restriction enzyme sites were incorporated for uni - directional cloning . the primer sequences were as follows ( nb : bamh1 and hindiii sites underlined ): sense 5 ′- aattcggatccgcaaggtgtcatggacctttttg - 3 ′ ( seq id no : 66 ); antisense , 5 ′- ccgcaagcttatatctttcatctgtgttgagtccaaac - 3 ′ ( seq id no : 67 ). the pcr step was performed as described above except that the annealing temperature was 58 ° c . and 30 cycles were used . the pcr product was purified as described above . the pet - 22b (+) vector and pcr product were digested with bamh1 and hindiii and ligation , using a 1 : 1 ratio of vector to pcr product , performed according to novagen &# 39 ; s protocol . plasmids were transformed into e . coli jm109 competent cells ( promega ) following manufacturer &# 39 ; s instructions and selected on ampicillin - agar . a selection of colonies was subjected to colony pcr to ensure the presence of the cdna encoding cy - gala - 1 . a colony which contained an insert of the correct estimated size was subjected to plasmid purification using a wizard plus sv miniprep kit ( promega ) and the purified plasmid was both sequenced and transformed into e . coli bl21 - codonplus ( de3 )- ril competent cells ( stratagene ) for expression of recombinant protein ( rcy - gala - 1 ). following induction with 1 mm isopropyl - beta - d - thiogalactopyranoside ( bioline ), soluble rcy - gala - 1 , present in the bacterial lysate supernatant , was purified on a his - trap hp column ( ge healthcare ), following manufacturer &# 39 ; s instructions . the purified protein was dialysed into phosphate buffered saline , ph 7 . 4 ( pbs ), using cellulose dialysis tubing ( sigma ) and stored at − 20 ° c . until required . purified rcy - gala - 1 ( 0 . 5 μg ) was separated by sds - page , and a band at the expected size excised and subjected to matrix - assisted laser desorption / ionization time - of - flight ( maldi - tof - tof ) mass spectrometry using an ultraflex ii maldi - tof - tof mass spectrometer ( bruker daltonics ). the identity of the protein was confirmed by comparing the peptide mass fingerprint ( pmf ) generated to the theoretical peptide mass fingerprint ( pmf ) of cy - gala - 1 . anti - rcy - gala - 1 antiserum was generated by injecting a rabbit with 50 μg of rcy - gala - 1 , in 0 . 5 mg / ml quila / pbs ( 1 ml total injection ). a secondary injection was administered three weeks later , after which a test bleed indicated a specific antibody response to the recombinant antigen . this experiment was performed under the legislation of a uk home office license . soluble somatic antigen extracts were prepared from cyathostomin stages ( il3 , el3 , dl and lp ) and adult worms of other helminth species ( a . perfoliata , strongylus equinus , strongylus edentatus , strongylus vulgaris and p . equorum ) as described previously [ 9 ]. however , il3 were disrupted using a ribolyser fast prep fp120 ( thermo scientific ) instead of a glass homogeniser . proteins were separated on 4 - 12 % polyacrylamide bis - tris gels ( nupage mes system , invitrogen ) according to the manufacturer &# 39 ; s protocol . for immunoblotting , proteins were transferred to nitrocellulose membranes . to assess cross - reactivity , 0 . 1 μg rcy - gala - 1 was loaded onto lanes of a 15 - well 12 % nupage gel using seeblue plus2 protein standards ( invitrogen ) for molecular weight estimations . in one lane , 0 . 4 μg was loaded and after electrophoresis was cut from the gel and stained with coomassie blue . after transfer , the blot was sliced into separate lanes and blocked in tntt ( 10 mm tris , 0 . 5m nacl , 0 . 05 % tween - 20 , 0 . 01 % thimerosal , ph 7 . 4 ). each of the following sera was used , diluted 1 : 200 in tntt : ci and hf sera pools ( described above ); a pool of 5 horses found to be cyathostomin - free ( cf ) from a local abattoir and a pool of 12 horses ( from the same abattoir ) with mucosal cyathostomin burdens of & gt ; 100 , 000 ( endemic infected — ei ); horses mono - specifically infected with s . edentatus or s . vulgaris [ 20 ], p . equorum or strongylus westeri [ 11 ]. also tested was rabbit antiserum ( and pre - immunisation samples ) generated to the native 20 kda cyathostomin complex [ 11 ]. sera were incubated at room temperature for 1 . 