Cocktail of Theileria equi antigens for detecting infection in equines

Shimaa Abd El-Salam El-Sayed，Mohamed Abdo Rizk，Mohamed Alaa Terkawi，Ahmed Mousa，El Said El Shirbini El Said，Gehad Elsayed，Mohamed Fouda，Naoaki Yokoyama，Ikuo Igarashi*National Research Center for Protozoan Diseases，Obihiro University of Agriculture and Veterinary Medicine，Inada-Cho，Obihiro，Hokkaido 080-8555，Japan

2Department of Biochemistry and Chemistry of Nutrition，Faculty of Veterinary Medicine，Mansoura University，Mansoura 35516，Egypt

3Department of Internal Medicine and Infectious Diseases，Faculty of Veterinary Medicine，Mansoura University，Mansoura 35516，Egypt

4Department of Biochemistry and Chemistry of Nutrition，Faculty of Veterinary Medicine，Sadat City University，Sadat City，Menoufyia，Egypt

Cocktail of Theileria equi antigens for detecting infection in equines

Shimaa Abd El-Salam El-Sayed1，2#，Mohamed Abdo Rizk1，3#，Mohamed Alaa Terkawi1，Ahmed Mousa4，El Said El Shirbini El Said2，Gehad Elsayed2，Mohamed Fouda2，Naoaki Yokoyama1，Ikuo Igarashi1*1National Research Center for Protozoan Diseases，Obihiro University of Agriculture and Veterinary Medicine，Inada-Cho，Obihiro，Hokkaido 080-8555，Japan

2Department of Biochemistry and Chemistry of Nutrition，Faculty of Veterinary Medicine，Mansoura University，Mansoura 35516，Egypt

3Department of Internal Medicine and Infectious Diseases，Faculty of Veterinary Medicine，Mansoura University，Mansoura 35516，Egypt

4Department of Biochemistry and Chemistry of Nutrition，Faculty of Veterinary Medicine，Sadat City University，Sadat City，Menoufyia，Egypt

ARTICLE INFO

Article history:

in revised form 3 Jul，2nd

revised form 10 Jul 2015

Accepted 26 Aug 2015

Available online 20 Oct 2015

Theileria equi

Equi merozoite antigen-2

Theileria equi protein 82

Theileria equi protein 43 Cocktail formula

Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain，Theileria equi（T.equi）protein 82（Te 82）and T.equi 104 kDa microneme-rhoptry antigen precursor（Te 43），to diagnose T.equi infection in horses as compared with equi merozoite antigen-2（EMA-2）.

Methods:In the current study，we applied a cocktail-ELISA containing two antigens（EMA-2+Te 82）to diagnose T.equi infection either in experimentally infected horses or in field infection.

Results:Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T.equi infection in horses as compared with Te 82 or Te 43 alone.

Conclusions:The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.09.001

1.Introduction

Equine piroplasmosis，a tick-borne parasitic disease that affects horses，mules，donkeys and zebras，is caused by Theileria equi（T.equi）and Babesia caballi and is typified by severe hemolysis that leads to hemoglobinuria，severe anemia，icterus and death［1］.The disease is associated with great economic loss due to the cost of treatment，loss of performance and restrictions in meeting international requirements related to exportation or participation in equestrian sports events［2］.Therefore，there is an urgent need to develop an effective diagnostic strategy for controlling the infection.

T.equi infection can be diagnosed directly by microscopic examination.However，detecting the parasite by microscopy is usually difficult during chronic infections because of low parasitemia，while serodiagnoses by immunofluorescent antibody test and ELISA are suitable for detecting antibodies in subclinical cases or in chronically infected animals with markedly low parasitemia［3，4］.

In view of the absence of equine piroplasmosis in Japan and the increasing number of horses imported to the country from places where the infection is endemic，the development of a highly specific and sensitive diagnostic antigen for T.equi is urgently required.Equi merozoite antigen-2（EMA-2）is a major surface protein of T.equi and is considered an important target for the development of an effective diagnostic reagent［5］.In addition，this antigen has highly antigenic properties and is recognized by ELISA in the sera of T.equi-infected animals worldwide［6］.However，further research on new antigens is extremely desirable for the development of a standard global diagnostic T.equi antigen.

