Research ArticleViral Serological and Antioxidant Investigations of EquineRhinitis A Virus in Serum and Nasal Swabs of CommerciallyUsed Horses in Poland

Barbara Bahanoacutew 1 Agnieszka Frdcka1 Natalia Jackulak1 Ewa Romuk 2

Tomasz Gwbarowski3 Aleksander Owczarek 4 and Dominika Stygar 5

1Faculty of Veterinary Medicine Department of Pathology Division of MicrobiologyWrocław University of Environmental and Life Sciences Wrocław Poland2Department of Biochemistry School of Medicine with Dentistry Division in Zabrze Medical University of Silesia Katowice Poland3Department of Basic Medical Sciences Wrocław Medical University Wrocław Poland4Department of Statistics Department of Instrumental Analysis School of Pharmacy with the Division of Laboratory Medicine inSosnowiec Medical University of Silesia Katowice Poland5Chair and Department of Physiology School of Medicine with Dentistry Division in ZabrzeMedical University of Silesia Katowice Poland

Correspondence should be addressed to Barbara Bazanow barbarabazanowupwredupl

Received 14 October 2017 Revised 19 February 2018 Accepted 15 March 2018 Published 22 April 2018

Academic Editor Paul M Tulkens

Copyright copy 2018 Barbara Bazanow et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Equine rhinitis A virus (ERAV) is considered to be an important pathogen in horses but relatively few studies areavailableAimsThe purpose of this study was to verify ERAV seroprevalence in selected horses in Poland in addition to correlationbetween ERAV and age and sex of analysed animals and the antioxidant status Methods The material collected from clinicallyhealthy horses was tested using the VNT (353 serum samples) and virus isolation method (44 nasal swabs) 27 serum samples withantibody titers between 0 and ge1 2048 were chosen for further analysis The study was conducted in group 1 (ERAV titer le 64)and group 2 (ERAV titer gt 64) Results Seroneutralisation tests showed positive results in 72 of serum samples No significantcorrelation between ERAV seropositive results and selected biochemical indicators was observed Group 2 had statistically higherconcentrations of SOD and CuZnSOD than the analysed group 1 Conclusions ERAV was not detected in the nasal swab samplesAntioxidant parameters did not significantly vary between horses of different breed sex or age The ERAV virus had an impact onplasma total SOD and CuZn SOD activity in horses in early stages of convalescence

1 Introduction

Equine rhinitis A virus (ERAV) transmitted through nasalsecretions and in urine causes mild to severe upper respira-tory tract disease including fever anorexia nasal dischargebronchitis pharyngitis coughing and swollen lymph nodes[1ndash4] Viral respiratory diseases are one of the most costlyproblems in equine breeding [5] Owners practitionersand virologists however usually devote little attention toERAV infections when compared to herpesvirusesrsquo infections

and equine influenza It is believed that ERAV causes atrivial illness but in many cases this virus weakens theathletic condition of the horse causing delays in training andalso hinders performance at an optimal level for prolongedperiods of time [6] Admittedly ERAV has been widelyisolated and serological investigations in horses have beencarried out in many countries but there is still a lack ofdetailed information about seroprevalenceThe role this virusplays in equine respiratory infections of a viral nature hasbeen marginalised and as a result relatively few studies

HindawiBioMed Research InternationalVolume 2018 Article ID 8719281 6 pageshttpsdoiorg10115520188719281

2 BioMed Research International

Table 1 Age and gender profile of horses included in the study

GenderNumber of animals Mares Stallions Geldingsin the groups 163 185 5

Age (years)1ndash3 y 4ndash6 y 7ndash9 y 10ndash12 13ndash15 y gt15 y

Number of animals 83 135 66 39 11 19Statistical significance was set at a p value below 005

about ERAV infections are available [5 7] However in lightof recent research ERAV is increasingly considered to bean important respiratory pathogen in horses [7] Reactiveoxygen species (ROS) are known to play a role in viraldiseases by influencing a hostrsquos defensive response Viralinfections influence the production of reactive oxygen species(ROS) in the infected cells and it has been proved that antiox-idant systems protect host cells against a variety of virusesby changing their oxidativeantioxidative status [8] ROScan kill pathogens directly by causing oxidative damage tobiocompounds or indirectly by stimulating pathogen elim-ination through various nonoxidative mechanisms [9] Thechanges in biochemical parameters and pathways involved inthe generation of ROS during viral infections are not fullyunderstood

To the best of our knowledge there have been no sero-logical investigations of equine rhinitis A virus in serum fromdifferent equine populations of horses in Poland to this pointThere was also no assessment of the relationship betweenequine rhinitis A virus infection and antioxidative responsein horsesThus the overall goal of this studywas to determinehow many from a group of selected animals in Polandthat were commercially used were ERAV seropositive andwhether there was any correlation between ERAV and the ageor sex of the analysed animals The second aim of this studywas to estimate the antioxidant status in ERAV seropositivehorses and whetherhow it changes in different stages ofconvalescence

2 Materials and Methods

Three hundred and fifty-three serum samples were collectedfrom clinically healthy equine populations originating fromall across Poland The age and gender profile of the selectedhorses is presented in Table 1 The study included horsesfrom horse riding clubs breeding farms purchasing centresand equine slaughterhousesThe collectedmaterial was testedusing a virus neutralisation test (VNT) in 96-well microtiterplates In each well 25 120583l of Minimum Essential Medium(MEM) (Sigma UK) supplemented with 2 foetal bovineserum (FBS Sigma USA) 4mM glutamine (Sigma UK)100Uml of penicillin and 100120583gml of streptomycin (SigmaGermany) was pipetted Serum samples were inactivatedfor 30min at 56∘C and then 25 120583l was added to a well inthe first column to obtain a starting dilution of 1 4 Fromthis well twofold dilutions were made by pipetting 25 120583l ofeach well into the next Then 25 120583l of hamster complement(Biomed Lublin Poland) and finally 25 120583l of 100 TCID50 of

Figure 1 CPE caused by ERAV replication Visible rounded cellsdetached from the bottom of the dish and formed aggregates Scalebar represents 20 120583m Mag 200x

Figure 2 Noninfected RK-13 cell line Scale bar represents 20120583mMag 200x

ERAV (V 172270 reference strain) were added to each wellSerum and virus were preincubated at 37∘C for 1 to 2 hours toallow for neutralisation of the virus Thereafter 50 120583l of RK-13 cell line suspension per well was added Plates were thenincubated for 5 days at 37∘C and under 5 CO2 The highestdilution of serum that neutralises the test dose of the viruswas determined as the titer of the serum

21 Nasal Swabs Among the serum samples 44 were col-lected together with nasal swabs in order to investigatethe presence of ERAV in nasal mucosa The virus isolationwas carried out in an RK-13 (rabbit kidney) cell line Allcollected materials were prepared using previously describedprocedures [10] and then inoculated (50ndash100 120583l per well) into24-well polystyrene plates containing an RK-13 cell line Theplates were then incubated in 37∘C5 CO2 and observeddaily for 5ndash10 days for the development of cytopathic effects(CPE) (Figures 1 and 2) using an inverted microscope

BioMed Research International 3

(Olympus Corp Hamburg Germany Axio Observer CarlZeiss MicroImaging GmbH) In the absence of visible CPE asecond passage was conducted

22 Antioxidative Status Twenty-seven serum samplesobtained from the group virological investigated werechosen for biochemical blood research The antibody titersreached between 0 and ge1 2048 In the case of 16 animalsthe titers were between the levels of 1 4 and 1 64 thus pairedsera tests were performed to confirmexclude the currentdisease Titers higher than 1 64 or a 4-fold increase inantibody titer obtained in a paired serum samples test wereconsidered to be indicators of recent infection Based onthese assumptions the investigated horses were dividedinto two groups seronegative or infected in the past (titerle 1 64) and group of horses which had had recent contactwith the virus had contracted infection shortly before theexperiment or tested positive in a paired serum sample test(titer gt 1 64)

3 Oxidative Stress Markers Analysis

An antioxidant system in the serum was analysed by deter-mining superoxide dismutase (SOD EC 11511) ceruloplas-min (CER) the total antioxidant capacity (TAC) and totaloxidant status (TOS) Lipid peroxidation was determined bymalondialdehyde (MDA) concentration

31 Protein Concentration Protein concentration was deter-mined using the Lowry et al methods [11]

32 Superoxide Dismutase Analysis (SOD) SOD isoenzymesrsquoactivity was determined according to the Oyanagui method[12] with KCN as the inhibitor of the CuZnSOD isoenzymeCuZnSOD activity was calculated as the difference betweentotal SOD activity and MnSOD activity SOD activity wascalculated against a blank probe containing bidistilled waterEnzyme activity was expressed as nitrite units (NU) per mLserum One NU exhibits 50 inhibition of the formation of anitrite ion under the methodrsquos condition [12] The inter- andintra-assay coefficients of variations (CV) were 28 and54 respectively

33 Ceruloplasmin Concentration of CER in serum wasdetermined spectrophotometrically according to Richterich[13] using the reaction with p-phenyl diamine CER catalysesthe oxidation of colourless p-phenylenediamine replacing itwith a blue-violet dye The absorbance of the samples wasread at 560 nm The measurement was conducted on aPerkinElmer VICTOR-X3 reader Interassay and intra-assaycoefficients of variations were respectively 13 and 40

34 Total Antioxidant Capacity (TAC) Plasma TAC wasmeasured using a commercial kit (Randox Co Eng-land) The 221015840azino-di-(3-ethylbenzothiazoline sulphonate)(ABTS) was incubated with a peroxidase (metmyoglobin)and hydrogen peroxide to produce the radical cation ABTS+which has a relatively stable blue-green colour and was mea-sured at 600 nmThe suppression of the colour was compared

to the standard for TAC measurement assays (Trolox) Theassay results are expressed as a Trolox equivalent (mmolL)[14]The inter- and intra-assay coefficients of variations (CV)were 11 and 38 respectively

35 Total Antioxidant Status The method according to Erel[15] is based on the oxidation of iron (II) ions to iron (III)ions in an acidic medium Then iron (III) ions with xyleneorange form a colourful complex ranging up to a blue-purple colouration Absorption readings were taken with the560 nm filter on the VICTOR-X3 from PerkinElmer Theconcentrationwas calculated from the calibration curve usingH2O2 as the standard Values are expressed in 120583moll

36 Lipid Peroxidation Malondialdehyde (MDA) concen-tration was measured in samples of serum according tothe method described by Ohkawa et al using the reactionwith thiobarbituric acid with spectrophotometric detectionemploying 515 nm excitation and 552 nm emission wave-lengths MDA concentration was calculated from the stan-dard curve and prepared from 1133-tetraethoxypropane[16]The inter- and intra-assay coefficients of variations (CV)were 21 and 83 respectively

