Veterinary Parasitology 200 (2014) 265 270

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Veterinary Parasitology

jo u r nal homep age: www.elsev ier .com/ locate /vetpar

Develo c sin natu

M.K. Nie . BellU.V. Andersenc

a M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, United Statesb Department of Statistics, George Mason University, Fairfax, VA, United Statesc Department of Large Animal Sciences, University of Copenhagen, Copenhagen, Denmark

a r t i c

Article history:Received 14 OReceived in re19 December Accepted 22 D

Keywords:Strongylus vulgELISAFoalsSero-conversio

1. Introdu

Strongylparasite infsite in the

CorresponE-mail add

0304-4017/$ http://dx.doi.ol e i n f o

ctober 2013vised form2013ecember 2013

aris

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a b s t r a c t

Strongylus vulgaris is regarded as the most pathogenic helminth parasite infecting horses.Migrating larvae cause pronounced endarteritis and thrombosis in the cranial mesentericartery and adjacent branches, and thromboembolism can lead to ischemia and infarc-tion of large intestinal segments. A recently developed serum ELISA allows detection ofS. vulgaris-specic antibodies during the six-month-long prepatent period. A population ofhorses has been maintained at the University of Kentucky without anthelmintic interven-tion since 1979, and S. vulgaris has been documented to be highly prevalent. In 2012, 12 foalswere born in this population, and were studied during a 12-month period (MarchMarch).Weekly serum samples were collected to monitor S. vulgaris specic antibodies with theELISA. Nine colts underwent necropsy at different time points between 90 and 300 days ofage. At necropsy, Strongylus spp. and Parascaris equorum were identied to species and stageand enumerated. Initial statistical ndings indicate a signicant interaction between foalage and ELISA results (p < 0.042). All foals had initial evidence of S. vulgaris-directed mater-nal antibodies transferred in the colostrum, but then remained ELISA negative during theirrst three months of life. Foals born in February and March became ELISA positive at about12 weeks of age, while those born in April and May went positive at about 15 and 21 weeks,respectively. Foal date of birth was signicantly associated with ELISA results (p < 0.0001).This could be explained by birth date-dependent differences in parasite exposure. One foalremained ELISA-negative throughout the course of 30 weeks during the study. A signi-cant association was found between ELISA values and larval S. vulgaris burdens (p < 0.0001)as well as a three-way interaction between S. vulgaris, S. edentatus, and P. equorum bur-dens (p < 0.001). A plateau with a subsequent decline in ELISA values corresponded with S.vulgaris larvae leaving the bloodstream and migrating back to the intestine.

2013 Elsevier B.V. All rights reserved.

ction

us vulgaris is regarded as the most pathogenicecting horses. Larval migrations of this para-cranial mesenteric artery and main branches

ding author. Tel.: +1 859 218 1103; fax: +1 859 257 8542.ress: martin.nielsen@uky.edu (M.K. Nielsen).

cause a pronounced brinous endarteritis with formationof aneurysms and thrombosis (Duncan, 1974). Thrombo-embolism has been described as the cause of a painfuland often fatal colic syndrome, where migrating S. vulgarisare associated with ischemia and infarction of intestinalsegments (Enigk, 1951; Duncan and Pirie, 1975). Overthe past ve decades, parasite control regimens involv-ing frequent and regular anthelmintic treatment of allhorses appear to have caused a decline in prevalence and

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.vetpar.2013.12.024pment of Strongylus vulgaris-specirally infected foals

lsena,, A.N. Vidyashankarb, H.S. Gravattea, Jerum antibodies

awa, E.T. Lyonsa,

266 M.K. Nielsen et al. / Veterinary Parasitology 200 (2014) 265 270

abundance of S. vulgaris, and the parasite is now consid-ered rare in managed horse populations (Herd, 1990; Loveand Duncan, 1991). However, surveys performed in man-aged horses in different places of the world, continue todocument tbasis (Hgl2012), and recommendment regimto suggest athese recenfarms for p

About 5order of Str(Lichtenfelstic strongylbetween spdifferences1993) and are availabvulgaris in the pathoglong prepatimmunosorvalidated foof horses (Asera from afew of thesefor interpre

At the Ukept naturaout anthelmhorses are with preval1991, 1994every year, cation andstudies.