Frequently associated in Theileria（FAINT）domains were first detected in Theileria annulata and Theileria parva as a stretch of 70 amino acids［7］.These domains were found to be overrepresented in proteins predicted to be secreted［7］.FAINT domain-containing genes are distributed throughout the T.equi genome，while they are not reported for Babesia bovis or Plasmodium falciparum［5］.In this study，two FAINT domains，including T.equi protein 82（Te 82）and T.equi 104 kDa microneme-rhoptry antigen precursor（Te 43），were used as diagnostic antigens for T.equi infection in comparison with EMA-2.Te 82 was previously used to diagnose T.equi infection［8］；however，this is the first time that Te 43 protein has been established for the diagnosis of T.equi infection in horses.

Cocktail-ELISA is a recently developed diagnostic tool that possesses high diagnostic sensitivity［9］.Its previous use in the diagnosis of tuberculosis and schistosomiasis has resulted in improved specificities，positive predictive values and kappa values as compared with the use of a single antigen［9，10］. Although，this technique is highly sensitive in diagnosing infection，it has never been used for the diagnosis of Babesia and Theileria infection.Therefore，the aim of this study was to use cocktail-ELISA to diagnose T.equi infection using two diagnostic antigens（EMA-2+Te 82）.This study is considered the first to use cocktail-ELISA for diagnosing T.equi infection.

2.Materials and methods

2.1.Parasites

U.S.Department of Agriculture strains of T.equi，kindly provided previously by the Equine Research Institute of the Japan Racing Association，were grown in equine erythrocytes in vitro，as described by Avarzed et al.，using a microaerophilic，stationary-phase culture system［11］.Briefly，medium 199 for T.equi（Sigma-Aldrich，Tokyo，Japan）supplemented with 40%equine serum，60 IU/mL of penicillin G，60μg/mL of streptomycin（Sigma）and 0.15μg/mL of amphotericin B（Sigma）was used as a maintaining medium.Additionally，13.6μg/mL of hypoxanthine（ICN Biomedicals Inc.，Costa Mesa，CA，USA）was added to the T.equi culture as a vital supplement.Cultures of parasitized red blood cells were incubated at 37°C in an atmosphere of 5%CO2，5%O2and 90%N2.The cultured parasite was harvested when the parasitemia reached 8%-10%.

2.2.Cloning，expression and purification of the recombinant bacterial proteins

Six oligonucleotide primers（Table 1）were used to amplify theEMA-2t，Te82t（23Y-660Saa）（accessionnumber: XP_004831145）and Te 43t（20I-389Caa）（accession number: AFZ81467）genes from the cDNA by PCR［12］.The amplified DNA was digested with EcoR I and Xho I and then ligated into the EcoR I and Xho I sites of a pGEX-4T Escherichia coli（E.coli）expression plasmid vector（Amersham Pharmacia Biotech，Little Chalfont，Buckinghamshire，United Kingdom）. The resulting plasmids，designated pGEX/EMA-2，pGEX/Te 82 and pGEX/Te 43，were used to transform into the E.coli DH5α strain and to express as glutathione S-transferase（GST）.Fused bacterial proteins were purified from the soluble fraction with glutathione-sepharose 4B（GE Healthcare Bio-sciences AB，Bj¨orkgatan 30，Sweden）.

2.3.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）and western blot analysis

The expressed recombinant proteins were verified by SDSPAGE and boiled for 5 min in a sodium dodecyl sulfate sample buffer［62.5 mmol/L Tris-HCl（pH 6.8），2%sodium dodecyl sulfate，5%mercaptoethanol，10%glycerol，0.02%bromophenol blue］and subjected to 12%SDS-PAGE（ATTO Corp.，Tokyo，Japan）；thereafter，the gel was stained with Coomassie blue stain for protein detection.

The T.equi proteins and control GST protein in SDS-PAGE gel were electrophoretically transferred onto polyvinylidene difluoride membranes（EMD Millipore，Billerica，MA，USA）. For western blotting，the membrane was blocked with 0.05% Tween 20 with phosphate buffered saline（PBS-T）plus 3%-5% skimmed milk and probed with the positive T.equi-infected sera diluted at 1:500 for 1 h and then washed three times with PBS-T. The membrane was incubated with horseradish peroxidaseconjugated goat anti-horse immunoglobulin G（IgG）antibody（1:8000 Bethyl）for 1 h.After three washes with PBS-T，reacted bandswerevisualizedusingasolutioncontaining3-diaminobenzidine tetrahydrochloride and H2O2（Dojindo Molecular Technologies，Inc.，Tokyo，Japan）.

