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Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance
Veterinary Parasitology 205 (2014) 490–498
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Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance P.P. Sengupta a,∗ , G.R. Rudramurthy a , M. Ligi a , M. Roy a , V. Balamurugan a , P. Krishnamoorthy a , M. Nagalingam a , L. Singh b , H. Rahman a a
National Institute of Veterinary Epidemiology and Disease Informatics, Hebbal, Bengaluru 560024, Karnataka, India Collaborating Unit, AICRP on ADMAS, State Disease Diagnostic Centre, Old B.P. Lab. Campus, Gopinath Marg, New Colony, Jaipur 302001, Rajasthan, India b
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 20 August 2014 Accepted 22 August 2014 Keywords: Trypanosoma evansi Surra Variable surface glycoprotein ELISA
a b s t r a c t Trypanosoma evansi, a haemoflagellate, causes “surra” an important chronic wasting disease of a wide range of wild and domestic herbivorous and carnivorous animals including cattle, buffaloes, camels, horses, etc. The untreated recovered animal can act as a carrier without exhibiting the disease symptoms and can be a source of infection to healthy animals. The diagnosis and subsequent treatment of the carrier animals is helpful to curb the disease. As the parasitaemia in carrier animals is very scanty, the conventional blood smear examination, which is widely practiced in the field, cannot detect such condition. For this purpose improved diagnostics are very much useful for mass sero-screening test such as ELISA. In the present study, the VSG of T. evansi was expressed in prokaryotic system (E. coli) and thereafter its immunoreactivity has been evaluated in immuno blot and enzyme immuno assay. The expressed protein showed 95.6% sensitivity, 98.0% specificity and 0.93 Cohen’s kappa value, when compared with standard antigens. The developed antigen has also been validated with field serum samples from bovine, camel and horse collected from different states of India. The data showed that the developed recombinant antigen can be a diagnostic tool to detect carrier animals as well as control of the disease. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma (T) evansi, a haemoflagellated parasite and a petite mutant of T. brucei (Field and Carrington, 2009) causes a fatal wasting disease called surra, results in havoc economical loss. Surra has a wide range of hosts such as cattle, buffaloes, camels, horses, etc. The mammalian hosts for surra in south east Asia are mostly cattle, buffaloes
∗ Corresponding author at: Parasitology Laboratory, National Institute of Veterinary Epidemiology and Disease Informatics, IVRI Campus, Hebbal, Bengaluru 560024, Karnataka, India. Tel.: +91 080 23419576; fax: +91 080 23415329. E-mail address: pinakiprasad [email protected] (P.P. Sengupta). http://dx.doi.org/10.1016/j.vetpar.2014.08.012 0304-4017/© 2014 Elsevier B.V. All rights reserved.
and horses (Holland et al., 2004). The disease is clinically characterized by anemia, recurrent fever, oedema, muscular weakness, loss of appetite and abortion, with a morbidity and mortality of 50–70%. The animals serve as carriers for the disease by exhibiting low levels of fluctuating parasites for years after recovery. The detection of carrier status in animal is very important in controlling the disease. Acute or subacute infection can be satisfactorily diagnosed by conventional parasitological techniques, but it becomes difficult in latent or chronic infection where parasitaemia is very low. The application of advanced laboratory techniques such as DNA amplification and sensitive serological tests can be helpful in the detection of carrier status of animal. Several genes have been analyzed/targeted for diagnosis of trypanosomosis, such as
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variable surface glycoprotein (VSG) (Sengupta et al., 2010), invariant surface glycoprotein (ISG) (Rudramurthy et al., 2013), kinetoplastid DNA, nuclear DNA and multilocus isoenzymes analysis (Gibson et al., 1980; Borst et al., 1987; Songa et al., 1990; Stevens and Godfrey, 1992; MathieuDaude and Tibayrenc, 1994; Zhang and Baltz, 1994). VSG is the primary immunogen of T. evansi in eliciting the antibody response in host. Potential serological tests have been developed for T. evansi based on the detection of either antigens or antibodies such as antigen detection ELISA for camels (Nantulya et al., 1989b; Diall et al., 1992; Verloo et al., 1998), buffaloes (Nantulya et al., 1989a; Davison et al., 1999; Verloo et al., 2000), antibody detection based on the predominant variant antigen type (VAT) RoTat 1.2 (Verloo et al., 2000), CATT/T. evansi (Bajyana Songa and Hamers, 1988; Verloo et al., 2000), LATEX/T. evansi (Verloo et al., 1998, 2000) and ELISA/T. evansi (Verloo et al., 2000). Earlier the VSG was expressed in different host cells, such as, insect cell line (Urakawa et al., 2001), yeast (Roge et al., 2013) and E. coli (Sengupta et al., 2012), etc. Further, it has also been reported that the immunoreactivity of the glycosylated and deglycosylated VSG remained same against anti-VSG antibody (Reinwald, 1985). In our earlier studies also, we showed that expressed N-terminal portion of VSG exhibits immunoreactivity. In the present study the whole VSG gene of T. evansi has been expressed in E. coli and assessed its immunoreactivity and further the rVSG has been evaluated for its potentiality to explore as a diagnostic antigen in ELISA for the serodiagnosis of surra using field sera samples. 