The aimopment of infected foaidentify theexposed foa

2. Materia

2.1. Horses

The Barnlished at U1968. Sinceanthelmintment stalliconsists of spring, and is carried oherd has benumerous levels of gaParascaris eeri, Oxyuris

1990, 1991, 1994, 1997). Large strongyles are particu-larly prevalent and abundant in this herd with S. vulgarisreported present in all necropsied foals (Lyons et al., 1990,1991, 1994, 1997).

2012 betweh, serughout

Necrop

e nineof agetion. M, S. edeibed (lgaris ered aating lthe veinth s

morp

ELISA

vulgarmeasu). Plate

g/ming an5 L

wells. ol as wwells. horse SA) w000. Tts weritive cts werntire gecropsree gro, interm

Statisti

atisticAS Inseen ELs wereixed med to ei) S. vul S. vuum coar mixandomnatizeo the ghat S. vulgaris is still encountered on a regularund et al., 1997; Boxell et al., 2004; Pilo et al.,one recent study reported an association withed surveillance-based selective therapy treat-ens (Nielsen et al., 2012). There is no evidencenthelmintic resistance in Strongylus species, butt ndings emphasize the need for monitoringresence of S. vulgaris.0 different parasite species belonging to theongylida have been described infecting horses

et al., 2008). They all shed the characteris-e type egg that does not allow differentiatingecies or genera based on morphological or size. Coprocultures (Russell, 1948; Bevilaqua et al.,a real-time PCR assay (Nielsen et al., 2008)le for diagnosing presence of adult female S.the intestinal tract, but these do not reectenic migrating larvae during their six-monthent period. Therefore, a serum enzyme-linkedbent assay (ELISA) was recently developed andr detecting migrating larvae in the bloodstreamndersen et al., 2013). Validation data includedbout 100 well-characterized horses, but very

were foals, so no solid information is availabletation of serum ELISA results in this age group.niversity of Kentucky, a herd of horses has beenlly exposed to mixed species of parasites with-intic intervention since 1979. As a result, thesesubstantially exposed to S. vulgaris infectionence rates at or close to 100% (Lyons et al., 1990,, 1997). About 812 foals are born into this herdand colts are most often necropsied with identi-

enumeration of parasites for various scientic

with this study was to evaluate the devel-S. vulgaris-specic antibody titers in naturallyls during their rst year of life. The goal was to

minimum sampling age at which S. vulgaris-ls can be expected to become ELISA positive.

ls and methods

10 parasitology research horse herd was estab-niversity of Kentuckys Maine Chance Farm in

1979, this herd has been maintained withoutic intervention except medication of replace-ons (Lyons et al., 1990). The herd currentlyabout 24 mixed light breed mares with off-is kept on pasture year-round. Natural breedingut with a stud stallion residing in the herd. Theen parasitologically well characterized throughstudies, and horses are known to harbor highstrointestinal parasites such as cyathostomins,quorum, Gasterophilus spp., Strongyloides west-equi, and Anoplocephala perfoliata (Lyons et al.,

InherdMarcthrou

2.2.

Thdays infecgarisdescrS. vurecovMigrting Helmusing

2.3.

S.cate 2013at 0.1blockand 7cate contrcate anti-TX, U1:40,resula posresulthe eing nto thearly

2.4.

St9.3 (Sbetwvalueto mformand (larvaequora lineas a reuthadue t, nine colts and three llies were born in theen February 19th and May 28. Beginning inm samples were collected weekly from all foals

one full year.

sies

colts underwent necropsy between 90 and 300 to document the different stages of S. vulgarisethods for recovering intestinal stages of S. vul-ntatus, and P. equorum have previously beenLyons et al., 1991). In addition, larval stages ofpresent in the cranial mesenteric artery werend counted by dissecting the arterial lumen.

arvae of S. edentatus were recovered by dissec-ntral abdominal walls and perirenal fat tissue.pecimens were identied to species and stagehological criteria (Lichtenfels et al., 2008).