37 Lipofuscin (LPS) Serum levels of LPS were determinedaccording to Tsuchida et al [17] Serum was added with3 1 vv ethanol-ether shaken and centrifuged The intensityof fluorescence was determined using a PerkinElmer spec-trophotometer LS45 at a wavelength of 345 nm (absorbance)and 430 nm (emission) in a dissolved solid The values areexpressed in relative lipid extract fluorescence (RF) where100 RF corresponds to the fluorescence of 01 120583gml quinidinesulphate in 01 N sulfuric acid LPS concentrations are shownin RF The inter- and intra-assay coefficients of variations(CV) are 28 and 97

4 Statistical Analysis

Statistical analysis was performed using STATISTICA 100PL (StatSoft Cracow Poland) and StataSE 120 (StataCorpLP TX USA) Statistical significance was set at a 119901 valuebelow 005 Data was expressed asmeansplusmn SD and the resultswere assessed for normality using the ShapirondashWilk methodand the quantile-quantile plot Correlations among analysedbiochemical parameters were assessed using Pearson corre-lation coefficient For comparison of selected biochemicalparameters the Student 119905-test was applied

5 Results

51 Viral and Serological Analyses Virus isolation tests didnot detect ERAV in any of the nasal swab samples Seroneu-tralisation tests showed positive results in 254 (72) serumsamples with titer levels from 1 4 to 1 ge2048 Statistical anal-ysis showed no significant relationship between gender orage and antibodies against ERAV (119901 gt 005)

52 Oxidative Status Analysis The median ERAV titer of 64was determined and the study group was divided into two

4 BioMed Research International

Table 2 Comparison of analyzed biochemical parameters for group 1 and group 2 Data are expressed as means plusmn SD CER ceruloplasminTAC total antioxidant capacity TAS total antioxidant status SOD superoxide dismutase MnSOD Mn superoxide dismutase CuZn SODsuperoxide dismutase MDA malondialdehyde LPS RF lipofuscin

Parameter Group IERAV le 64

Group IIERAV gt 64 119901

Protein gl 6894 plusmn 1237 7302 plusmn 729 0311CER mgdl 1086 plusmn 353 1062 plusmn 455 0878TAC mmoll 094 plusmn 011 09 plusmn 014 0441TOS umoll 1024 plusmn 428 835 plusmn 587 0345Total SOD NUml 1332 plusmn 284 1601 plusmn 247 lt005Mn SOD NUml 718 plusmn 178 808 plusmn 205 0234CuZn SOD NUml 614 plusmn 25 794 plusmn 196 lt005LPS RF 1561 plusmn 341 1775 plusmn 448 0173MDA umoll 621 plusmn 309 659 plusmn 282 0742Statistical significance was set at a 119901 value below 005

ERAV lt 64 ERAV gt 6411

12

13

14

15

16

17

18

SOD

(NU

ml)

MeanMean plusmn standard error Mean plusmn 95 confidence interval

Figure 3 Total superoxide dismutase serum levels of horses fromgroup 1 (ERAV le 64) and group 2 (ERAV gt 64) Statisticalsignificance was set at a 119901 value below 005

subgroups group 1 (ERAV titer le 64) and group 2 (ERAVtiter gt 64) (Table 2) There was no statistically significantcorrelation between ERAV seropositive results and selectedbiochemical indicators The high titer group (ERAV gt 64)had statistically higher concentrations of SODandCuZnSODthan the low titer group (119901 lt 005 Figures 3 and 4 andTable 2) For other biochemical indicators there were nostatistically significant differences between the studied groups(Table 2)

6 Discussion

ERAV is commonly found in populations of horses world-wide nevertheless to the best of our knowledge in thepopulations of horses in the territory of Poland this virus hasnot been investigated yet In this study 72 of animals were

ERAV lt 64 ERAV gt 6445

50

55

60

65

70

75

80

85

90

95

CuZN

SOD

(NU

ml)

MeanMean plusmn standard error Mean plusmn 95 confidence interval

Figure 4 CuZn superoxide dismutase serum levels of horsesfrom group 1 (ERAV le 64) and group 2 (ERAV gt 64) Statisticalsignificance was set at a 119901 value below 005

ERAV seropositive including 46 of horses with serum titerhigher than 1 64 and 13 of horses with serum titer higherthan 1 1024whichmay suggest that those horses had recentlybeen infected In general the studies on ERVs show that thepercentages of prevalence may vary from 0 to 100 [18 19]

In European countries the prevalence of ERAV wasreported as high at more than 60 In the United Kingdom100 of serologically tested horses had a significant titer toERAV [19] Similar percentage of adult seropositive animals(73) was obtained in Ireland (1998) nevertheless most ofthe examined horses were febrile [20] A high prevalence ofERAV was confirmed in Austria in 2005 where 90 of theequine sera and 27 of the human sera showed reactivity toERAV [21] and in France the percentage was 794 [22] Ininvestigations carried out in the United States of America theprevalence of ERAV neutralising antibodies was reported atthe level of 77 [23] Reports from central-eastern Asia all

BioMed Research International 5

provinces ofMongolia show that this percentage has reachedonly 342 out of a total of 300 horses [24] According to theauthors this was probably because the blood samples werecollected mainly from young horses Age was also related tothe ERAV in the study of Burrows where 60 of adult horseswere seropositive in comparison with 125 of seropositivefoals and 83 of yearlings [18] Nevertheless we did notconfirm a significant relationship between age or gender andERAV antibodies in this study Unfortunately we cannotconfirmour result concerning the gender because correlationbetween sex and ERAV antibodies has not been widelydescribed in the literature on the subject yet

The prevalence of ERBV the second equine rhinitis virusbelonging to the Picornaviridae family in equine sera inPoland is close to 70 [25] Interestingly available studiesreport that ERBV seems to be more commonly isolated thanERAV [7] In our study we obtained a similar percentageof ERAV seropositive horses which means that ERAV andERBV are equally distributed among the horses in Poland

The above results have demonstrated that equine rhi-noviruses are commonly circulated among horses globallyand their clinical importance may have been underestimated[5] This study shows that ERAV antibodies are highly rep-resented in analysed equine subjects which suggests that sub-clinical infectionsmanifested by high seroprevalence in clini-cally healthy animals are quite possibly a very commonoccur-rence Nasopharyngeal swabs are best collected from infectedhorses suspected of being infected within 24ndash48 hours ofinfection to detect the virus [7] In our investigation theresults of virus isolation were negative probably because theswabs were taken more than two days after infection Furtherinvestigations need to be carried out for a better understand-ing of pathogenesis and the epidemiology of ERAV virus

Oxidative stress is considered to play a role in the progres-sion of viral infections [26] contributing to viral replicationinflammation and decreased immune cell proliferation [27]It is known that oxidative stress caused via viral infectionmay lead to several aspects of viral pathogenesis includinginflammatory response and viral replication [28] The oxi-dantantioxidant status in different groups of horses is shownto be unbalanced in several physiological situations [29] Theinfection by different types of virus results in an imbalancedoxidantantioxidant relation The main forces of cell pro-tection against ROS are the enzymatic antioxidant systems(superoxide dismutase glutathione peroxidase and catalase)and nonenzymatic antioxidant systems (glutathione ascor-bate and uric acid) which prevent lipid peroxidation andDNA damage [30] The strategy of defence against hydroxylradicals is the dismutation process of superoxide anions(O2minus) conducted by superoxide dismutase disproportiona-

tion of H2O2 catalysed by CAT and reduction with GPx [31]The synthesis of SOD is stimulated by a reduction of molecu-lar oxygen TNF interleukins endotoxins and chemicals andhypoxia [32 33] In our study horses currently infected byequine rhinitis A virus showed increased serum levels of totalSOD and ZnCu SOD but not Mn SOD when compared tosubjects with past infections or seronegative subjects TotalSOD can be considered as one of the most sensitive markersof oxidative stress [32 33] The fact that total SOD increased

but not Mn SOD was considered to be a positive trend in thephysiological mechanisms of cell protection Increased totalactivity of SOD in plasma may be understood as an adaptiveresponse of the cells to elevated oxidative stress markersRadakovic et al [34] showed that horses naturally infected byTheileria equi present increased erythrocytes levels of CuZnSOD concentrations which suggests that T equi could havemodified isoenzyme activity via free radical production Inour study the general trend of total oxidant status reductionand lack of a significant increase in MDA serum levelsshows the positive effects of antioxidative processes Otherstudies show increased levels of blood MDA and decreasedantioxidant enzymes under conditions of stress in racinghorses following jumping activity [35] On the other handOnmaz et al [36] found that decreased total SOD levels andincreased MDA concentrations occur in horses exposed tolong-term transport stress which confirms the presence ofindependent changes in intra- and extracellular activities ofantioxidant systems in response to different stress stimulatorsand environmental markers

7 Conclusions

ERAV may have an influence on the condition of horsescausing delays in training and hindering performance at anoptimal level for prolonged periods of time [6] ERAV virusantibodies were present in approximately 72 of commer-cially used horses analysed which shows that this virus iswidely distributed in PolandThe age of horses had no signif-icant effect on antioxidant and virus parameters The ERAVvirus as a stress marker had a significant impact on plasmatotal SOD and CuZn SOD activity in horses in early stagesof convalescence when compared with other subjects Abetter understanding of the basic physiological processesmayenhance the identification of animals at the risk of oxidativestress which may be accompanied by poor health and hasimportance for welfare outcomes

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors wish to thank Professor Dr Reto Zanoni fromInstitut fur Virologie und Immunologie (Universitat Bern)for making ERAV reference strain available Mr ScottRichards is acknowledged for reviewing the language of thismanuscript

References

[1] S E Lynch J R Gilkerson S J Symes J-A Huang and CA Hartley ldquoPersistence and chronic urinary shedding of theaphthovirus equine rhinitis A virusrdquo Comparative ImmunologyMicrobiology amp Infectious Diseases vol 36 no 1 pp 95ndash1032013

6 BioMed Research International

[2] W H McCollum and P J Timoney ldquoStudies on the seropreva-lence and frequency of equine rhinovirus 1 and 2 infection innormal horse urinerdquo in Sixth International Conference onEquineDiseases pp 83ndash87 1992

[3] J Horsington S E Lynch J R Gilkerson M J Studdert andC A Hartley ldquoEquine picornaviruses Well known but poorlyunderstoodrdquo Veterinary Microbiology vol 167 no 1-2 pp 78ndash85 2013

[4] F Li H E Drummer N Ficorilli M J Studdert and B SCrabb ldquoIdentification of noncytopathic equine rhinovirus 1 asa cause of acute febrile respiratory disease in horsesrdquo Journal ofClinical Microbiology vol 35 no 4 pp 937ndash943 1997