5 h . washing consisted of three , 5 min incubations in tntt . the secondary and tertiary steps were as described for the immunoscreening ( above ), with the exception that the anti - 20 kda antiserum blots were incubated with goat anti - rabbit ig : hrp ( dako ) at 1 : 500 . the blots were developed as for the library screen . for detection of cy - gala - 1 protein in somatic extracts of cyathostomins and other helminth species , somatic extracts ( 9 μg each antigen ) were loaded onto 10 - well , 4 - 12 % nupage gels , using 10 ng rcy - gala - 1 for comparison . after transfer to nitrocellulose , periodate treatment of the blots was performed as described previously [ 9 ]. the blots were probed with pre - immunisation rabbit serum and anti - rcy - gala - 1 serum at 1 : 300 in tntt , followed by goat anti - rabbit ( ig ): hrp ( dako ) at 1 : 500 , and developed as described above . three 5 min washes were applied between steps . to test reactivity of experimentally infected pony sera ( ci ) to rcy - gala - 1 over the course of infection , the following conditions were used . each well of a microlon high binding plate ( greiner bio - one ) was coated with 100 μl of rcy - gala - 1 ( 1 μgml − 1 in bicarbonate coating buffer , 0 . 1 m , ph 9 . 6 ) overnight at 4 ° c . plates were washed with 0 . 05 % tween - 20 in pbs ( pbst ), six times . block solution ( 2 % soya infant powder ( w / v ) in pbst ) was added , 200 μl per well , and incubated for 1 h at 37 ° c . plates were washed six times and ci and hf sera ( 1 : 200 in block solution ), from weekly time points 2 weeks before infection to 16 weeks pi , added and incubated for 2 h at 37 ° c . after washing , 100 μl goat anti - equine igg ( t ) were added , diluted 1 : 200 in blocking solution . after 1 h at 37 ° c . and washing , 100 μl rabbit anti - goat ( ig ): hrp were added , diluted in block at 1 : 500 , and incubated for 1 h at 37 ° c . to develop the reaction , sigmafast opd tablets ( sigma ) were dissolved in h 2 o according to the manufacturer &# 39 ; s instructions and 100 μl added to each well and incubated for 15 min . fifty μl of 2 . 5 m h 2 so 4 were added to stop the reaction and the absorbance read at 490 nm . the same conditions were used to measure the anti - rcy - gala - 1 antiserum response to somatic extracts of cyathostomin stages and other adult helminth species extracts , except that these were coated at 2 μgml − 1 . the antiserum and goat anti - rabbit : hrp were used at 1 : 500 . cyathostomin dl were fixed in 10 % formal saline and immobilised in a solidified gelatin plug by mixing with molten 5 % gelatin / pbs (& lt ; 30 ° c .) and allowing to set . the plugs were then dehydrated with alcohol and xylene and embedded in paraffin wax . sections were cut at 3 μm using a microtome and the slides stored at 4 ° c . immunolocalisation was performed using an envision + system - hrp for rabbit primary antibodies ( dakocytomation ) in a sequenza slide rack ( thermo scientific ) at room temperature . after de - waxing , the slides were incubated in 0 . 5 % tween - 80 / pbs ( pbst80 ) with 0 . 3 % h 2 o 2 for 20 min , to inactivate endogenous peroxidises . blocking was performed using 100 μl 25 % normal goat serum ( ngs ) in pbst80 for 1 h . rabbit antisera obtained prior to and after two immunisations with rcy - gala - 1 were diluted 1 : 100 in 10 % ngs / pbst80 , and 100 μl incubated on the slides for 1 h . after two washes in pbs at room temperature , 100 μl of hrp - labelled polymer conjugated to goat anti - rabbit ig , was incubated ( neat ) for 30 min . the reactions were developed in neat 3 - amino - 9 - ethylcarbazole substrate chromogen for 7 . 5 min . slides were washed in h 2 o and counterstained using haematoxylin . single worm pcr to identify the gene encoding gala in different cyathostomin species genomic dna was isolated from 54 individually identified adult cyathostomins using the dneasy blood and tissue kit ( qiagen ) according to their protocol , but with the addition of a homogenisation step before the proteinase k digestion step ; each individual was disrupted briefly using a 1 . 