is-specic ELISAs were performed with dupli-res as previously described (Andersen et al.,s were coated with 75 L of recombinant SvSXPl in PBS and incubated overnight at 4 C. Afterd washing wells, horse sera were diluted 1:50of diluted serum added to each of two dupli-Diluted serum from a positive and a negativeell as a blank sample were included in dupli-

Horseradish peroxidase (HRP)-conjugated goatIgG(T) (Bethyl Laboratories, Inc., Montgomery,as used as secondary antibodies at a dilution ofhe optic density was recorded at 450 nm ande reported as normalized values, percentage ofontrol (PP) (Andersen et al., 2013). The ELISAe evaluated graphically both as mean values forroup of foals and for the nine foals undergo-y. For the latter evaluation, foals were assignedups of three foals each to distinguish betweenediate, and late stages of S. vulgaris infection.

cal analyses

al analyses were performed, using SAS softwaretitute Inc., Cary, NC, USA) to identify associationISA PP values and date of birth (DOB). ELISA PP

log-transformed to reduce the skewness priorodel analyses. Additional analyses were per-

valuate the association between ELISA PP valueslgaris burdens and (ii) three-way interaction oflgaris, larval S. edentatus, and total intestinal P.unts. All these analyses were performed usinged effects model with foal effect being treated

effect after adjusting for DOB. Since foals wered at different time points during the study andeneral sparsity in the data, all the variables were

M.K. Nielsen et al. / Veterinary Parasitology 200 (2014) 265 270 267

Table 1General information on the 12 participating foals with corresponding Strongylus spp. and Parascaris equorum necropsy data, where available.

Foal Sex Born Necropsy agea Intestinal S.vulgaris

Migrating S.vulgarisb

Intestinal S.edentatus

Migrating S.edentatusc

Total P.equorumd

A F e

B M C M D M E F F F G M H F I M J M K M L M

a Age at necb S. vulgaris in branc S. edentatu ue.d All intestine Females w

treated as cevaluated a

3. Results

The 12 fcorrespondaddition towere infectnot generat

Mean S.weeks of thpoint, onlyis not inclumigrating vmean serumat necropsydisplayed Ecutoff valuethese for upone and thrbelow the cthe initial wFig. 3 preseoff value inborn in Febconversion half of Aprilfoals born iby 17 weekconverted aPP values b30 weeks oin the cranismaller incfoals born a

Statistiction betwevulgaris burtistically sigand three-w

ntatusting fo

scussi

is studlopmeted foarnal ane withe rempsy d(131lgaris arteriee wee

is thaostic itions.e at se

interes foalse ELI

born eld be fo19 FEB 10 MAR 94 0 162 17 MAR 102 0 292 5 APR 204 46 76 14 APR 15 APR 22 APR 131 0 158 22 APR 271 99 296 25 APR 293 324 77 5 MAY 214 0 249 15 MAY 309 188 9 28 MAY 337 447 77

ropsy given in days.larvae dissected out of the cranial mesenteric artery, celiac artery and mas larvae dissected out of the ventral abdominal wall and perirenal fat tissal stages of P. equorum encountered.ere not necropsied, so no worm count data available for these.

lass variables. All statistical signicances weret the 5% signicance level.

oals, their date of birth, age at necropsy, anding worm counts are presented in Table 1. In

the parasites reported in the table, all foalsed with cyathostomins, but total counts wereed.

vulgaris antibody ELISA values for the rst 41e study are depicted in Fig. 1. After this time-

one foal was left in the study and that dataded in the gure. Fig. 2 summarizes counts ofersus intestinal S. vulgaris and corresponding

ELISA PP values for the foals grouped by age. At their rst week of life, eight of the foalsLISA PP-values above the identied diagnostic

of 8.75% (Andersen et al., 2013) and maintained to 13 weeks of age (minimum and medium ofee weeks, respectively), before dropping downutoff. The mean half-life of ELISA titers duringeeks of the study was estimated to 19 days.

nts the percentage of foal-weeks above the cut- different foal age groups. In general, the foals