[5] A Diaz-Mendez ldquoCharacterization of an Equine Rhinitis AVirus (ERAVON05) and development of an experimentalinfectionmodel in horsesrdquohttpsatriumlibuoguelphcaxmluihandle102143645

[6] J A Mumford and P D Rossdale ldquoVirus and its relationship tothe ldquopoor performancerdquo syndromerdquo Equine Veterinary Journalvol 12 no 1 pp 3ndash9 1980

[7] L Viel and A Diaz-Mendez ldquoEquine rhinitis viruses An over-looked cause of respiratory infection Boehringer IngelheimVetmedica Inc 2013rdquo

[8] E Gaudernak J Seipelt A Triendl A Grassauer and EKuechler ldquoAntiviral effects of pyrrolidine dithiocarbamate onhuman rhinovirusesrdquo Journal of Virology vol 76 no 12 pp6004ndash6015 2002

[9] C N Paiva andM T Bozza ldquoAre reactive oxygen species alwaysdetrimental to pathogensrdquoAntioxidantsampRedox Signaling vol20 no 6 pp 1000ndash1034 2014

[10] J Timoney J H Gillespie F W Scott and J E BarloughLaboratory diagnosis of viral infection in Hagan and Brunerrsquosmicrobiology and infectious diseases of domestic animals CornellUniv Press Ithaca NY USA 8th edition 1988

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1951

[12] Y Oyanagui ldquoReevaluation of assaymethods and establishmentof kit for superoxide dismutase activityrdquo Analytical Biochem-istry vol 142 no 2 pp 290ndash296 1984

[13] R Richterich Clinical Chemistry PZWLWarsaw Poland 1971[14] F Fathiazad A Khaki M Nouri and A A Khaki ldquoEffect of

cinnamon zeylanicum on serum testosterone and anti-oxidantslevels in ratsrdquo International Journal of Womenrsquos Health andReproduction Sciences vol 1 no 1 pp 29ndash35 2013

[15] O Erel ldquoA new automated colorimetric method for measuringtotal oxidant statusrdquo Clinical Biochemistry vol 38 no 12 pp1103ndash1111 2005

[16] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxides inanimal tissues by thiobarbituric acid reactionrdquo Analytical Bio-chemistry vol 95 no 2 pp 351ndash358 1979

[17] M Tsuchida TMiura KMizutani andKAibara ldquoFluorescentsubstances in mouse and human serum as a parameter of vivolipid peroxidationrdquo Biochimica et Biophysica Acta vol 834 no2 pp 196ndash204 1985

[18] R Burrows ldquoLaboratory diagnosis of some virus infections ofthe upper respiratory tract of the horserdquo Equine VeterinaryJournal vol 1 no 1 pp 32ndash38 1968

[19] G Plummer and J B Kerry ldquoStudies on an equine respiratoryvirusrdquo Veterinary Record vol 74 pp 967ndash970 1962

[20] M Klaey M Sanchez-Higgins D P Leadon A Cullinane RStraub and H Gerber ldquoField case study of equine rhinovirus

1 infection Clinical signs and clinicopathologyrdquo Equine Veteri-nary Journal vol 30 no 3 pp 267ndash269 1998

[21] G Kriegshauser A Deutz E Kuechler T Skern H Lussy andN Nowotny ldquoPrevalence of neutralizing antibodies to Equinerhinitis A and B virus in horses and manrdquo Veterinary Microbi-ology vol 106 no 3-4 pp 293ndash296 2005

[22] E Plateau and E Levy ldquoSerological prevalence of equineadenovirus and rhinovirus among horse populations in thedistrict of Parisrdquo Recueil De Medecine Veterinaire vol 164 no4 pp 413ndash418 1990

[23] D F Holmes M J Kemen and L Coggins ldquoEquine rhinovirusinfection - serologic evidence of infection in selected UnitedStates horse populationsrdquo Equine Infectious Diseases vol IV pp315ndash319 1978

[24] O Pagamjav K Kobayashi H Murakami et al ldquoSerologicalsurvey of equine viral diseases in Mongoliardquo Microbiology andImmunology vol 55 no 4 pp 289ndash292 2011

[25] Z Gradzki L Boguta and Z Grądzki ldquoBadania serologicznenad wystepowaniem zakazen wywolywanych przez wirusyzapalenia nosa koni typu b w PolscerdquoMedycynaWeterynaryjnavol 65 no 2 pp 119ndash123 2009

[26] AMandas E L IorioMGCongiu et al ldquoOxidative imbalancein HIV-1 infected patients treated with antiretroviral therapyrdquoJournal of Biomedicine and Biotechnology vol 7 Article ID749575 2009

[27] S Aquaro F Scopelliti M Pollicita and C F Perno ldquoOxidativestress and HIV infection Target pathways for novel therapiesrdquoFuture HIVTherapy vol 2 no 4 pp 327ndash338 2008

[28] M L Reshi Y-C Su and J-R Hong ldquoRNA viruses ROS-mediated cell deathrdquo International Journal of Cell Biology vol2014 Article ID 467452 2014

[29] N Kirschvink B D Moffarts and P Lekeux ldquoThe oxidantantioxidant equilibrium in horsesrdquo The Veterinary Journal vol177 no 2 pp 178ndash191 2008

[30] P M Clarkson and H S Thompson ldquoAntioxidants what roledo they play in physical activity and healthrdquo American Journalof Clinical Nutrition vol 72 no 2 pp 637Sndash646S 2000

[31] A Łuszczewski E Matyska-Piekarska J Trefler I Wawer JŁacki and P Sliwinska-Stanczyk ldquoReactive oxygen speciesmdashphysiological and pathological function in the human bodyrdquoReumatologıa Clınica vol 45 no 5 pp 284ndash289 2007

[32] S S Percival and E D Harris ldquoRegulation of CuZn superoxidedismutase with copper Caeruloplasmin maintains levels offunctional enzyme activity during differentiation of K562 cellsrdquoBiochemical Journal vol 274 no 1 pp 153ndash158 1991

[33] R J Shephard and P N Shek ldquoCold exposure and immunefunctionrdquo Canadian Journal of Physiology and Pharmacologyvol 76 no 9 pp 828ndash836 1998

[34] M Radakovic D Davitkov S Borozan et al ldquoOxidative stressand DNA damage in horses naturally infected with TheileriaequirdquoThe Veterinary Journal vol 217 pp 112ndash118 2016

[35] G Maranon B Munoz-Escassi W Manley et al ldquoThe effect ofmethyl sulphonyl methane supplementation on biomarkers ofoxidative stress in sport horses following jumping exerciserdquoActa Veterinaria Scandinavica vol 50 45 pages 2008

[36] A C Onmaz R Van Den Hoven V Gunes M Cinar and OKucuk ldquoOxidative stress in horses after a 12-hours transportperiodrdquo Revue de Medecine Veterinaire vol 162 no 4 pp 213ndash217 2011

BioMed Research International 3

(Olympus Corp Hamburg Germany Axio Observer CarlZeiss MicroImaging GmbH) In the absence of visible CPE asecond passage was conducted

22 Antioxidative Status Twenty-seven serum samplesobtained from the group virological investigated werechosen for biochemical blood research The antibody titersreached between 0 and ge1 2048 In the case of 16 animalsthe titers were between the levels of 1 4 and 1 64 thus pairedsera tests were performed to confirmexclude the currentdisease Titers higher than 1 64 or a 4-fold increase inantibody titer obtained in a paired serum samples test wereconsidered to be indicators of recent infection Based onthese assumptions the investigated horses were dividedinto two groups seronegative or infected in the past (titerle 1 64) and group of horses which had had recent contactwith the virus had contracted infection shortly before theexperiment or tested positive in a paired serum sample test(titer gt 1 64)

3 Oxidative Stress Markers Analysis

An antioxidant system in the serum was analysed by deter-mining superoxide dismutase (SOD EC 11511) ceruloplas-min (CER) the total antioxidant capacity (TAC) and totaloxidant status (TOS) Lipid peroxidation was determined bymalondialdehyde (MDA) concentration

31 Protein Concentration Protein concentration was deter-mined using the Lowry et al methods [11]

32 Superoxide Dismutase Analysis (SOD) SOD isoenzymesrsquoactivity was determined according to the Oyanagui method[12] with KCN as the inhibitor of the CuZnSOD isoenzymeCuZnSOD activity was calculated as the difference betweentotal SOD activity and MnSOD activity SOD activity wascalculated against a blank probe containing bidistilled waterEnzyme activity was expressed as nitrite units (NU) per mLserum One NU exhibits 50 inhibition of the formation of anitrite ion under the methodrsquos condition [12] The inter- andintra-assay coefficients of variations (CV) were 28 and54 respectively

33 Ceruloplasmin Concentration of CER in serum wasdetermined spectrophotometrically according to Richterich[13] using the reaction with p-phenyl diamine CER catalysesthe oxidation of colourless p-phenylenediamine replacing itwith a blue-violet dye The absorbance of the samples wasread at 560 nm The measurement was conducted on aPerkinElmer VICTOR-X3 reader Interassay and intra-assaycoefficients of variations were respectively 13 and 40

34 Total Antioxidant Capacity (TAC) Plasma TAC wasmeasured using a commercial kit (Randox Co Eng-land) The 221015840azino-di-(3-ethylbenzothiazoline sulphonate)(ABTS) was incubated with a peroxidase (metmyoglobin)and hydrogen peroxide to produce the radical cation ABTS+which has a relatively stable blue-green colour and was mea-sured at 600 nmThe suppression of the colour was compared

to the standard for TAC measurement assays (Trolox) Theassay results are expressed as a Trolox equivalent (mmolL)[14]The inter- and intra-assay coefficients of variations (CV)were 11 and 38 respectively

35 Total Antioxidant Status The method according to Erel[15] is based on the oxidation of iron (II) ions to iron (III)ions in an acidic medium Then iron (III) ions with xyleneorange form a colourful complex ranging up to a blue-purple colouration Absorption readings were taken with the560 nm filter on the VICTOR-X3 from PerkinElmer Theconcentrationwas calculated from the calibration curve usingH2O2 as the standard Values are expressed in 120583moll

36 Lipid Peroxidation Malondialdehyde (MDA) concen-tration was measured in samples of serum according tothe method described by Ohkawa et al using the reactionwith thiobarbituric acid with spectrophotometric detectionemploying 515 nm excitation and 552 nm emission wave-lengths MDA concentration was calculated from the stan-dard curve and prepared from 1133-tetraethoxypropane[16]The inter- and intra-assay coefficients of variations (CV)were 21 and 83 respectively