5 ml microfuge tube homogeniser in 50 μl atl buffer supplied with the kit . the following 10 species were examined ( nb : numbers of worms used for each species is shown in parenthesis ): cyathostomum catinatum ( 10 ), cylicostephanus goldi ( 8 ), coronocyclus coronatus ( 6 ), cyathostomum pateratum ( 6 ), cylicocyclus nassatus ( 6 ), cylicostephanus longibursatus ( 5 ), cylicocyclus ashworthi ( 4 ), cylicocyclus leptostomum ( 3 ), coronocyclus labiatus ( 1 ) and cylicostephanus minutus ( 1 ). the same primers used in section 2 . 3 for rt - pcr were used to amplify a conserved fragment of cy - gala in each species . the cycling conditions were : 2 min at 94 ° c ., followed by 40 cycles of 15 sec at 94 ° c ., 30 sec at 58 ° c . and 60 sec at 72 ° c ., and a final extension at 72 ° c . for 7 min . pcr products were analysed on agarose gels as described above and pcr products from each of the 54 individuals cloned into pgemt - easy ( promega ) according to manufacturer &# 39 ; s instructions . each clone was sequenced in forward and reverse directions with vector - specific primers using the commercial sequence facility described above . immunoscreening of the cyathostomin dl cdna library and sequence analysis of cy - gala - 1 the primary immunoscreening yielded 33 positive clones ; five of which were excluded as false positives on the basis of the secondary screen using hf sera . the remaining 28 clones contained inserts ranging in size from approximately 500 to 1500 bp . sequence analysis indicated that 15 of these showed high identity to one another ( 73 - 100 % at the amino acid [ aa ] level ). one of these ( cy - gala - 1 ) represented a full - length coding sequence : i . e . it contained a putative initiation codon , signal peptide and termination codon upstream of a poly - a tail . the entire coding sequence was 223 aa which , after cleavage of the signal peptide , would result in a 206 aa mature protein estimated at 25 . 6 kda . cy - gala - 1 contains a highly conserved domain as revealed by a domain search via blastp analysis [ 28 ]. the function of this domain is unknown and in caenorhabditis elegans is designated domain of unknown function 148 ( duf148 ). the cy - gala - 1 sequence displayed highest aa identity to a sequence from nippostrongylus brasiliensis ( accession number : bab68205 ; 35 % identity over 128 residues ), also identified via immunoscreening [ 38 ]. two predicted proteins from c . elegans showed 34 % identity over 105 residues to cy - gala - 1 . these proteins were 44 . 5 % identical to each other . also identified , were two trichostrongyloid ests : one from teladorsagia circumcincta l3 ( accession number : aam45145 ), which displayed 32 % identity to cy - gala - 1 over 102 aa , and one from ostertagia osteragi adult worms ( accession number : cad22110 ) with showed 32 % identity over 140 aa . the two c . elegans orthologues ( referred to here as ce - klp - 1 ( np — 502026 ) and ce - klp - 2 ( np — 501448 )) contain glycine - rich domains which gives them homology to keratin sequences and hence their designation as ‘ keratin - like ’ proteins ( klp ). all the parasitic nematode sequences described here lack this glycine rich sequence , despite some being previously designated as ‘ klp - like ’ proteins [ 38 ]. rather than classifying cy - gala - 1 as a klp , it was instead named to reflect its localisation to the gut ( see below ). an alignment of cy - gala - 1 with its orthologous sequences in n . brasiliensis and c . elegans is depicted in fig1 . in all the parasitic nematode sequences , except that of t . circumcincta , a histidine - rich motif precedes duf148 ( fig1 ); its function is unknown . in addition , four potential n - linked glycosylation sites were identified . searching cy - gala - 1 at nembase gave additional significant hits . all of these est sequences contained regions with high identity to duf148 and some had glycine - rich regions . the closest matches were to sequences identified in adult ancylostoma ceylanicum ( accession numbers : cb176510 , cb190303 and cb339159 ), with 45 - 46 % aa identity to cy - gala - 1 over 110 residues . cy - gala - 1 transcript was detected in dl and el3 cdna and not in cdna from lp parasites ( fig2 ). after 40 cycles , similar levels of coxi pcr product were observed in dl and lp cdna . however , a coxi pcr product from el3 was less intense , indicating low quality of el3 cdna . this was due to degradation of el3 rna caused by the extensive digestion method required to harvest these larvae . these results indicate the apparent specificity of this transcript for mucosal stages ; hence the gene was selected for expression of recombinant protein for assessment as a diagnostic marker . rcy - gala - 1 was obtained from the soluble fraction of the e . coli lysate ; the purified protein was approximately 28 kda ( fig3 a ). the identity of this protein as rcy - gala - 1 was confirmed by maldi - tof - tof ( data not shown ). its molecular weight was slightly higher than the expected size of native cy - gala , calculated to be 25 . 6 kda , and was due to addition of the his - tag and e . coli signal peptide . anti - rcy - gala - 1 antiserum predominantly recognised the expected size band in somatic dl extracts ( section 3 . 4 and fig4 ). the immunoreactivity of the recombinant antigen is shown in ( fig3 b ). only igg ( t ) in ci and ei sera equine sera bound rcy - gala - 1 , indicating that both experimentally and naturally infected horses recognise this antigen . sera from horses harbouring other parasitic helminths did not contain igg ( t ) that bound cy - gala - 1 . the rabbit antiserum to the cyathostomin larval anti - 20 kda complex generated previously [ 11 ], showed strong reactivity to rcy - gala - 1 . levels of rcy - gala - 1 - specific igg ( t ) in sera from infected vs . non - infected ponies [ 29 ] were measured by elisa ( fig3 b ). increases in rcy - gala - 1 - specific igg ( t ) levels were observed in all infected ponies by 6 weeks pi . a more rapid increase was observed in pony 104 . antigen - specific igg ( t ) levels plateaued at 8 weeks pi for 104 and 12 weeks pi for 101 and 105 ; these levels remained elevated until the end of the measurement period at 16 weeks pi . no significant increases in rcy - gala - 1 - specific igg ( t ) levels were observed in any of the hf ponies throughout the experiment . murphy and love ( 1997 ) [ 29 ] described clinical signs in the infected animals from 4 - 6 weeks pi . while all showed a slower increase in percentage weight gain than the control group , pony 104 showed a drop in weight gain over weeks 4 - 8 pi . these signs may indicate a higher level of infection in 104 . anti - rcy - gala - 1 antiserum reactivity was tested against somatic extracts from a . perfoliata , p . equorum , s . edentatus , s . vulgaris and s . equorum [ fig4 ]. no reactivity was observed except to a band at 38 kda in the p . equorum extract . binding to this band was less than that seen in the cyathostomin dl lane ( fig4 ). antiserum raised to rcy - gala - 1 was used to investigate the presence of the native protein in different cyathostomin stages ( fig4 ). this antiserum bound the 28 kda recombinant antigen ( fig4 , lane 1 ): an additional band at 53 kda was bound and may represent a dimeric form of cy - gala - 1 . the anti - rcy - gala - 1 antisera showed reactivity to el3 and dl somatic extracts but not to adult extract ( fig4 ). immunoreactivity to antigens in el3 and dl stages was primarily directed at molecules of approximately 26 kda , corresponding to the calculated molecular mass of cy - gala - 1 . two other el3 and dl antigens were bound by igg in anti - cy - gala - 1 antisera , one at approximately 45 kda and the other at 55 kda . the elisa results indicated high reactivity to dl , however no binding was observed in el3 or adult extract . dl were subjected to immunolocalisation studies ( fig5 ). reactivity was detected in the gut of individual worms , where considerable staining was observed on the gut epithelium and in the gut lumen . no reactivity was detected to any other structures in the nematodes . single worm pcr to identify the gene encoding cy - gala in different cyathostomin species single worm pcr experiments were performed using primers to amplify a 220 bp fragment of cy - gala - 1 from 50 morphologically - identified adult worms encompassing 10 species . a pcr product was obtained from all nematodes tested and sequencing confirmed that pcr products representative of each species encoded cy - gala sequence . fig6 shows a schematic representation of this fragment and pcr products from each species . there was variation in size of the pcr product obtained from different species ; from 267 bp ( for all c . coronatus individuals ) to 284 bp ( for one c . goldi individual ). this variation was due to a difference in intron size at this site amongst the species . the precise location of the intron was conserved as indicated by splice site analysis ( fig6 ). nucleotide identities between individuals from different species ranged from 78 . 