2.4.ELISAs

EMA-2，Te 82 and Te 43 proteins were used separately as antigens for ELISA or as a cocktail of EMA-2+Te 82.These antigens were evaluated using a panel of the T.equi experimentally infected sera that had been collected serially from one horse during the course of infection.In addition to 44 serum samples from horses experimentally infected with T.equi，376 field samples were collected from T.equi infection-endemic areas，including South Africa（262 samples）and Ghana（114 samples）.The 96-well microtiter plates（Nunc，Roskilde，Denmark）were coated overnight at 4°C with 50μL of each recombinant protein at a concentration of 2μg/mL per well in acoating buffer（50 mmol/L carbonate-bicarbonate buffer，pH 9.6）.The plates were washed once with 0.05%PBS-T and then incubated with 100μL of a blocking solution（3%skim milk in PBS）for 1 h at 37°C.After washing，the plates were incubated with 50μL of the serum samples diluted 1:100 with the blocking solution for 1 h at 37°C.The plates were washed six times with PBS-Tandthenincubatedwith50μLofhorseradish peroxidase-conjugated goat anti-horse IgG antibody（Bethyl）diluted 1:4000 with the blocking solution for 1 h at 37°C as a secondary antibody.The plates were washed six times as described above and 100μL of a substrate solution［0.1 mol/L citric acid，0.2 mol/L sodium phosphate，0.3 mg/mL of 2，2-azide-bis（3-ethylbenzthiazoline-6-sulfonic acid）（Sigma）and 0.01%of 30%H2O2］was then added to each well.After incubation for 1 h at room temperature，the optical density（OD）was measured with an MTP-500 microplate reader（Corona Electric，Tokyo，Japan）at a wavelength of 415 nm.The ELISA result was determined for each sample by subtracting the mean OD value of two readings with GST protein from the mean OD value of two readings with the EMA-2，Te 43 and Te 82 proteins and a cocktail of EMA-2+Te 82.Using receiver operating characteristic curve analysis with MedCalc statistical software（version 11.4），cutoff values were calculated on the basis of 25 noninfected equine sera.

Table 1 Primers used in the study.

3.Results

The genes encoding EMA-2，Te 43 and Te 82 were successfully expressed as soluble GST fusion proteins in E.coli with molecular masses of 50 kDa，74 kDa and 85 kDa，respectively（Figure 1）.Sera collected from horses experimentally infected with T.equi specifically reacted to all of the recombinant proteins but not to the control GST protein in western blot analysis，suggesting their high antigenicity in the infected sera（Figure 2）.Furthermore，the specificity and sensitivity of EMA-2，Te 43 and Te 82 proteins and a cocktail of EMA-2+Te 82 were evaluated in a standard ELISA with experimentally infected and negative-control equine sera and the cutoff OD values were determined to be 0.059，0.088，0.084 and 0.069，respectively（Figure 3）.

The diagnostic performances of these recombinant proteins were evaluated with sequential sera from a horse infected with T.equi that had developed a high titer of antibody response to recombinant protein EMA-2 and a cocktail formula of antigens at Day 6 post infection，and this high antibody titer was maintained until 36 days post infection（Figure 4）.Next，the sensitivity of the assay was evaluated using 44 serum samples from horses experimentally infected with T.equi and 376 field samples collected from endemic areas of T.equi infection，as previously mentioned.Of44experimentallyinfectedserum samples，28，5 and 39 samples were found to be positive for Te 82，Te 43 and a cocktail formula of EMA-2 and Te 82，respectively（Table 2）.While，for the field samples，224，213，9 and 220 sera samples showed higher OD than cutoff values for EMA-2，Te 82，Te 43 and a cocktail formula of EMA-2 and Te 82，respectively（Table 3）.The fewer number of positive samples detected by Te 43 indicated the low potential of this protein as a diagnostic antigen.The specificity and sensitivity of ELISAs with serum samples were determined on the basis of EMA-2 results.The specificity results were 79.87%，96.90%and 90.00%，while the sensitivity results were 79.35%，13.63%and 83.48%for Te 82，Te 43 and a cocktail of（EMA-2+Te 82），respectively（Table 4）.Additionally，a cocktail formula（EMA-2+Te 82）showed the highest kappa value among the antigens used（Table 4）.These results revealed the high diagnostic performance of the cocktail formula for diagnosing T.equi infection in horses.