37 Lipofuscin (LPS) Serum levels of LPS were determinedaccording to Tsuchida et al [17] Serum was added with3 1 vv ethanol-ether shaken and centrifuged The intensityof fluorescence was determined using a PerkinElmer spec-trophotometer LS45 at a wavelength of 345 nm (absorbance)and 430 nm (emission) in a dissolved solid The values areexpressed in relative lipid extract fluorescence (RF) where100 RF corresponds to the fluorescence of 01 120583gml quinidinesulphate in 01 N sulfuric acid LPS concentrations are shownin RF The inter- and intra-assay coefficients of variations(CV) are 28 and 97

4 Statistical Analysis

Statistical analysis was performed using STATISTICA 100PL (StatSoft Cracow Poland) and StataSE 120 (StataCorpLP TX USA) Statistical significance was set at a 119901 valuebelow 005 Data was expressed asmeansplusmn SD and the resultswere assessed for normality using the ShapirondashWilk methodand the quantile-quantile plot Correlations among analysedbiochemical parameters were assessed using Pearson corre-lation coefficient For comparison of selected biochemicalparameters the Student 119905-test was applied

5 Results

51 Viral and Serological Analyses Virus isolation tests didnot detect ERAV in any of the nasal swab samples Seroneu-tralisation tests showed positive results in 254 (72) serumsamples with titer levels from 1 4 to 1 ge2048 Statistical anal-ysis showed no significant relationship between gender orage and antibodies against ERAV (119901 gt 005)

52 Oxidative Status Analysis The median ERAV titer of 64was determined and the study group was divided into two

4 BioMed Research International

Table 2 Comparison of analyzed biochemical parameters for group 1 and group 2 Data are expressed as means plusmn SD CER ceruloplasminTAC total antioxidant capacity TAS total antioxidant status SOD superoxide dismutase MnSOD Mn superoxide dismutase CuZn SODsuperoxide dismutase MDA malondialdehyde LPS RF lipofuscin

Parameter Group IERAV le 64

Group IIERAV gt 64 119901

Protein gl 6894 plusmn 1237 7302 plusmn 729 0311CER mgdl 1086 plusmn 353 1062 plusmn 455 0878TAC mmoll 094 plusmn 011 09 plusmn 014 0441TOS umoll 1024 plusmn 428 835 plusmn 587 0345Total SOD NUml 1332 plusmn 284 1601 plusmn 247 lt005Mn SOD NUml 718 plusmn 178 808 plusmn 205 0234CuZn SOD NUml 614 plusmn 25 794 plusmn 196 lt005LPS RF 1561 plusmn 341 1775 plusmn 448 0173MDA umoll 621 plusmn 309 659 plusmn 282 0742Statistical significance was set at a 119901 value below 005

ERAV lt 64 ERAV gt 6411

12

13

14

15

16

17

18

SOD

(NU

ml)

MeanMean plusmn standard error Mean plusmn 95 confidence interval

Figure 3 Total superoxide dismutase serum levels of horses fromgroup 1 (ERAV le 64) and group 2 (ERAV gt 64) Statisticalsignificance was set at a 119901 value below 005

subgroups group 1 (ERAV titer le 64) and group 2 (ERAVtiter gt 64) (Table 2) There was no statistically significantcorrelation between ERAV seropositive results and selectedbiochemical indicators The high titer group (ERAV gt 64)had statistically higher concentrations of SODandCuZnSODthan the low titer group (119901 lt 005 Figures 3 and 4 andTable 2) For other biochemical indicators there were nostatistically significant differences between the studied groups(Table 2)

6 Discussion

ERAV is commonly found in populations of horses world-wide nevertheless to the best of our knowledge in thepopulations of horses in the territory of Poland this virus hasnot been investigated yet In this study 72 of animals were

ERAV lt 64 ERAV gt 6445

50

55

60

65

70

75

80

85

90

95

CuZN

SOD

(NU

ml)

MeanMean plusmn standard error Mean plusmn 95 confidence interval

Figure 4 CuZn superoxide dismutase serum levels of horsesfrom group 1 (ERAV le 64) and group 2 (ERAV gt 64) Statisticalsignificance was set at a 119901 value below 005

ERAV seropositive including 46 of horses with serum titerhigher than 1 64 and 13 of horses with serum titer higherthan 1 1024whichmay suggest that those horses had recentlybeen infected In general the studies on ERVs show that thepercentages of prevalence may vary from 0 to 100 [18 19]

In European countries the prevalence of ERAV wasreported as high at more than 60 In the United Kingdom100 of serologically tested horses had a significant titer toERAV [19] Similar percentage of adult seropositive animals(73) was obtained in Ireland (1998) nevertheless most ofthe examined horses were febrile [20] A high prevalence ofERAV was confirmed in Austria in 2005 where 90 of theequine sera and 27 of the human sera showed reactivity toERAV [21] and in France the percentage was 794 [22] Ininvestigations carried out in the United States of America theprevalence of ERAV neutralising antibodies was reported atthe level of 77 [23] Reports from central-eastern Asia all

BioMed Research International 5

provinces ofMongolia show that this percentage has reachedonly 342 out of a total of 300 horses [24] According to theauthors this was probably because the blood samples werecollected mainly from young horses Age was also related tothe ERAV in the study of Burrows where 60 of adult horseswere seropositive in comparison with 125 of seropositivefoals and 83 of yearlings [18] Nevertheless we did notconfirm a significant relationship between age or gender andERAV antibodies in this study Unfortunately we cannotconfirmour result concerning the gender because correlationbetween sex and ERAV antibodies has not been widelydescribed in the literature on the subject yet

The prevalence of ERBV the second equine rhinitis virusbelonging to the Picornaviridae family in equine sera inPoland is close to 70 [25] Interestingly available studiesreport that ERBV seems to be more commonly isolated thanERAV [7] In our study we obtained a similar percentageof ERAV seropositive horses which means that ERAV andERBV are equally distributed among the horses in Poland

The above results have demonstrated that equine rhi-noviruses are commonly circulated among horses globallyand their clinical importance may have been underestimated[5] This study shows that ERAV antibodies are highly rep-resented in analysed equine subjects which suggests that sub-clinical infectionsmanifested by high seroprevalence in clini-cally healthy animals are quite possibly a very commonoccur-rence Nasopharyngeal swabs are best collected from infectedhorses suspected of being infected within 24ndash48 hours ofinfection to detect the virus [7] In our investigation theresults of virus isolation were negative probably because theswabs were taken more than two days after infection Furtherinvestigations need to be carried out for a better understand-ing of pathogenesis and the epidemiology of ERAV virus

Oxidative stress is considered to play a role in the progres-sion of viral infections [26] contributing to viral replicationinflammation and decreased immune cell proliferation [27]It is known that oxidative stress caused via viral infectionmay lead to several aspects of viral pathogenesis includinginflammatory response and viral replication [28] The oxi-dantantioxidant status in different groups of horses is shownto be unbalanced in several physiological situations [29] Theinfection by different types of virus results in an imbalancedoxidantantioxidant relation The main forces of cell pro-tection against ROS are the enzymatic antioxidant systems(superoxide dismutase glutathione peroxidase and catalase)and nonenzymatic antioxidant systems (glutathione ascor-bate and uric acid) which prevent lipid peroxidation andDNA damage [30] The strategy of defence against hydroxylradicals is the dismutation process of superoxide anions(O2minus) conducted by superoxide dismutase disproportiona-

tion of H2O2 catalysed by CAT and reduction with GPx [31]The synthesis of SOD is stimulated by a reduction of molecu-lar oxygen TNF interleukins endotoxins and chemicals andhypoxia [32 33] In our study horses currently infected byequine rhinitis A virus showed increased serum levels of totalSOD and ZnCu SOD but not Mn SOD when compared tosubjects with past infections or seronegative subjects TotalSOD can be considered as one of the most sensitive markersof oxidative stress [32 33] The fact that total SOD increased

but not Mn SOD was considered to be a positive trend in thephysiological mechanisms of cell protection Increased totalactivity of SOD in plasma may be understood as an adaptiveresponse of the cells to elevated oxidative stress markersRadakovic et al [34] showed that horses naturally infected byTheileria equi present increased erythrocytes levels of CuZnSOD concentrations which suggests that T equi could havemodified isoenzyme activity via free radical production Inour study the general trend of total oxidant status reductionand lack of a significant increase in MDA serum levelsshows the positive effects of antioxidative processes Otherstudies show increased levels of blood MDA and decreasedantioxidant enzymes under conditions of stress in racinghorses following jumping activity [35] On the other handOnmaz et al [36] found that decreased total SOD levels andincreased MDA concentrations occur in horses exposed tolong-term transport stress which confirms the presence ofindependent changes in intra- and extracellular activities ofantioxidant systems in response to different stress stimulatorsand environmental markers

7 Conclusions

ERAV may have an influence on the condition of horsescausing delays in training and hindering performance at anoptimal level for prolonged periods of time [6] ERAV virusantibodies were present in approximately 72 of commer-cially used horses analysed which shows that this virus iswidely distributed in PolandThe age of horses had no signif-icant effect on antioxidant and virus parameters The ERAVvirus as a stress marker had a significant impact on plasmatotal SOD and CuZn SOD activity in horses in early stagesof convalescence when compared with other subjects Abetter understanding of the basic physiological processesmayenhance the identification of animals at the risk of oxidativestress which may be accompanied by poor health and hasimportance for welfare outcomes

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors wish to thank Professor Dr Reto Zanoni fromInstitut fur Virologie und Immunologie (Universitat Bern)for making ERAV reference strain available Mr ScottRichards is acknowledged for reviewing the language of thismanuscript

References

[1] S E Lynch J R Gilkerson S J Symes J-A Huang and CA Hartley ldquoPersistence and chronic urinary shedding of theaphthovirus equine rhinitis A virusrdquo Comparative ImmunologyMicrobiology amp Infectious Diseases vol 36 no 1 pp 95ndash1032013

6 BioMed Research International

[2] W H McCollum and P J Timoney ldquoStudies on the seropreva-lence and frequency of equine rhinovirus 1 and 2 infection innormal horse urinerdquo in Sixth International Conference onEquineDiseases pp 83ndash87 1992

[3] J Horsington S E Lynch J R Gilkerson M J Studdert andC A Hartley ldquoEquine picornaviruses Well known but poorlyunderstoodrdquo Veterinary Microbiology vol 167 no 1-2 pp 78ndash85 2013

[4] F Li H E Drummer N Ficorilli M J Studdert and B SCrabb ldquoIdentification of noncytopathic equine rhinovirus 1 asa cause of acute febrile respiratory disease in horsesrdquo Journal ofClinical Microbiology vol 35 no 4 pp 937ndash943 1997

[5] A Diaz-Mendez ldquoCharacterization of an Equine Rhinitis AVirus (ERAVON05) and development of an experimentalinfectionmodel in horsesrdquohttpsatriumlibuoguelphcaxmluihandle102143645