2. Materials and methods 2.1. T. evansi stabilates and whole cell lysate (WCL) antigen preparation Different isolates of T. evansi such as buffalo, dog, lion and leopard, maintained in the Parasitology Laboratory, NIVEDI, Bengaluru, India, were used in the present study. The different isolates of T. evansi were propagated in rats as mentioned earlier (Sengupta et al., 2010) and purified using diethyl amino ethyl (DEAE) cellulose (DE.52-Whatman) column (Lanham and Godfrey, 1970). The WCL antigens from different isolates were prepared as mentioned earlier (Sengupta et al., 2012), the protein concentration of the supernatant was estimated (Lowry et al., 1951) and kept in aliquots at −80 ◦ C till further use. 2.2. VSG RoTat 1.2 antigen The VSG RoTat 1.2 antigen was obtained from the Koning Leopold Institute of Tropical Medicine, Antwerp, Belgium (OIE reference laboratory of surra) and used as per manufacturer’s instruction. The antigen was used in ELISA at 600 ng/well to compare the immunoreactivity with expressed protein. 2.3. Preparation of hyper immune/immune sera in animals The hyper immune sera against T. evansi were raised in rabbits (buffalo, dog, lion and leopard isolates) and also
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in cattle (buffalo isolate); while immune serum was raised in buffalo (buffalo isolate) (Sengupta et al., 2012) and sera available in the parasitology laboratory were used in the present study. 2.4. Sera collection from field The bovine field sera (n = 1051) were collected randomly from different states of India, such as Karnataka, Tamil Nadu, Maharashtra, Odisha, West Bengal and Uttarakhand, which covers southern, northern, eastern and western regions of India, between 2010 and 2013. The sera from camel (n = 100) were collected from Rajasthan (India) from the field, while horse sera (n = 51) were collected from Karnataka state in India from an organized herd in 2014. All the animals were apparently healthy. However, the disease status of the animals was not available. The sera separated after the sampling was preserved at −80 ◦ C for further use. The sera included in the present study were screened for trypanosomosis by serological tests such as CATT/T. evansi and ELISA in duplicate. The ELISA was carried out using rVSG and VSG RoTat 1.2 antigens for comparative evaluation. 2.5. Amplification of VSG gene by RT-PCR A set of expression primers TEVSG-F (5 -CATGAATTCCAAGGCGCTCGTTGG-3 ) corresponding to 10–24 bp and TEVSG-R (5 -GCCTGTAAGCTTTTTGTTTTTTGCATCTGATTC3 ) to 1216–1196 bp were designed from our published sequence EF495337. The forward and reverse primers were respectively introduced with EcoRI and HindIII restriction site for cloning into expression vector. The histidine (His) sequences were introduced in the forward as well as the reverse primers to purify the expressed protein. The total RNA was isolated from purified trypanosomes (buffalo isolate) and preserved at −80 ◦ C, till further use. RT-PCR was carried out from the total RNA to synthesize VSG specific cDNA using random nanomer by following the standard protocol described earlier (Sengupta et al., 2010). The cDNA obtained was subjected to PCR for the amplification of the VSG gene. The 25 l PCR reaction mixture contained Taq buffer, 10 mm dNTPS, 20 Pmol each of TEVSG-F/R primers and 3 units (U) Taq DNA polymerase (MBI fermentas). The PCR cycle was carried out with initial denaturation at 94 ◦ C for 3 min, followed by 35 cycles of one min, denaturation at 94 ◦ C for 1 min., primer annealing at 55 ◦ C for 1 min, and extension at 72 ◦ C for 1.20 min with a final extension at 72 ◦ C for 10 min. The amplified product was analyzed in 1% agarose gel and documented in gel doc analyzer (BioRad). 2.6. Construction of expression cassette in pET33 (b) vector The amplicon run on the agarose gel was extracted using Qiagen gel extraction kit as per manufacturer’s instruction. The purified product was initially ligated into pGEMT@ Easy Cloning vector (Promega) and transformed into E. coli Top10 competent cells. The transformed cells were plated on Luria Bertini (LB) agar containing ampicillin (50 g/ml),
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isoprophyl -thiogalacto pyranoside (IPTG) (1 mM) and X-.gal (20 l/ml) for the selection of recombinant clones. The white positive clones were selected and confirmed by colony lysis, colony touch PCR, restriction enzyme digestion (EcoRI single digestion) and DNA sequencing using T7 and Sp6 universal primers. The VSG gene from pGEMT@ Easy vector was released using EcoRI and HindIII digestion and cloned into EcoRI and HindIII site of pET33(b) expression vector system (Novagen) and the recombinant plasmid was named as pET33(b)VSG. The plasmid pET33 (b) VSG was transformed into E. coli (Top 10) competent cells by following the standard protocol (Sambrook and Russell, 2001). The transformed cells were plated on the LB agar medium containing kanamycin (50 g/ml) and incubated at 37 ◦ C for overnight. The clones appeared on the plate were screened as mentioned earlier. The recombinant plasmid DNA isolated from the positive clones was sequenced using vector specific (T7 promoter/terminator primers) and gene specific (TEVSGF/R) primers to determine the orientation of the insert in the vector backbone.