S. edeadjus

4. Di

Thdeveinfecmateof liferwisNecrodays S. vuteric beforstudydiagninfec

Agand whenbecamfoals shouruary and March (n = 3), demonstrated sero-at about 12 weeks of age, those born in the rst

(n = 3) sero-converted at about 15 weeks of age,n the second half of April (n = 3) sero-converteds of age, and foals born in May (n = 3) sero-t 21 weeks of age. One foal (J) maintained ELISAelow 7% throughout the study until necropsy atf age, despite harboring 249 S. vulgaris larvaeal mesenteric artery. However, it did exhibit arease in ELISA values at the same age as othert the same time.al analyses identied a signicant associa-en S. vulgaris ELISA PP values and larval S.dens (p < 0.0001) and DOB (p < 0.0001). A sta-nicant association between ELISA PP valuesay interaction between larval S. vulgaris, larval

between dicentral Kenand August40 C for thcipitation wduring the www.nws.ndevelopmetrates that pconditions (sible that fhave ingestmonths, whhave exposthe followinobserved EL 0 0 00 0 2620 30 66 0 0 4380 57 256 251 680 0 288 129 209

10 131 3

ches.

, and total intestinal P. equorum burdens afterr DOB was also determined (p < 0.001).

on

y generated new and useful information on thent of S. vulgaris-specic antibodies in naturallyls. Collectively, the foals displayed evidence oftibodies being present during the rst weeks

a half-life of about three weeks, but oth-ained ELISA negative until 12 weeks of age.ata from the youngest three foals aging 941319 weeks) documented that large numbers oflarvae had established in the cranial mesen-s at early age without causing sero-conversionks later. Therefore, one conclusion from thist the serum ELISA does not generate reliablenformation in the younger foals despite heavy

ro-conversion varied between 12 and 21 weekstingly, this was statistically associated with

were born. Those born later in the springSA positive at a much later age, compared toarlier in the year. We believe the explanationund in different levels of parasite transmissionfferent seasons. In 2012, weather conditions intucky were very dry and hot during June, July,

with temperatures cycling between 30 ande large majority of the time. In addition, pre-as signicantly below the norms for the regionsummer months (National Weather Service,oaa.gov). Available evidence on survival and

nt of free-living strongyle stages clearly illus-asture transmission is very limited under suchNielsen et al., 2007). Therefore, it appears plau-oals born earlier in the spring were likely toed S. vulgaris larvae already during the springile foals born just before summer would noture to high numbers of infective larvae untilg autumn months later. This could explain theISA results.

268 M.K. Nielsen et al. / Veterinary Parasitology 200 (2014) 265 270

Fig. 1. Mean serum SvSXP ELISA values reported as percent of positive control (Y-axis) as a function of weeks (X-axis) during the course of the study. Yerror bars indicate 95% condence intervals for each mean value.

The serum ELISA is developed to detect antibodiesagainst a polypeptide in the excretory/secretory fractionof the migrating larvae, named SvSXP (Andersen et al.,2013). When this assay was developed, the polypeptidewas initially identied in a cDNA library constructed from amixture of migrating late L4 and early L5 larvae, reectinglater stagesunknown tolarval stageis primarilywould helpwhere infe

before sero-conversion. It is also possible that maturationof the immune system played a role, with the younger foalsbeing incapable of mounting an antibody response to theinfection until a later age. However, this would not explainthe observed differences between foals born at differenttimes during the spring.

othere obsersionghoutne waecline

Fig. 2. Numbeconsisting of tmean serum S of S. vulgaris larval infection. Thus, it remains which extent the gene is expressed in youngers, and it is possible that production of SvSXP

associated with later stage of infection. This explain the observations made in this study,cted foals remained sero-negative for months

Anphasconvthroudeclivae drs of arterial (white columns) and intestinal (gray columns) S. vulgaris specimenhree foals necropsied at three different age intervals. Age intervals of necropsievSXP ELISA values reported as percent of positive control for each group (values notable nding was the apparent plateau-erved with the ELISA results. After sero-, foals continued to maintain high ELISA levels

the study. A tendency toward a graduates observed as the numbers of migrating lar-d and adult intestinal worm burdens increaseds (values on left Y-axis) encountered in three groups eachd foals are given on the X-axis. Stippled line indicates theon right Y-axis).