[6] J A Mumford and P D Rossdale ldquoVirus and its relationship tothe ldquopoor performancerdquo syndromerdquo Equine Veterinary Journalvol 12 no 1 pp 3ndash9 1980

[7] L Viel and A Diaz-Mendez ldquoEquine rhinitis viruses An over-looked cause of respiratory infection Boehringer IngelheimVetmedica Inc 2013rdquo

[8] E Gaudernak J Seipelt A Triendl A Grassauer and EKuechler ldquoAntiviral effects of pyrrolidine dithiocarbamate onhuman rhinovirusesrdquo Journal of Virology vol 76 no 12 pp6004ndash6015 2002

[9] C N Paiva andM T Bozza ldquoAre reactive oxygen species alwaysdetrimental to pathogensrdquoAntioxidantsampRedox Signaling vol20 no 6 pp 1000ndash1034 2014

[10] J Timoney J H Gillespie F W Scott and J E BarloughLaboratory diagnosis of viral infection in Hagan and Brunerrsquosmicrobiology and infectious diseases of domestic animals CornellUniv Press Ithaca NY USA 8th edition 1988

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1951

[12] Y Oyanagui ldquoReevaluation of assaymethods and establishmentof kit for superoxide dismutase activityrdquo Analytical Biochem-istry vol 142 no 2 pp 290ndash296 1984

[13] R Richterich Clinical Chemistry PZWLWarsaw Poland 1971[14] F Fathiazad A Khaki M Nouri and A A Khaki ldquoEffect of

cinnamon zeylanicum on serum testosterone and anti-oxidantslevels in ratsrdquo International Journal of Womenrsquos Health andReproduction Sciences vol 1 no 1 pp 29ndash35 2013

[15] O Erel ldquoA new automated colorimetric method for measuringtotal oxidant statusrdquo Clinical Biochemistry vol 38 no 12 pp1103ndash1111 2005

[16] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxides inanimal tissues by thiobarbituric acid reactionrdquo Analytical Bio-chemistry vol 95 no 2 pp 351ndash358 1979

[17] M Tsuchida TMiura KMizutani andKAibara ldquoFluorescentsubstances in mouse and human serum as a parameter of vivolipid peroxidationrdquo Biochimica et Biophysica Acta vol 834 no2 pp 196ndash204 1985

[18] R Burrows ldquoLaboratory diagnosis of some virus infections ofthe upper respiratory tract of the horserdquo Equine VeterinaryJournal vol 1 no 1 pp 32ndash38 1968

[19] G Plummer and J B Kerry ldquoStudies on an equine respiratoryvirusrdquo Veterinary Record vol 74 pp 967ndash970 1962

[20] M Klaey M Sanchez-Higgins D P Leadon A Cullinane RStraub and H Gerber ldquoField case study of equine rhinovirus

1 infection Clinical signs and clinicopathologyrdquo Equine Veteri-nary Journal vol 30 no 3 pp 267ndash269 1998

[21] G Kriegshauser A Deutz E Kuechler T Skern H Lussy andN Nowotny ldquoPrevalence of neutralizing antibodies to Equinerhinitis A and B virus in horses and manrdquo Veterinary Microbi-ology vol 106 no 3-4 pp 293ndash296 2005

[22] E Plateau and E Levy ldquoSerological prevalence of equineadenovirus and rhinovirus among horse populations in thedistrict of Parisrdquo Recueil De Medecine Veterinaire vol 164 no4 pp 413ndash418 1990

[23] D F Holmes M J Kemen and L Coggins ldquoEquine rhinovirusinfection - serologic evidence of infection in selected UnitedStates horse populationsrdquo Equine Infectious Diseases vol IV pp315ndash319 1978

[24] O Pagamjav K Kobayashi H Murakami et al ldquoSerologicalsurvey of equine viral diseases in Mongoliardquo Microbiology andImmunology vol 55 no 4 pp 289ndash292 2011

[25] Z Gradzki L Boguta and Z Grądzki ldquoBadania serologicznenad wystepowaniem zakazen wywolywanych przez wirusyzapalenia nosa koni typu b w PolscerdquoMedycynaWeterynaryjnavol 65 no 2 pp 119ndash123 2009

[26] AMandas E L IorioMGCongiu et al ldquoOxidative imbalancein HIV-1 infected patients treated with antiretroviral therapyrdquoJournal of Biomedicine and Biotechnology vol 7 Article ID749575 2009

[27] S Aquaro F Scopelliti M Pollicita and C F Perno ldquoOxidativestress and HIV infection Target pathways for novel therapiesrdquoFuture HIVTherapy vol 2 no 4 pp 327ndash338 2008

[28] M L Reshi Y-C Su and J-R Hong ldquoRNA viruses ROS-mediated cell deathrdquo International Journal of Cell Biology vol2014 Article ID 467452 2014

[29] N Kirschvink B D Moffarts and P Lekeux ldquoThe oxidantantioxidant equilibrium in horsesrdquo The Veterinary Journal vol177 no 2 pp 178ndash191 2008

[30] P M Clarkson and H S Thompson ldquoAntioxidants what roledo they play in physical activity and healthrdquo American Journalof Clinical Nutrition vol 72 no 2 pp 637Sndash646S 2000

[31] A Łuszczewski E Matyska-Piekarska J Trefler I Wawer JŁacki and P Sliwinska-Stanczyk ldquoReactive oxygen speciesmdashphysiological and pathological function in the human bodyrdquoReumatologıa Clınica vol 45 no 5 pp 284ndash289 2007

[32] S S Percival and E D Harris ldquoRegulation of CuZn superoxidedismutase with copper Caeruloplasmin maintains levels offunctional enzyme activity during differentiation of K562 cellsrdquoBiochemical Journal vol 274 no 1 pp 153ndash158 1991

[33] R J Shephard and P N Shek ldquoCold exposure and immunefunctionrdquo Canadian Journal of Physiology and Pharmacologyvol 76 no 9 pp 828ndash836 1998

[34] M Radakovic D Davitkov S Borozan et al ldquoOxidative stressand DNA damage in horses naturally infected with TheileriaequirdquoThe Veterinary Journal vol 217 pp 112ndash118 2016

[35] G Maranon B Munoz-Escassi W Manley et al ldquoThe effect ofmethyl sulphonyl methane supplementation on biomarkers ofoxidative stress in sport horses following jumping exerciserdquoActa Veterinaria Scandinavica vol 50 45 pages 2008

[36] A C Onmaz R Van Den Hoven V Gunes M Cinar and OKucuk ldquoOxidative stress in horses after a 12-hours transportperiodrdquo Revue de Medecine Veterinaire vol 162 no 4 pp 213ndash217 2011

4 BioMed Research International

Table 2 Comparison of analyzed biochemical parameters for group 1 and group 2 Data are expressed as means plusmn SD CER ceruloplasminTAC total antioxidant capacity TAS total antioxidant status SOD superoxide dismutase MnSOD Mn superoxide dismutase CuZn SODsuperoxide dismutase MDA malondialdehyde LPS RF lipofuscin

Parameter Group IERAV le 64

Group IIERAV gt 64 119901

Protein gl 6894 plusmn 1237 7302 plusmn 729 0311CER mgdl 1086 plusmn 353 1062 plusmn 455 0878TAC mmoll 094 plusmn 011 09 plusmn 014 0441TOS umoll 1024 plusmn 428 835 plusmn 587 0345Total SOD NUml 1332 plusmn 284 1601 plusmn 247 lt005Mn SOD NUml 718 plusmn 178 808 plusmn 205 0234CuZn SOD NUml 614 plusmn 25 794 plusmn 196 lt005LPS RF 1561 plusmn 341 1775 plusmn 448 0173MDA umoll 621 plusmn 309 659 plusmn 282 0742Statistical significance was set at a 119901 value below 005

ERAV lt 64 ERAV gt 6411

12

13

14

15

16

17

18

SOD

(NU

ml)

MeanMean plusmn standard error Mean plusmn 95 confidence interval

Figure 3 Total superoxide dismutase serum levels of horses fromgroup 1 (ERAV le 64) and group 2 (ERAV gt 64) Statisticalsignificance was set at a 119901 value below 005

subgroups group 1 (ERAV titer le 64) and group 2 (ERAVtiter gt 64) (Table 2) There was no statistically significantcorrelation between ERAV seropositive results and selectedbiochemical indicators The high titer group (ERAV gt 64)had statistically higher concentrations of SODandCuZnSODthan the low titer group (119901 lt 005 Figures 3 and 4 andTable 2) For other biochemical indicators there were nostatistically significant differences between the studied groups(Table 2)

6 Discussion

ERAV is commonly found in populations of horses world-wide nevertheless to the best of our knowledge in thepopulations of horses in the territory of Poland this virus hasnot been investigated yet In this study 72 of animals were

ERAV lt 64 ERAV gt 6445

50

55

60

65

70

75

80

85

90

95

CuZN

SOD

(NU

ml)

MeanMean plusmn standard error Mean plusmn 95 confidence interval

Figure 4 CuZn superoxide dismutase serum levels of horsesfrom group 1 (ERAV le 64) and group 2 (ERAV gt 64) Statisticalsignificance was set at a 119901 value below 005

ERAV seropositive including 46 of horses with serum titerhigher than 1 64 and 13 of horses with serum titer higherthan 1 1024whichmay suggest that those horses had recentlybeen infected In general the studies on ERVs show that thepercentages of prevalence may vary from 0 to 100 [18 19]

In European countries the prevalence of ERAV wasreported as high at more than 60 In the United Kingdom100 of serologically tested horses had a significant titer toERAV [19] Similar percentage of adult seropositive animals(73) was obtained in Ireland (1998) nevertheless most ofthe examined horses were febrile [20] A high prevalence ofERAV was confirmed in Austria in 2005 where 90 of theequine sera and 27 of the human sera showed reactivity toERAV [21] and in France the percentage was 794 [22] Ininvestigations carried out in the United States of America theprevalence of ERAV neutralising antibodies was reported atthe level of 77 [23] Reports from central-eastern Asia all

BioMed Research International 5

provinces ofMongolia show that this percentage has reachedonly 342 out of a total of 300 horses [24] According to theauthors this was probably because the blood samples werecollected mainly from young horses Age was also related tothe ERAV in the study of Burrows where 60 of adult horseswere seropositive in comparison with 125 of seropositivefoals and 83 of yearlings [18] Nevertheless we did notconfirm a significant relationship between age or gender andERAV antibodies in this study Unfortunately we cannotconfirmour result concerning the gender because correlationbetween sex and ERAV antibodies has not been widelydescribed in the literature on the subject yet