2.6.1. Expression of VSG in E. coli host [BL-21(DE3) pLys S] The recombinant plasmid pET33(b)VSG isolated was transformed into E. coli BL21(DE3)PLysS (Invitrogen) expression host system by following the standard protocol (Sambrook and Russell, 2001). The transformed cells were plated on the LB agar medium containing chloramphenicol (34 g/ml) and kanamycin (50 g/ml) and incubated at 37 ◦ C for overnight. The positive BL21 recombinant clones appeared in the plate were selected as mentioned earlier to express the protein.
2.7. Expression and purification of rVSG Eight recombinant positive clones were analysed for the expression of VSG. The inoculum for the protein expression was prepared by inoculating a single positive colony into 5 ml LB broth containing chloramphenicol (34 g/ml) and kanamycin (30 g/ml) and incubated in a shaker incubator at 37 ◦ C for overnight. Two hundred and fifty microlitre of the overnight culture was further inoculated into 25 ml LB broth containing respective antibiotics and incubated at 37 ◦ C in a shaker incubator for 3 h. After 3 h incubation the gene expression was induced by adding IPTG to a final concentration of 1 mM and incubated at 37 ◦ C in a shaker incubator. The optimal post induction time (PIT) was determined by the SDS PAGE analysis of culture collected (1 ml) at every 1 h interval (0–8 h). The protein was expressed in bulk after the determination of optimal PIT and the His tagged recombinant VSG protein (rVSG) was purified using NiNTA agarose column (Qiagen, USA) by following the manufacturer’s instructions. The purified protein was dialyzed against phosphate buffered saline (PBS) pH 7.2 and stored in aliquots at −20 ◦ C after estimating the protein concentration (Lowry et al., 1951) till further use.
2.8. Characterization of rVSG 2.8.1. SDS PAGE and immunoblot SDS PAGE was carried out by loading 40 g and 60 g of purified and cell lysate samples respectively into the 10% polyacrylamide gel as described by Sambrook and Russell (2001). Prior to loading, the protein samples were boiled in electrophoresis sample buffer (50 mM Tris pH 6.8, 10% glycerol, 5% -mercaptoethanol, 2% SDS and 0.1% bromophenol blue) for 5 min. The gel was stained for overnight on a shaker with page blue staining solution (Fermentas USA) followed by destaining. For immunoblot assay the electrophoresed protein was transferred on to nitrocellulose membrane following the standard protocol and then it was developed by treating the membrane with anti Histagged antibodies (1:1000 dilutions). The membrane was later treated with substrate solution containing diamino benzidine tetrahydrochloride (DAB) as a substrate. Further, the immuno reactivity of the expressed protein was determined using hyper immune/immune sera. Later the membrane was treated with respective secondary antibody conjugated with horse radish peroxidase and then treated with substrate solution to develop the blot.