9 - 99 . 1 % for the whole fragment . higher nucleotide identities were observed in the coding region ; interspecies variation ranged from 82 . 2 - 98 . 9 % over 180 nt , while the amino acid identities were 80 - 100 % over 60 residues . at the aa level , intra - species variation was as follows : c . catinatum 93 . 3 - 100 %; cs . goldi 90 . 0 - 100 . 0 %; co . coronatus 96 . 7 - 100 . 0 %; c . pateratum 93 . 3 - 100 %; cc . nassatus 88 . 3 - 98 . 3 %; cs . longibursatus 91 . 7 - 100 %; cc . ashworthi 90 . 0 - 100 . 0 %; cc . leptostomum 88 . 3 - 100 . 0 %. in an attempt to assign a species for the library clone , cy - gala - 1 , the coding sequence from each individual was compared against cy - gala - 1 in this 220 bp fragment . the highest identity was found to a c . pateratum individual ( 97 . 8 % nt identity and 98 . 3 % aa identity ). therefore , with the available sequence data for each species , we have provisionally identified cy - gala - 1 as belonging to c . pateratum . the optimum concentration of antigen to use in an elisa using a cocktail of antigens was evaluated using sera from cyathostomin infected ( positive ) and non - infected ( negative ) animals . fig7 a shows the serum antibody response to varying concentrations of antigen in two different cocktails of antigen ( ct1 and ct2 ). ct1 contains gala - 1 , - gala 2 and - 3 . ct2 contains these three antigens plus cid1 . individual antigen concentration is shown on the x - axis and optical density ( o . d ) on the y - axis . fig7 b shows the ratio of the od signal obtained om cyathostomin infected ( positive ) vs . uninfected ( negative ) animals in an elisa . individual antigen concentration is on the x - axis and ratio of positive to negative optical density on the y - axis . two different cocktails of antigen were tested in an elisa to assess their potential for discriminating different levels of mucosal infection . fig7 c and d shows mean serum antibody response to cocktail 1 ( ct1 ) and cocktail 2 ( ct2 ) respectively in groups of horses with varying infection levels . ct1 and ct2 were as described above . horses were grouped as follows according to total mucosal parasite burden ( tmb ). neg ; uninfected horses tmb = 0 ( n = 5 ), low ; tmb = 0 - 20000 , ( n = 8 ), medium ; tmb = 20000 - 100000 , ( n = 7 ), high ; tmb =& gt ; 100000 ( n = 26 ). error bars show +/− standard error of the mean . o . d = optical density . identification of the cyathostomin gala sequence is an advance in the development of an elisa for the diagnosis of larval cyathostominosis . three important criteria were met by this protein : 1 ) it appeared to be specific to larval stages ; 2 ) there was no cross reactivity with the other equine helminth species assessed here and 3 ) the gene encoding the protein was isolated from all cyathostomin species examined with a relatively low level of sequence variation amongst the species . furthermore , serum igg ( t ) responses to rcy - gala - 1 increased within 5 weeks of the administration of an experimental infection and the protein was also the target of igg ( t ) responses in naturally infected horses . the rt - pcr , immunoblot and elisa results indicated that cy - gala - 1 is restricted to parasitic larval stages , particularly dl stages . this is a vital feature for a diagnostic marker that specifically indicates mucosal larval burden . despite numerous attempts , rna extracted from el3 was of relatively poor quality so it was difficult to judge precise levels of transcription in these stages . el3 require extensive digestion in pepsin / hcl at 37 ° c . to remove