The prevalence of ERBV the second equine rhinitis virusbelonging to the Picornaviridae family in equine sera inPoland is close to 70 [25] Interestingly available studiesreport that ERBV seems to be more commonly isolated thanERAV [7] In our study we obtained a similar percentageof ERAV seropositive horses which means that ERAV andERBV are equally distributed among the horses in Poland

The above results have demonstrated that equine rhi-noviruses are commonly circulated among horses globallyand their clinical importance may have been underestimated[5] This study shows that ERAV antibodies are highly rep-resented in analysed equine subjects which suggests that sub-clinical infectionsmanifested by high seroprevalence in clini-cally healthy animals are quite possibly a very commonoccur-rence Nasopharyngeal swabs are best collected from infectedhorses suspected of being infected within 24ndash48 hours ofinfection to detect the virus [7] In our investigation theresults of virus isolation were negative probably because theswabs were taken more than two days after infection Furtherinvestigations need to be carried out for a better understand-ing of pathogenesis and the epidemiology of ERAV virus

Oxidative stress is considered to play a role in the progres-sion of viral infections [26] contributing to viral replicationinflammation and decreased immune cell proliferation [27]It is known that oxidative stress caused via viral infectionmay lead to several aspects of viral pathogenesis includinginflammatory response and viral replication [28] The oxi-dantantioxidant status in different groups of horses is shownto be unbalanced in several physiological situations [29] Theinfection by different types of virus results in an imbalancedoxidantantioxidant relation The main forces of cell pro-tection against ROS are the enzymatic antioxidant systems(superoxide dismutase glutathione peroxidase and catalase)and nonenzymatic antioxidant systems (glutathione ascor-bate and uric acid) which prevent lipid peroxidation andDNA damage [30] The strategy of defence against hydroxylradicals is the dismutation process of superoxide anions(O2minus) conducted by superoxide dismutase disproportiona-

tion of H2O2 catalysed by CAT and reduction with GPx [31]The synthesis of SOD is stimulated by a reduction of molecu-lar oxygen TNF interleukins endotoxins and chemicals andhypoxia [32 33] In our study horses currently infected byequine rhinitis A virus showed increased serum levels of totalSOD and ZnCu SOD but not Mn SOD when compared tosubjects with past infections or seronegative subjects TotalSOD can be considered as one of the most sensitive markersof oxidative stress [32 33] The fact that total SOD increased

but not Mn SOD was considered to be a positive trend in thephysiological mechanisms of cell protection Increased totalactivity of SOD in plasma may be understood as an adaptiveresponse of the cells to elevated oxidative stress markersRadakovic et al [34] showed that horses naturally infected byTheileria equi present increased erythrocytes levels of CuZnSOD concentrations which suggests that T equi could havemodified isoenzyme activity via free radical production Inour study the general trend of total oxidant status reductionand lack of a significant increase in MDA serum levelsshows the positive effects of antioxidative processes Otherstudies show increased levels of blood MDA and decreasedantioxidant enzymes under conditions of stress in racinghorses following jumping activity [35] On the other handOnmaz et al [36] found that decreased total SOD levels andincreased MDA concentrations occur in horses exposed tolong-term transport stress which confirms the presence ofindependent changes in intra- and extracellular activities ofantioxidant systems in response to different stress stimulatorsand environmental markers

7 Conclusions

ERAV may have an influence on the condition of horsescausing delays in training and hindering performance at anoptimal level for prolonged periods of time [6] ERAV virusantibodies were present in approximately 72 of commer-cially used horses analysed which shows that this virus iswidely distributed in PolandThe age of horses had no signif-icant effect on antioxidant and virus parameters The ERAVvirus as a stress marker had a significant impact on plasmatotal SOD and CuZn SOD activity in horses in early stagesof convalescence when compared with other subjects Abetter understanding of the basic physiological processesmayenhance the identification of animals at the risk of oxidativestress which may be accompanied by poor health and hasimportance for welfare outcomes

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors wish to thank Professor Dr Reto Zanoni fromInstitut fur Virologie und Immunologie (Universitat Bern)for making ERAV reference strain available Mr ScottRichards is acknowledged for reviewing the language of thismanuscript

References

[1] S E Lynch J R Gilkerson S J Symes J-A Huang and CA Hartley ldquoPersistence and chronic urinary shedding of theaphthovirus equine rhinitis A virusrdquo Comparative ImmunologyMicrobiology amp Infectious Diseases vol 36 no 1 pp 95ndash1032013

6 BioMed Research International

[2] W H McCollum and P J Timoney ldquoStudies on the seropreva-lence and frequency of equine rhinovirus 1 and 2 infection innormal horse urinerdquo in Sixth International Conference onEquineDiseases pp 83ndash87 1992

[3] J Horsington S E Lynch J R Gilkerson M J Studdert andC A Hartley ldquoEquine picornaviruses Well known but poorlyunderstoodrdquo Veterinary Microbiology vol 167 no 1-2 pp 78ndash85 2013

[4] F Li H E Drummer N Ficorilli M J Studdert and B SCrabb ldquoIdentification of noncytopathic equine rhinovirus 1 asa cause of acute febrile respiratory disease in horsesrdquo Journal ofClinical Microbiology vol 35 no 4 pp 937ndash943 1997

[5] A Diaz-Mendez ldquoCharacterization of an Equine Rhinitis AVirus (ERAVON05) and development of an experimentalinfectionmodel in horsesrdquohttpsatriumlibuoguelphcaxmluihandle102143645

[6] J A Mumford and P D Rossdale ldquoVirus and its relationship tothe ldquopoor performancerdquo syndromerdquo Equine Veterinary Journalvol 12 no 1 pp 3ndash9 1980

[7] L Viel and A Diaz-Mendez ldquoEquine rhinitis viruses An over-looked cause of respiratory infection Boehringer IngelheimVetmedica Inc 2013rdquo

[8] E Gaudernak J Seipelt A Triendl A Grassauer and EKuechler ldquoAntiviral effects of pyrrolidine dithiocarbamate onhuman rhinovirusesrdquo Journal of Virology vol 76 no 12 pp6004ndash6015 2002

[9] C N Paiva andM T Bozza ldquoAre reactive oxygen species alwaysdetrimental to pathogensrdquoAntioxidantsampRedox Signaling vol20 no 6 pp 1000ndash1034 2014

[10] J Timoney J H Gillespie F W Scott and J E BarloughLaboratory diagnosis of viral infection in Hagan and Brunerrsquosmicrobiology and infectious diseases of domestic animals CornellUniv Press Ithaca NY USA 8th edition 1988

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1951

[12] Y Oyanagui ldquoReevaluation of assaymethods and establishmentof kit for superoxide dismutase activityrdquo Analytical Biochem-istry vol 142 no 2 pp 290ndash296 1984

[13] R Richterich Clinical Chemistry PZWLWarsaw Poland 1971[14] F Fathiazad A Khaki M Nouri and A A Khaki ldquoEffect of

cinnamon zeylanicum on serum testosterone and anti-oxidantslevels in ratsrdquo International Journal of Womenrsquos Health andReproduction Sciences vol 1 no 1 pp 29ndash35 2013

[15] O Erel ldquoA new automated colorimetric method for measuringtotal oxidant statusrdquo Clinical Biochemistry vol 38 no 12 pp1103ndash1111 2005

[16] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxides inanimal tissues by thiobarbituric acid reactionrdquo Analytical Bio-chemistry vol 95 no 2 pp 351ndash358 1979

[17] M Tsuchida TMiura KMizutani andKAibara ldquoFluorescentsubstances in mouse and human serum as a parameter of vivolipid peroxidationrdquo Biochimica et Biophysica Acta vol 834 no2 pp 196ndash204 1985

[18] R Burrows ldquoLaboratory diagnosis of some virus infections ofthe upper respiratory tract of the horserdquo Equine VeterinaryJournal vol 1 no 1 pp 32ndash38 1968

[19] G Plummer and J B Kerry ldquoStudies on an equine respiratoryvirusrdquo Veterinary Record vol 74 pp 967ndash970 1962

[20] M Klaey M Sanchez-Higgins D P Leadon A Cullinane RStraub and H Gerber ldquoField case study of equine rhinovirus

1 infection Clinical signs and clinicopathologyrdquo Equine Veteri-nary Journal vol 30 no 3 pp 267ndash269 1998

[21] G Kriegshauser A Deutz E Kuechler T Skern H Lussy andN Nowotny ldquoPrevalence of neutralizing antibodies to Equinerhinitis A and B virus in horses and manrdquo Veterinary Microbi-ology vol 106 no 3-4 pp 293ndash296 2005

[22] E Plateau and E Levy ldquoSerological prevalence of equineadenovirus and rhinovirus among horse populations in thedistrict of Parisrdquo Recueil De Medecine Veterinaire vol 164 no4 pp 413ndash418 1990

[23] D F Holmes M J Kemen and L Coggins ldquoEquine rhinovirusinfection - serologic evidence of infection in selected UnitedStates horse populationsrdquo Equine Infectious Diseases vol IV pp315ndash319 1978

[24] O Pagamjav K Kobayashi H Murakami et al ldquoSerologicalsurvey of equine viral diseases in Mongoliardquo Microbiology andImmunology vol 55 no 4 pp 289ndash292 2011

[25] Z Gradzki L Boguta and Z Grądzki ldquoBadania serologicznenad wystepowaniem zakazen wywolywanych przez wirusyzapalenia nosa koni typu b w PolscerdquoMedycynaWeterynaryjnavol 65 no 2 pp 119ndash123 2009

[26] AMandas E L IorioMGCongiu et al ldquoOxidative imbalancein HIV-1 infected patients treated with antiretroviral therapyrdquoJournal of Biomedicine and Biotechnology vol 7 Article ID749575 2009

[27] S Aquaro F Scopelliti M Pollicita and C F Perno ldquoOxidativestress and HIV infection Target pathways for novel therapiesrdquoFuture HIVTherapy vol 2 no 4 pp 327ndash338 2008

[28] M L Reshi Y-C Su and J-R Hong ldquoRNA viruses ROS-mediated cell deathrdquo International Journal of Cell Biology vol2014 Article ID 467452 2014

[29] N Kirschvink B D Moffarts and P Lekeux ldquoThe oxidantantioxidant equilibrium in horsesrdquo The Veterinary Journal vol177 no 2 pp 178ndash191 2008

[30] P M Clarkson and H S Thompson ldquoAntioxidants what roledo they play in physical activity and healthrdquo American Journalof Clinical Nutrition vol 72 no 2 pp 637Sndash646S 2000

[31] A Łuszczewski E Matyska-Piekarska J Trefler I Wawer JŁacki and P Sliwinska-Stanczyk ldquoReactive oxygen speciesmdashphysiological and pathological function in the human bodyrdquoReumatologıa Clınica vol 45 no 5 pp 284ndash289 2007