2.8.2. Enzyme linked immunosorbent assay (ELISA) The antigen concentration, sera dilution and conjugate dilution for ELISA were optimized by checker board titration. ELISA was carried out by overnight coating of ELISA plates (Maxisorp, Nunc) at 4 ◦ C with purified recombinant VSG, WCL of T. evansi (Buffalo isolate), and RoTat 1.2 antigens respectively at a concentration of 12.5 g/well, 600 ng/well, and 600 ng/well in PBS (pH 7.2) respectively, whereas antigen free control wells received 100 l/well of PBS. After overnight incubation and in subsequent incubation steps the plates were washed 4 times with washing buffer [0.25% (v/v) Tween-20 in PBS, pH 7.2]. One hundred micro liters of blocking buffer [3% w/v lactalbumin enzymatic hydrolysate, 5% w/v skimmed milk powder & 0.05% v/v tween 20 in PBS (pH 7.2)] was added to each well and plates were incubated for 1 h at 37 ◦ C on a plate shaker (Heidoltch titramax 101). Hyperimmune/immune sera from rabbit/bovine/buffalo and field sera were diluted in dilution buffer (1:1 diluted blocking buffer in PBS, pH 7.2) and added (100 l/well) followed by incubation for 1 h at 37 ◦ C in a shaker incubator. Secondary antibody, anti bovine (for bovine sera)/anti rabbit (for rabbit sera)/anti horse (for horse sera)/protein G (for camel sera) conjugated with horseradish peroxidase (1:10,000) was added to each well (100 l/well) and further incubated for 1 h on a plate shaker. Hundred micro liters of substrate-chromogen (ortho phenylene di-amine) solution containing 0.03% H2 O2 was added to each well after four washes, and incubated for 15–20 min for the development of color. The reaction was stopped after the development of color by adding 100 l/well stopping solution (1 M H2 SO4 ) and plate was shaken for 10 s to read OD at 492 nm in ELISA reader (Biorad micro plate reader, bench mark). The specificity/cross reactivity of rVSG was evaluated with cattle serum samples clinically infected with Theileria annulata and Babesia bigemina in ELISA.
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Fig. 1. (A) PCR, (B); colony PCR (C) restriction enzyme digestion (RED) of plasmid (pGEMT-Easy+VSG) with EcoRI, (D) RED of plasmid (pET 33b+VSG) with EcoRI and NotI: Lane M: 1 kb DNA ladder, A; lane 1 – PCR control, lane 2 – PCR product, B; lane 1 – PCR control, lanes 2, 3 and 4 – clones 1, 2 and 3 respectively with plasmid PGEMT® Easy+VSG, lanes 4, 5 and 6 – clones 1, 2 and 3 respectively with plasmid pET 33b+VSG, C: lane 1 – undigested plasmid DNA, lanes 2, 3 and 4 – RED of plasmid DNA from clone 1 and 2 respectively, D; lane 1 and 2 – undigested plasmid DNA from clone 1 and 2 respectively, lanes 3 and 4 – RED plasmid DNA from clones 1 and 2 respectively.
The field/herd serum samples were subjected to ELISA in duplicate using rVSG and VSG RoTat 1.2 antigens. 2.8.3. CATT/T. evansi CATT/T. evansi, the rapid direct agglutination test, uses freeze dried trypanosomes of T. evansi VAT RoTat 1.2 (Bajyana Songa and Hamers, 1988; Verloo et al., 2001). All the reagents were used as per instruction of manufacturer. The hyperimmune/immune sera samples were diluted 1:4 dilutions up to 1:32 (double dilutions) with CATT buffer. The test card was rotated for 5 min at 70 rpm and later the card was observed for agglutination to read the results. The field/herd serum samples were tested in duplicate at 1:4 dilutions. 2.8.4. Statistical analysis The optimum combination of diagnostic specificity/sensitivity (Thrushfield, 2005) and cut off positive percentivity (PP) value were determined by frequency analysis graph (Wright et al., 1993). The raw data (OD values) obtained were converted into PP using the formula PP = (test sample OD value − negative control OD value)/(positive control OD value − negative control OD value) × 100. The cut off PP was determined from frequency distribution graph plotted with PP values obtained from 255 known positive and 259 known negative sera (confirmed with CATT/T. evansi) using rVSG. The sensitivity, specificity and cut-off PP value of VSG RoTat 1.2 antigen was determined by keeping CATT/T. evansi as gold standard. Thereafter, sensitivity, specificity and cut-off