M.K. Nielsen et al. / Veterinary Parasitology 200 (2014) 265 270 269

Fig. 3. Percen t of a postudy.

(Fig. 2). Howvariable to aeral, it remafor ELISA vaeffective anthis would

The Barnhistorical aout anthelmpresence oequine parcannot be rprivately oments. Althoccur in ma2004; Pilo eunlikely tofound in thiprevalence carried out of the ELISAdifferent an

The stattion betweeS. vulgaris dation worwith arteriathe signicaedentatus, aciation betwin all the these specitwo speciesObservationelaborate stthat other pand cause finfected wi

uggestot ser

infect summc anttectaber, thebe depdition,

antibo positi

ict of

e Univse of rSris. Tw. Andtage of foal weeks above the diagnostic cutoff ELISA value of 8.75 percen

ever, data observations were too few and toollow any rm conclusions in this regard. In gen-ins unknown how long time would be neededlues to decline to negative levels following anthelmintic treatment and studies investigatingbe of great value.

10 parasitology herd utilized in this study isnd unique by having been maintained with-intic treatment for decades. This allows a rm

f S. vulgaris as well as a wide array of otherasites. Consequently, the foals studied hereinegarded as representative of managed foals onwned farms using regular anthelmintic treat-ough S. vulgaris infection has been shown tonaged horses (Hglund et al., 1997; Boxell et al.,t al., 2012; Nielsen et al., 2012), they are highly

encounter the same high infection levels ass study. Therefore, studies monitoring the sero-of S. vulgaris on managed horse farms should be

not sdid nwere

Inspecito deFurthmay In adfer ofwere

Con

Ththe uvulgaUlla Vto provide more information on interpretation in different age groups of horses exposed tothelmintic regimens.istical analyses revealed a signicant associa-n ELISA PP-values and the numbers of arteriallarvae. This is in accordance with prior vali-k, where ELISA values associated signicantlyl larval burdens (Andersen et al., 2013). Further,nt three-way interaction between S. vulgaris, S.nd P. equorum merely indicates a positive asso-een these three parasites that were present

studied foals. In other words, when one ofes were present in high numbers, the other

were likely to also be numerous in the foals.s in this data set were too few to allow moreatistical analyses. Although it remains possiblearasites can cross-react with the serum ELISAalse-positives, a validation study using horsesth and without different parasite categories did

References

Andersen, U.VC.R., LyonsSvSXP: a Snosis. Para

Bevilaqua, C.Minfective laMed. Vet.

Boxell, A.C., Giof gastroinAustralia.

Duncan, J.L., 13437.

Duncan, J.L., Pinfections

Enigk, K., 1951Pferdes. M

Herd, R.P., 199tomes andVet. 12, 73

Hglund, J., LjuA., 1997. Onematodesitive control in different age intervals (X-axis) during the

that (Andersen et al., 2013). The present studyve to address this question further, as all foalsed with all major gastrointestinal nematodes.ary, this study illustrated that S. vulgaris-

ibodies in heavily exposed foals do not develople levels before 12 weeks of age at the earliest.

study suggested that age of sero-conversionendent on parasite exposure in individual foals.

the study provided evidence of maternal trans-dies early in life. Finally, serum ELISA PP-valuesvely associated with arterial S. vulgaris burdens.

interest statement

ersity of Kentucky has applied for a patent forvSXP and relevant epitopes in the diagnosis of S.o of the co-inventors, Drs. Martin K. Nielsen andersen, declare to have no conicts of interest.., Howe, D.K., Dangoudoubiyam, S., Toft, N., Reinemeyer,, E.T., Olsen, S.N., Monrad, J., Nejsum, P., Nielsen, M.K., 2013.trongylus vulgaris antigen with potential for prepatent diag-sit. Vectors 6, 84..L., Rodrigues, M.L., Concordet, D., 1993. Identication ofrvae of some common nematode strongylids of horses. Rev.