[32] S S Percival and E D Harris ldquoRegulation of CuZn superoxidedismutase with copper Caeruloplasmin maintains levels offunctional enzyme activity during differentiation of K562 cellsrdquoBiochemical Journal vol 274 no 1 pp 153ndash158 1991

[33] R J Shephard and P N Shek ldquoCold exposure and immunefunctionrdquo Canadian Journal of Physiology and Pharmacologyvol 76 no 9 pp 828ndash836 1998

[34] M Radakovic D Davitkov S Borozan et al ldquoOxidative stressand DNA damage in horses naturally infected with TheileriaequirdquoThe Veterinary Journal vol 217 pp 112ndash118 2016

[35] G Maranon B Munoz-Escassi W Manley et al ldquoThe effect ofmethyl sulphonyl methane supplementation on biomarkers ofoxidative stress in sport horses following jumping exerciserdquoActa Veterinaria Scandinavica vol 50 45 pages 2008

[36] A C Onmaz R Van Den Hoven V Gunes M Cinar and OKucuk ldquoOxidative stress in horses after a 12-hours transportperiodrdquo Revue de Medecine Veterinaire vol 162 no 4 pp 213ndash217 2011

BioMed Research International 5

provinces ofMongolia show that this percentage has reachedonly 342 out of a total of 300 horses [24] According to theauthors this was probably because the blood samples werecollected mainly from young horses Age was also related tothe ERAV in the study of Burrows where 60 of adult horseswere seropositive in comparison with 125 of seropositivefoals and 83 of yearlings [18] Nevertheless we did notconfirm a significant relationship between age or gender andERAV antibodies in this study Unfortunately we cannotconfirmour result concerning the gender because correlationbetween sex and ERAV antibodies has not been widelydescribed in the literature on the subject yet

The prevalence of ERBV the second equine rhinitis virusbelonging to the Picornaviridae family in equine sera inPoland is close to 70 [25] Interestingly available studiesreport that ERBV seems to be more commonly isolated thanERAV [7] In our study we obtained a similar percentageof ERAV seropositive horses which means that ERAV andERBV are equally distributed among the horses in Poland

The above results have demonstrated that equine rhi-noviruses are commonly circulated among horses globallyand their clinical importance may have been underestimated[5] This study shows that ERAV antibodies are highly rep-resented in analysed equine subjects which suggests that sub-clinical infectionsmanifested by high seroprevalence in clini-cally healthy animals are quite possibly a very commonoccur-rence Nasopharyngeal swabs are best collected from infectedhorses suspected of being infected within 24ndash48 hours ofinfection to detect the virus [7] In our investigation theresults of virus isolation were negative probably because theswabs were taken more than two days after infection Furtherinvestigations need to be carried out for a better understand-ing of pathogenesis and the epidemiology of ERAV virus

Oxidative stress is considered to play a role in the progres-sion of viral infections [26] contributing to viral replicationinflammation and decreased immune cell proliferation [27]It is known that oxidative stress caused via viral infectionmay lead to several aspects of viral pathogenesis includinginflammatory response and viral replication [28] The oxi-dantantioxidant status in different groups of horses is shownto be unbalanced in several physiological situations [29] Theinfection by different types of virus results in an imbalancedoxidantantioxidant relation The main forces of cell pro-tection against ROS are the enzymatic antioxidant systems(superoxide dismutase glutathione peroxidase and catalase)and nonenzymatic antioxidant systems (glutathione ascor-bate and uric acid) which prevent lipid peroxidation andDNA damage [30] The strategy of defence against hydroxylradicals is the dismutation process of superoxide anions(O2minus) conducted by superoxide dismutase disproportiona-

tion of H2O2 catalysed by CAT and reduction with GPx [31]The synthesis of SOD is stimulated by a reduction of molecu-lar oxygen TNF interleukins endotoxins and chemicals andhypoxia [32 33] In our study horses currently infected byequine rhinitis A virus showed increased serum levels of totalSOD and ZnCu SOD but not Mn SOD when compared tosubjects with past infections or seronegative subjects TotalSOD can be considered as one of the most sensitive markersof oxidative stress [32 33] The fact that total SOD increased

but not Mn SOD was considered to be a positive trend in thephysiological mechanisms of cell protection Increased totalactivity of SOD in plasma may be understood as an adaptiveresponse of the cells to elevated oxidative stress markersRadakovic et al [34] showed that horses naturally infected byTheileria equi present increased erythrocytes levels of CuZnSOD concentrations which suggests that T equi could havemodified isoenzyme activity via free radical production Inour study the general trend of total oxidant status reductionand lack of a significant increase in MDA serum levelsshows the positive effects of antioxidative processes Otherstudies show increased levels of blood MDA and decreasedantioxidant enzymes under conditions of stress in racinghorses following jumping activity [35] On the other handOnmaz et al [36] found that decreased total SOD levels andincreased MDA concentrations occur in horses exposed tolong-term transport stress which confirms the presence ofindependent changes in intra- and extracellular activities ofantioxidant systems in response to different stress stimulatorsand environmental markers

7 Conclusions

ERAV may have an influence on the condition of horsescausing delays in training and hindering performance at anoptimal level for prolonged periods of time [6] ERAV virusantibodies were present in approximately 72 of commer-cially used horses analysed which shows that this virus iswidely distributed in PolandThe age of horses had no signif-icant effect on antioxidant and virus parameters The ERAVvirus as a stress marker had a significant impact on plasmatotal SOD and CuZn SOD activity in horses in early stagesof convalescence when compared with other subjects Abetter understanding of the basic physiological processesmayenhance the identification of animals at the risk of oxidativestress which may be accompanied by poor health and hasimportance for welfare outcomes

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors wish to thank Professor Dr Reto Zanoni fromInstitut fur Virologie und Immunologie (Universitat Bern)for making ERAV reference strain available Mr ScottRichards is acknowledged for reviewing the language of thismanuscript

References

[1] S E Lynch J R Gilkerson S J Symes J-A Huang and CA Hartley ldquoPersistence and chronic urinary shedding of theaphthovirus equine rhinitis A virusrdquo Comparative ImmunologyMicrobiology amp Infectious Diseases vol 36 no 1 pp 95ndash1032013

6 BioMed Research International

[2] W H McCollum and P J Timoney ldquoStudies on the seropreva-lence and frequency of equine rhinovirus 1 and 2 infection innormal horse urinerdquo in Sixth International Conference onEquineDiseases pp 83ndash87 1992

[3] J Horsington S E Lynch J R Gilkerson M J Studdert andC A Hartley ldquoEquine picornaviruses Well known but poorlyunderstoodrdquo Veterinary Microbiology vol 167 no 1-2 pp 78ndash85 2013

[4] F Li H E Drummer N Ficorilli M J Studdert and B SCrabb ldquoIdentification of noncytopathic equine rhinovirus 1 asa cause of acute febrile respiratory disease in horsesrdquo Journal ofClinical Microbiology vol 35 no 4 pp 937ndash943 1997

[5] A Diaz-Mendez ldquoCharacterization of an Equine Rhinitis AVirus (ERAVON05) and development of an experimentalinfectionmodel in horsesrdquohttpsatriumlibuoguelphcaxmluihandle102143645

[6] J A Mumford and P D Rossdale ldquoVirus and its relationship tothe ldquopoor performancerdquo syndromerdquo Equine Veterinary Journalvol 12 no 1 pp 3ndash9 1980

[7] L Viel and A Diaz-Mendez ldquoEquine rhinitis viruses An over-looked cause of respiratory infection Boehringer IngelheimVetmedica Inc 2013rdquo

[8] E Gaudernak J Seipelt A Triendl A Grassauer and EKuechler ldquoAntiviral effects of pyrrolidine dithiocarbamate onhuman rhinovirusesrdquo Journal of Virology vol 76 no 12 pp6004ndash6015 2002

[9] C N Paiva andM T Bozza ldquoAre reactive oxygen species alwaysdetrimental to pathogensrdquoAntioxidantsampRedox Signaling vol20 no 6 pp 1000ndash1034 2014

[10] J Timoney J H Gillespie F W Scott and J E BarloughLaboratory diagnosis of viral infection in Hagan and Brunerrsquosmicrobiology and infectious diseases of domestic animals CornellUniv Press Ithaca NY USA 8th edition 1988

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1951

[12] Y Oyanagui ldquoReevaluation of assaymethods and establishmentof kit for superoxide dismutase activityrdquo Analytical Biochem-istry vol 142 no 2 pp 290ndash296 1984

[13] R Richterich Clinical Chemistry PZWLWarsaw Poland 1971[14] F Fathiazad A Khaki M Nouri and A A Khaki ldquoEffect of

cinnamon zeylanicum on serum testosterone and anti-oxidantslevels in ratsrdquo International Journal of Womenrsquos Health andReproduction Sciences vol 1 no 1 pp 29ndash35 2013

[15] O Erel ldquoA new automated colorimetric method for measuringtotal oxidant statusrdquo Clinical Biochemistry vol 38 no 12 pp1103ndash1111 2005

[16] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxides inanimal tissues by thiobarbituric acid reactionrdquo Analytical Bio-chemistry vol 95 no 2 pp 351ndash358 1979

[17] M Tsuchida TMiura KMizutani andKAibara ldquoFluorescentsubstances in mouse and human serum as a parameter of vivolipid peroxidationrdquo Biochimica et Biophysica Acta vol 834 no2 pp 196ndash204 1985

[18] R Burrows ldquoLaboratory diagnosis of some virus infections ofthe upper respiratory tract of the horserdquo Equine VeterinaryJournal vol 1 no 1 pp 32ndash38 1968

[19] G Plummer and J B Kerry ldquoStudies on an equine respiratoryvirusrdquo Veterinary Record vol 74 pp 967ndash970 1962

[20] M Klaey M Sanchez-Higgins D P Leadon A Cullinane RStraub and H Gerber ldquoField case study of equine rhinovirus

1 infection Clinical signs and clinicopathologyrdquo Equine Veteri-nary Journal vol 30 no 3 pp 267ndash269 1998

[21] G Kriegshauser A Deutz E Kuechler T Skern H Lussy andN Nowotny ldquoPrevalence of neutralizing antibodies to Equinerhinitis A and B virus in horses and manrdquo Veterinary Microbi-ology vol 106 no 3-4 pp 293ndash296 2005

[22] E Plateau and E Levy ldquoSerological prevalence of equineadenovirus and rhinovirus among horse populations in thedistrict of Parisrdquo Recueil De Medecine Veterinaire vol 164 no4 pp 413ndash418 1990

[23] D F Holmes M J Kemen and L Coggins ldquoEquine rhinovirusinfection - serologic evidence of infection in selected UnitedStates horse populationsrdquo Equine Infectious Diseases vol IV pp315ndash319 1978