them in sufficient quantity from the intestinal mucosa and submucosa and so it is technically difficult to obtain sufficient high quality rna . the el3 somatic protein extracts also contained a small amount of contaminating host protein ( it is impossible to totally separate every single worm from its host capsule ), and this may have resulted in the lower levels of reactivity of el3 extracts to cy - gala - 1 antiserum as indicated by the elisa results . immunolocalisation was also attempted in el3 , but degradation resulted in a lack of distinct morphology and no specific binding was observed ( data not shown ). therefore it remains to be fully elucidated if cy - gala is a significant immunogen of el3 , or is predominantly an antigen of the later larval stages . immunolocalisation studies of diseased equine mucosa are planned , to provide el3 embedded in their mucosal cysts . serum igg ( t ) responses to rcy - gala - 1 over the time course of an experimental infection showed that the antigen is a reasonably early indicator of infection and these responses were identified whilst the infections were not patent [ 29 ]. indeed , in these ponies , the infections never progressed to patency even though the experiment was continued until 60 and 62 weeks pi in two of the animals . substantial increases in reactivity were observed at 5 weeks pi in one animal ( pony 104 ) and by 6 weeks in all ponies . cyathostomin larval - specific serum igg ( t ) responses were analysed previously in these animals and similar dynamics of responses were observed to the 20 and 25 kda complexes purified from el3 / dl mixtures [ 11 ]. furthermore , serum igg ( t ) reactivity to crude larval antigen was also observed to increase only after 6 weeks pi in these ponies [ 9 ], suggesting that only by this time point do larvae stimulate a detectable serum igg ( t ) response . pony 104 had the most pronounced increase in igg ( t ) to rcy - gala - 1 and this is similar to its response to crude larval antigen and the purified 20 - and 25 - kda antigen complexes [ 9 , 11 ]. the clinical signs observed in this pony ( reduced weight gain , lowest plasma fructosamine ) indicate that it may have had a greater burden of mucosal larvae [ 29 ]. indeed , when this animal was euthanized at 20 weeks pi it was found to have a high cyathostomin burden . unfortunately the other two ponies in the group were necropsied at 60 and 62 weeks pi so their burdens cannot be directly compared with pony 104 . nevertheless , the data provides preliminary evidence that this recombinant antigen may be able to distinguish varying degrees of disease . as mentioned above , there is similarity of the igg ( t ) response to rcy - gala - 1 and to the two larval antigen complexes purified and shown to have diagnostic potential previously [ 10 , 11 ]. the molecular mass of cy - gala , estimated at 25 . 6 kda , means that it could feasibly be a component of the 25 kda antigen complex , an observation supported by the results using anti - rcy - gala - 1 against el and dl somatic extracts in western blots . antiserum generated to the 20 kda complex in rabbits also bound rcy - gala - 1 indicating its presence in this complex also . this is not altogether surprising as these complexes were excised rather crudely from sds - polyacrylamide gels [ 10 , 11 ]. specificity of cy - gala - 1 in the cyathostominae was confirmed by probing the recombinant protein with sera from horses infected mono - specifically with heterologous helminth species . while experimentally infected ( ci ) and naturally infected ( ei ) horses recognised rcy - gala - 1 , igg ( t ) in serum from horses with large strongyle infections ( s . edentatus , s . westeri or s . vulgaris ) and p . equorum infection , did not bind the antigen . cross - reactivity was further explored by probing somatic extracts of other equine parasites with anti - rcy - gala - 1 serum : extracts from a . perfoliata , p . equorum , s . edentatus , s . vulgaris and s . equorum were analysed . in the elisa no binding above background levels was observed in any of the five other parasite extracts . in the immunoblot , there was a degree of binding to a band of approximately 38 kda in the p . equorum