Toulouse 12, 989995.bson, K.T., Hobbs, R.P., Thompson, R.C.A., 2004. Occurrencetestinal parasites in horses in metropolitan Perth, WesternAust. Vet. J. 82, 9195.974. Strongylus vulgaris infection in the horse. Vet. Rec. 95,

irie, H.M., 1975. The pathogenesis of single experimental with Strongylus vulgaris in foals. Res. Vet. Sci. 18, 8293.. Die Pathogenese der thrombotisch-embolische Kolik desonatsh. Tierheilk. 3, 6574.0. The changing world of worms the rise of the cyathos-

the decline of Strongylus vulgaris. Comp. Cont. Educ. Pract.2736.ngstrom, B.L., Nilsson, O., Lundquist, H., Osterman, E., Uggla,ccurrence of Gasterophilus intestinalis and some parasitic

s of horses in Sweden. Acta Vet. Scand. 38, 157165.

270 M.K. Nielsen et al. / Veterinary Parasitology 200 (2014) 265 270

Lichtenfels, J.R., Kharchenko, V.A., Dvojnos, G.M., 2008. Illustrated iden-tication keys to strongylid parasites (Strongylidae: Nematoda) ofhorses, zebras and asses (Equidae). Vet. Parasitol. 156, 4161.

Love, S., Duncan, J.L., 1991. Could the worms have turned? Equine Vet. J.23, 152154.

Lyons, E.T., Drudge, J.H., Tolliver, S.C., 1990. Prevalence of some inter-nal parasites found (19711989) in horses born on a farm in CentralKentucky. J. Equine Vet. Sci. 10, 99107.

Lyons, E.T., Tolliver, S.C., Drudge, J.H., Granstrom, D.E., Stamper, S., Collins,S.S., 1991. Transmission of some internal parasites in horses born in1989 on a farm in Central Kentucky. J. Helminthol. Soc. Wash. 58,213219.

Lyons, E.T., Tolliver, S.C., Stamper, S., Drudge, J.H., Granstrom, D.E., Collins,S.S., 1994. Transmission of some species of internal parasites in horsesborn in 1990, 1991, and 1992 in the same pasture on a farm in CentralKentucky. Vet. Parasitol. 52, 257269.

Lyons, E.T., Tolliver, S.C., Collins, S.S., Drudge, J.H., Granstrom, D.E., 1997.Transmission of some species of internal parasites in horses born in

1993, 1994, and 1995 on the same pasture on a farm in Central Ken-tucky. Vet. Parasitol. 70, 225240.

Nielsen, M.K., Kaplan, R.M., Thamsborg, S.M., Monrad, J., Olsen, S.N., 2007.Climatic inuences on development and survival of free-living stagesof equine strongyles: implications for worm control strategies andmanaging anthelmintic resistance. Vet. J. 174, 2332.

Nielsen, M.K., Peterson, D.S., Monrad, J., Thamsborg, S.M., Olsen, S.N.,Kaplan, R.M., 2008. Detection and semi-quantication of Strongylusvulgaris DNA in equine faeces by real-time quantitative PCR. Int. J.Parasitol. 38, 443453.

Nielsen, M.K., Vidyashankar, A.N., Olsen, S.N., Monrad, J., Thamsborg, S.M.,2012. Strongylus vulgaris associated with usage of selective therapyon Danish horse farms is it reemerging? Vet. Parasitol. 189, 260266.

Pilo, C., Altea, A., Pirino, S., Nicolussi, P., Varcasia, A., Genchi, M., Scala, A.,2012. Strongylus vulgaris (Looss, 1900) in horses in Italy: is it still aproblem? Vet. Parasitol. 184, 161167.

Russell, A.F., 1948. The development of helminthiasis in thoroughbredfoals. J. Comp. Pathol. 58, 107127.

Development of Strongylus vulgaris-specific serum antibodies in naturally infected foals1 Introduction2 Materials and methods2.1 Horses2.2 Necropsies2.3 ELISA2.4 Statistical analyses

3 Results4 DiscussionConflict of interest statementReferences