[24] O Pagamjav K Kobayashi H Murakami et al ldquoSerologicalsurvey of equine viral diseases in Mongoliardquo Microbiology andImmunology vol 55 no 4 pp 289ndash292 2011

[25] Z Gradzki L Boguta and Z Grądzki ldquoBadania serologicznenad wystepowaniem zakazen wywolywanych przez wirusyzapalenia nosa koni typu b w PolscerdquoMedycynaWeterynaryjnavol 65 no 2 pp 119ndash123 2009

[26] AMandas E L IorioMGCongiu et al ldquoOxidative imbalancein HIV-1 infected patients treated with antiretroviral therapyrdquoJournal of Biomedicine and Biotechnology vol 7 Article ID749575 2009

[27] S Aquaro F Scopelliti M Pollicita and C F Perno ldquoOxidativestress and HIV infection Target pathways for novel therapiesrdquoFuture HIVTherapy vol 2 no 4 pp 327ndash338 2008

[28] M L Reshi Y-C Su and J-R Hong ldquoRNA viruses ROS-mediated cell deathrdquo International Journal of Cell Biology vol2014 Article ID 467452 2014

[29] N Kirschvink B D Moffarts and P Lekeux ldquoThe oxidantantioxidant equilibrium in horsesrdquo The Veterinary Journal vol177 no 2 pp 178ndash191 2008

[30] P M Clarkson and H S Thompson ldquoAntioxidants what roledo they play in physical activity and healthrdquo American Journalof Clinical Nutrition vol 72 no 2 pp 637Sndash646S 2000

[31] A Łuszczewski E Matyska-Piekarska J Trefler I Wawer JŁacki and P Sliwinska-Stanczyk ldquoReactive oxygen speciesmdashphysiological and pathological function in the human bodyrdquoReumatologıa Clınica vol 45 no 5 pp 284ndash289 2007

[32] S S Percival and E D Harris ldquoRegulation of CuZn superoxidedismutase with copper Caeruloplasmin maintains levels offunctional enzyme activity during differentiation of K562 cellsrdquoBiochemical Journal vol 274 no 1 pp 153ndash158 1991

[33] R J Shephard and P N Shek ldquoCold exposure and immunefunctionrdquo Canadian Journal of Physiology and Pharmacologyvol 76 no 9 pp 828ndash836 1998

[34] M Radakovic D Davitkov S Borozan et al ldquoOxidative stressand DNA damage in horses naturally infected with TheileriaequirdquoThe Veterinary Journal vol 217 pp 112ndash118 2016

[35] G Maranon B Munoz-Escassi W Manley et al ldquoThe effect ofmethyl sulphonyl methane supplementation on biomarkers ofoxidative stress in sport horses following jumping exerciserdquoActa Veterinaria Scandinavica vol 50 45 pages 2008

[36] A C Onmaz R Van Den Hoven V Gunes M Cinar and OKucuk ldquoOxidative stress in horses after a 12-hours transportperiodrdquo Revue de Medecine Veterinaire vol 162 no 4 pp 213ndash217 2011

6 BioMed Research International

[2] W H McCollum and P J Timoney ldquoStudies on the seropreva-lence and frequency of equine rhinovirus 1 and 2 infection innormal horse urinerdquo in Sixth International Conference onEquineDiseases pp 83ndash87 1992

[3] J Horsington S E Lynch J R Gilkerson M J Studdert andC A Hartley ldquoEquine picornaviruses Well known but poorlyunderstoodrdquo Veterinary Microbiology vol 167 no 1-2 pp 78ndash85 2013

[4] F Li H E Drummer N Ficorilli M J Studdert and B SCrabb ldquoIdentification of noncytopathic equine rhinovirus 1 asa cause of acute febrile respiratory disease in horsesrdquo Journal ofClinical Microbiology vol 35 no 4 pp 937ndash943 1997

[5] A Diaz-Mendez ldquoCharacterization of an Equine Rhinitis AVirus (ERAVON05) and development of an experimentalinfectionmodel in horsesrdquohttpsatriumlibuoguelphcaxmluihandle102143645

[6] J A Mumford and P D Rossdale ldquoVirus and its relationship tothe ldquopoor performancerdquo syndromerdquo Equine Veterinary Journalvol 12 no 1 pp 3ndash9 1980

[7] L Viel and A Diaz-Mendez ldquoEquine rhinitis viruses An over-looked cause of respiratory infection Boehringer IngelheimVetmedica Inc 2013rdquo

[8] E Gaudernak J Seipelt A Triendl A Grassauer and EKuechler ldquoAntiviral effects of pyrrolidine dithiocarbamate onhuman rhinovirusesrdquo Journal of Virology vol 76 no 12 pp6004ndash6015 2002

[9] C N Paiva andM T Bozza ldquoAre reactive oxygen species alwaysdetrimental to pathogensrdquoAntioxidantsampRedox Signaling vol20 no 6 pp 1000ndash1034 2014

[10] J Timoney J H Gillespie F W Scott and J E BarloughLaboratory diagnosis of viral infection in Hagan and Brunerrsquosmicrobiology and infectious diseases of domestic animals CornellUniv Press Ithaca NY USA 8th edition 1988

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo Journalof Biological Chemistry vol 193 pp 265ndash275 1951

[12] Y Oyanagui ldquoReevaluation of assaymethods and establishmentof kit for superoxide dismutase activityrdquo Analytical Biochem-istry vol 142 no 2 pp 290ndash296 1984

[13] R Richterich Clinical Chemistry PZWLWarsaw Poland 1971[14] F Fathiazad A Khaki M Nouri and A A Khaki ldquoEffect of

cinnamon zeylanicum on serum testosterone and anti-oxidantslevels in ratsrdquo International Journal of Womenrsquos Health andReproduction Sciences vol 1 no 1 pp 29ndash35 2013

[15] O Erel ldquoA new automated colorimetric method for measuringtotal oxidant statusrdquo Clinical Biochemistry vol 38 no 12 pp1103ndash1111 2005

[16] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxides inanimal tissues by thiobarbituric acid reactionrdquo Analytical Bio-chemistry vol 95 no 2 pp 351ndash358 1979

[17] M Tsuchida TMiura KMizutani andKAibara ldquoFluorescentsubstances in mouse and human serum as a parameter of vivolipid peroxidationrdquo Biochimica et Biophysica Acta vol 834 no2 pp 196ndash204 1985

[18] R Burrows ldquoLaboratory diagnosis of some virus infections ofthe upper respiratory tract of the horserdquo Equine VeterinaryJournal vol 1 no 1 pp 32ndash38 1968

[19] G Plummer and J B Kerry ldquoStudies on an equine respiratoryvirusrdquo Veterinary Record vol 74 pp 967ndash970 1962

[20] M Klaey M Sanchez-Higgins D P Leadon A Cullinane RStraub and H Gerber ldquoField case study of equine rhinovirus

1 infection Clinical signs and clinicopathologyrdquo Equine Veteri-nary Journal vol 30 no 3 pp 267ndash269 1998

[21] G Kriegshauser A Deutz E Kuechler T Skern H Lussy andN Nowotny ldquoPrevalence of neutralizing antibodies to Equinerhinitis A and B virus in horses and manrdquo Veterinary Microbi-ology vol 106 no 3-4 pp 293ndash296 2005

[22] E Plateau and E Levy ldquoSerological prevalence of equineadenovirus and rhinovirus among horse populations in thedistrict of Parisrdquo Recueil De Medecine Veterinaire vol 164 no4 pp 413ndash418 1990

[23] D F Holmes M J Kemen and L Coggins ldquoEquine rhinovirusinfection - serologic evidence of infection in selected UnitedStates horse populationsrdquo Equine Infectious Diseases vol IV pp315ndash319 1978

[24] O Pagamjav K Kobayashi H Murakami et al ldquoSerologicalsurvey of equine viral diseases in Mongoliardquo Microbiology andImmunology vol 55 no 4 pp 289ndash292 2011

[25] Z Gradzki L Boguta and Z Grądzki ldquoBadania serologicznenad wystepowaniem zakazen wywolywanych przez wirusyzapalenia nosa koni typu b w PolscerdquoMedycynaWeterynaryjnavol 65 no 2 pp 119ndash123 2009

[26] AMandas E L IorioMGCongiu et al ldquoOxidative imbalancein HIV-1 infected patients treated with antiretroviral therapyrdquoJournal of Biomedicine and Biotechnology vol 7 Article ID749575 2009

[27] S Aquaro F Scopelliti M Pollicita and C F Perno ldquoOxidativestress and HIV infection Target pathways for novel therapiesrdquoFuture HIVTherapy vol 2 no 4 pp 327ndash338 2008

[28] M L Reshi Y-C Su and J-R Hong ldquoRNA viruses ROS-mediated cell deathrdquo International Journal of Cell Biology vol2014 Article ID 467452 2014

[29] N Kirschvink B D Moffarts and P Lekeux ldquoThe oxidantantioxidant equilibrium in horsesrdquo The Veterinary Journal vol177 no 2 pp 178ndash191 2008

[30] P M Clarkson and H S Thompson ldquoAntioxidants what roledo they play in physical activity and healthrdquo American Journalof Clinical Nutrition vol 72 no 2 pp 637Sndash646S 2000

[31] A Łuszczewski E Matyska-Piekarska J Trefler I Wawer JŁacki and P Sliwinska-Stanczyk ldquoReactive oxygen speciesmdashphysiological and pathological function in the human bodyrdquoReumatologıa Clınica vol 45 no 5 pp 284ndash289 2007

[32] S S Percival and E D Harris ldquoRegulation of CuZn superoxidedismutase with copper Caeruloplasmin maintains levels offunctional enzyme activity during differentiation of K562 cellsrdquoBiochemical Journal vol 274 no 1 pp 153ndash158 1991

[33] R J Shephard and P N Shek ldquoCold exposure and immunefunctionrdquo Canadian Journal of Physiology and Pharmacologyvol 76 no 9 pp 828ndash836 1998

[34] M Radakovic D Davitkov S Borozan et al ldquoOxidative stressand DNA damage in horses naturally infected with TheileriaequirdquoThe Veterinary Journal vol 217 pp 112ndash118 2016

[35] G Maranon B Munoz-Escassi W Manley et al ldquoThe effect ofmethyl sulphonyl methane supplementation on biomarkers ofoxidative stress in sport horses following jumping exerciserdquoActa Veterinaria Scandinavica vol 50 45 pages 2008

[36] A C Onmaz R Van Den Hoven V Gunes M Cinar and OKucuk ldquoOxidative stress in horses after a 12-hours transportperiodrdquo Revue de Medecine Veterinaire vol 162 no 4 pp 213ndash217 2011

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