Table 2 Summary of ELISA results with recombinant proteins with serum samples from horses experimentally infected with T.equi.

Table 3 Summary of ELISA results with recombinant proteins with 376 field sera collected from T.equi-endemic areas，South Africa and Ghana.

Table 4 Specificity，sensitivity and kappa value of ELISA.

4.Discussion

Equine piroplasmosis is considered a serious threat due to the international movement of infected horses［13］.Therefore，developinganeffectivediagnosticcandidateforsuch infections is urgently required.Serological tests，particularly ELISA，seemtobethemostpracticalandeconomical technique for epidemiological investigation［15］.Indeed，as compared to other serological tests，the ELISA technique offers greater sensitivity，objectivity and capacity for rapid adaptation in examining a large number of serum samples. The crude antigen prepared from merozoites has traditionally beenutilizedfortheserologicaldetectionofparasite antibodies.However，recombinantproteincanbean alternative source，allowing better standardization of the test［9］.

The high antigenicity of the EMA-2 antigen for detecting T.equi infection might be attributable to the fact that EMA-2 is a unique protein that was not expressed during any of the asexual erythrocytic developmental stages of T.equi merozoites but was expressed only during the early developmental stage［13］.These findings indicate that the expression of EMA-2 on the merozoite surface is highly stage-specific.Additionally，the EMA-2 antigen was mutually expressed on the surface of extra-erythrocytic merozoites，and the intra-erythrocytic merozoites shed only EMA-2 on the erythrocytic cytoplasm and/or inside membrane surface，which is responsible for its erythrocytic binding behavior［13］.

Our results reveal the good diagnostic performance of the Te 82 antigen for detecting the infection with high specificity and sensitivity values.The Te 82 antigen used in this study exhibited higher sensitivity and lower specificity values than those obtained previously with the Be 82/1-235 antigen［14］.Moreover，the Te 82 antigen used in this study has the ability to detect infection 12 days earlier than Be 82［8］，confirming the good diagnostic performance of this antigen for detecting T.equi infection.

Although our findings revealed that the cocktail formula of EMA-2 and Te 82 antigens demonstrated the same performance as EMA-2 alone，the cocktail formula has several advantages over the single antigens.

The cocktail formula requires less volume of antigens than single antigens do.Moreover，until now，no single recombinant ELISA containing a single antigen has been able to detect all IgG-or immunoglobulin M-positive samples examined across different stages of the disease.There are different theories as to why this might be.First，the humoral immune response varies with the stage of infection［16］.Hence，some antibodies present at one stage of infection may be absent in other stages and vice versa.This requires that multiple epitopes from different antigensarepresentinanimmunoassaytodetectthe antibodies present at different disease stages.Second，the ability to reconstitute native epitopes in recombinant proteins produced in E.coli is dependent on the expression vector and the protein purification employed.Therefore，combining two or more recombinant proteins will increase the sensitivity of recombinant-ELISAs，as has been previously determined for toxoplasmosis［17］，tuberculosis and schistosomiasis，resulting in improving specificities，positive predictive values and kappa values as compared with using a single antigen［9，10］.

In conclusion，our study has revealed that the ELISA technique using a cocktail formula of EMA-2+Te 82 provides a new，practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and helps better manage such disease conditions.A cocktail-ELISA of different antigens might help improve the diagnostic strategy of equine Babesia and Theileria parasites.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

This study was supported by the Ministry of Higher Education Egypt.

［1］Garba UM，Sackey AKB，Tekdek LB，Agbede RIS，Bisalla M. Clinical manifestations and prevalence of piroplasmosis in Nigerian royal horses.J Vet Adv 2011；1（1）:11-5.

［2］Hussain MH，Saqib M，Raza F，Muhammad G，Asi MN，Mansoor MK，et al.Seroprevalence of Babesia caballi and Theileria equi in five draught equine populated metropolises of Punjab，Pakistan.Vet Parasitol 2014；202（3-4）:248-56.