extract , but this was of far less intensity than binding observed in the cyathostomin dl samples . furthermore , there was no cross reactivity to p . equorum antigens when the samples were assessed using the elisa . the presence of sequences encoding gala - like proteins was confirmed in 10 cyathostomin species , indicating ubiquity of this gene in the group . there are currently 50 recognised cyathostomin species [ 23 ], and while a large number of species are often found in infected individuals [ 6 , 7 ], the bulk of the burden is consistently found to comprise 5 - 10 species [ 26 , 27 , 36 ]. nine of the species explored in this study belong to the 10 most common cyathostomins as identified by reinemeyer et al . ( 1984 ) [ 36 ], ogbourne ( 1976 ) [ 30 ] and lictenfels et al 2001 [ 22 ]. the presence of cy - gala in these species indicates it is likely to be present in most , if not all , cyathostomins . an analysis of the sequence of cy - gala - 1 amongst the cyathostomins indicated a low level of sequence diversity across the selected 220 bp region . it is possible that greater diversity exists outside this region and the full - length cdna sequences of cy - gala are currently being isolated from a number of species to investigate this further . promisingly , for development of a specific immunoassay , the levels of sequence diversity identified thus far are substantially lower among cyathostomins than they are when the cy - gala sequences are compared to orthologous sequences in other nematode species , i . e . 80 - 100 % vs . 25 - 35 % identity . the nematodes that were present in the ci pony group unfortunately had not been identified , so it is difficult to compare levels of rcy - gala - 1 igg ( t ) with the species present . a factor that must be considered in the development of any helminth immunodiagnostic assay is the length of time that circulating specific immunoglobulin levels take to return to normal values after anthelmintic treatment . since the ponies used in the experimental infection were not treated with anthelmintic before necropsy , this could not be assessed here . studies on a commercially - available serological elisa for a . perfoliata [ 33 , 34 ], which is based on the specific binding of igg ( t ) to a purified 12 / 13 kda antigen complex , indicated that post - treatment igg ( t ) levels can take months to reduce to ‘ non - infection ’ levels [ 2 , 4 ]. also , kjaer et al . ( 2007 ) [ 18 ] found that two thirds of horses which had no visible signs of tapeworm infection at necropsy had elisa ods higher than the current accepted cut - off for infection ( 0 . 2 ). despite this , the a . perfoliata 12 / 13 kda antigen elisa is still regarded as the most useful diagnostic tool for infection [ 1 , 18 ]. these observations suggest that circulating igg ( t ) levels may remain high for a time after treatment and this will be considered when designing how a cyathostomin diagnostic assay , based on igg ( t ), could be used in future . no function has been ascribed to orthologues of cy - gala in other nematode species and only nb - klp has been characterised in any detail [ 38 ]. it was speculated that nb - klp may be a cuticular protein , based on its identity to ce - klps , which are described as ‘ keratin - like ’. however the authors did not explore this further . ce - klp - 1 and - 2 encode hypothetical proteins , and some information regarding these is available in wormbase ( www . wormbase . org ). both are predicted to be alpha - helical proteins , and ce - klp - 1 has been confirmed by transcript evidence , while ce - klp - 2 has been partially confirmed . ce - klp - 1 shows no rnai phenotype , while ce - klp - 2 displays ‘ embryonic lethal ’, indicating that it may play a role in development . an anatomic expression plan is available for ce - klp - 2 , showing expression in pharyngeal muscles and tail neurons which is different to what was observed here with localisation of cy - gala to the worm intestinal lumen . the function of this molecule remains to be elucidated . abbott j . b ., barrett e . j ., the problem of diagnosing tapeworm infections in horses , equine vet . j . 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