［3］Mosqueda J，Olvera-Ramirez A，Aguilar-Tipacamu G，Canto GJ. Current advances in detection and treatment of babesiosis.Curr Med Chem 2012；19（10）:1504-18.

［4］Goo YK，Jia H，Aboge GO，Terkawi MA，Kuriki K，Nakamura C，et al.Babesia gibsoni:serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP. Exp Parasitol 2008；118（4）:555-60.

［5］FreemanJM，KappmeyerLS，UetiMW，McElwainTF，Baszler TV，Echaide I，et al.A Babesia bovis gene syntenic to Theileria parva p67 is expressed in blood and tick stage parasites. Vet Parasitol 2010；173（3-4）:211-8.

［6］KumarS，KumarR，GuptaAK，YadavSC，GoyalSK，Khurana SK，et al.Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population.Vet Parasitol 2013；198（1-2）:10-7.

［7］Pain A，Renauld H，Berriman M，Murphy L，Yeats CA，Weir W，et al.Genome of the host-cell transforming parasite Theileria annulata compared with T.parva.Science 2005；309（5731）:131-3.

［8］Hirata H，Ikadai H，Yokoyama N，Xuan X，Fujisaki K，Suzuki N，et al.Cloning of a truncated Babesia equi gene encoding an 82-kilodalton protein and its potential use in an enzyme-linked immunosorbent assay.J Clin Microbiol 2002；40（4）:1470-4.

［9］Moendeg KJ，Angeles JM，Goto Y，Leonardo LR，Kirinoki M，Villacorte EA，et al.Development and optimization of cocktail-ELISA for a unified surveillance of zoonotic schistosomiasis in multiple host species.Parasitol Res 2015；114（3）:1225-8.

［10］Shin AR，Shin SJ，Lee KS，Eom SH，Lee SS，Lee BS，et al. Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K-strain of Mycobacterium tuberculosis.Clin Vaccine Immunol 2008；15（12）: 1788-95.

［11］Avarzed A，Igarashi I，Kanemaru T，Hirumi K，Omata Y，Saito A，et al.Improved in vitro cultivation of Babesia caballi.J Vet Med Sci 1997；59（6）:479-81.

［12］Nishisaka M，Yokoyama N，Xuan XN，Inoue N，Nagasawa H，Fujisaki K，et al.Characterisation of the gene encoding a protective antigen from Babesia microti identified it asηsubunit of chaperonin containing T-complex protein 1.Int J Parasitol 2001；31（14）: 1673-9.

［13］Kumar S，Yokoyama N，Kim JY，Huang X，Inoue N，Xuan X，et al. Expression of Babesia equi EMA-1 and EMA-2 during merozoite developmental stages in erythrocyte and their interaction with erythrocytic membrane skeleton.Mol Biochem Parasitol 2004；133（2）:221-7.

［14］Hirata H，Xuan X，Yokoyama N，Nishikawa Y，Fujisaki K，Suzuki N，et al.Identification of a specific antigenic region of the P82 protein of Babesia equi and its potential use in serodiagnosis. J Clin Microbiol 2003；41（2）:547-51.

［15］Mahmoud MS，Kandil OM，Nasr SM，Hendawy SH，Habeeb SM，Mabrouk DM，et al.Serological and molecular diagnostic surveys combinedwithexamininghematologicalprofilessuggests increased levels of infection and hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt.Parasit Vectors 2015；8:319.

［16］Moir IL，Davidson MM，Ho-Yen DO.Comparison of IgG antibody profiles by immunoblotting in patients with acute and previous Toxoplasma gondii infection.J Clin Pathol 1991；44（7）: 569-72.

［17］Johnson AM，Roberts H，Tenter AM.Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosis and comparison with traditional ELISAs.J Med Microbiol 1992；37: 404-9.

16 Jun 2015

Ikuo Igarashi，DVM，PhD，National Research Center for Protozoan Diseases，Obihiro University of Agriculture and Veterinary Medicine，Inada-Cho，Obihiro，Hokkaido 080-8555，Japan.

Tel:+81 155 49 5641

Fax:+81 155 49 5643

E-mail:igarcpmi@obihiro.ac.jp

Peer review under responsibility of Hainan Medical University.

#These authors contributed equally to this work.