PP value of rVSG was determined by keeping VSG RoTat 1.2 antigen as gold standard. Cohen’s kappa test (Cohen, 1960) was used to determine a correlation/agreement between the different diagnostic tests. The Cohen’s kappa was determined using on-line free soft ware KappaVassarStats (cited at: www.vassarstats.net/kappa.html). The Chi-square (2 ) test of independence was done as per Snedecor and Cochran (1968). 3. Results 3.1. Amplification and cloning of VSG gene of T. evansi The expression primer set amplified 1.3 kb of VSG by PCR (Fig. 1A). The recombinant clones were identified by colony touch PCR (Fig. 1B) and restriction enzyme digestion (Fig. 1C and D). The cloned VSG sequence is of 1206 nucleotides (nt) encoding a polypeptide of 402 amino acids (aa), with an apparent molecular weight of 42.7 kDa. The sequencing results showed that VSG in pET 33(b) is in correct orientation with respect to promoter sequence and also in frame with the ATG codon. 3.2. Expression, purification and characterization of recombinant VSG Out of the six positive recombinant clones, five clones showed high expression level. The optimal post induction time (PIT) was found to be 6 h. The concentration of recombinant protein remained same even after 6 h of
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Fig. 2. (A and B) SDS PAGE analysis of unpurified cell lysate samples and purified protein samples respectively, (C) immunoblot analysis: Lane M – prestained protein ladder, A; lane 1 – induced BL 21 DE3 pLys S, lane 2 – induced BL 21 DE3 pLys S with pET33b, lane 3 – uninduced BL 21 DE3 pLys S with pET33b+VSG, lanes 4, 5, 6, 7, 8, 9, 10, 11 – induced pET33b+VSG at 0, 1, 2, 3, 4, 5, 6, 7 h post induction respectively, B; lanes 1, 2 and 4 – induced clones, BL 21 DE3 pLys S, BL21 DE3 pLys S with pET33b, BL 21 DE3 pLys S with pET33b+VSG respectively, lane 3 – uninduced BL21 DE3 pLys S with pET33b+VSG. C; lanes 1, 2, 3, 4 – induced rVSG v/s rabbit hyperimmune sera of T. evansi buffalo, dog, lion and leopard isolates respectively, (lanes 5–8) uninduced rVSG v/s rabbit hyperimmune sera of T. evansi buffalo, dog, lion and leopard isolates respectively, (lane 9 – induced rVSG vs rabbit control serum.
PIT (Fig. 2A). The yield of the purified rVSG ranged from 30 to 50 mg/l of bacterial culture. The SDS PAGE analysis of the induced cell lysates and purified products revealed the presence of rVSG at 48.3 kDa [rVSG (42.7 kDa) + vector protein fusion (5.6 kDa) = 48.3 kDa]. However the protein band corresponding to 48.3 kDa was not found in the cell lysates/purified products from controls such as induced BL21(DE-3) pLys S, induced BL-21(DE-3) pLys S with pET 33(b) DNA and uninduced BL-21(DE-3) pLys S with pET 33(b) VSG (Fig. 2B). 3.2.1. Immunoblot and ELISA Western blot analysis showed that the recombinant VSG is highly immunoreactive. The recombinant protein reacted with bovine hyper immune serum and buffalo immune serum raised against T. evansi (buffalo isolate). The reactivity of buffalo immune serum was from 21 days onwards till 60 days. Rabbit hyper immune serum raised against the buffalo, dog, lion and leopard isolates of T. evansi also recognized the recombinant protein (Fig. 2C). The recombinant protein was found to be immunoreactive against different hyper immune and immune sera raised in experimental animals, while the recombinant protein was not reactive with sera from non-infected control animals. The hyper immune sera raised against WCL antigens showed higher OD values against WCL/RoTat 1.2
antigens than rVSG. Moreover the cattle samples infected with infected with T. annulata and B. bigemina did not react with rVSG/VSG RoTat 1.2 antigens in ELISA and CATT/T. evansi test. The comparative performance of the expressed and WCL/VSG RoTat 1.2 antigens in ELISA has been demonstrated in Fig. 3. 3.2.2. Serodiagnosis of surra The optimum combination of diagnostic sensitivity and specificity of rVSG was found to be 95.6% (95% CI, 93.2–97.9) and 98.0% (95% CI, 97–99) respectively in ELISA at >25 PP value, keeping VSG Ro Tat 1.2 as gold standard antigen (Fig. 4). While the sensitivity and specificity of VSG RoTat 1.2 was found to be respectively 95.2% (95% CI, 92.8–97.7) and 94.38% (95% CI, 92.7–96.0) respectively at >30 PP value by keeping CATT/T. evansi as gold standard (fig not shown). The epidemiology revealed that the disease is more prevalent in West Bengal (eastern India) with seropositivity (SP) up to 38.9% (2 = 0.502, df = 2, p > 0.05) followed by, Tamil Nadu (SP = 32.5%, 2 = 0.264, df = 2, p > 0.05), Karnataka (SP = 31.3%, 2 = 2.228, df = 2, p > 0.05), Odisha (SP = 28.5%, 2 = 1.165, df = 2, p > 0.05) and Maharashtra (SP = 19.8%, 2 = 0.094, df = 2, p > 0.05). Uttarakhand experienced very low prevalence of 12.2% seropositivity (2 = 3.305, df = 2, p > 0.05). The central, eastern and southern parts of India experiences high prevalence of surra in
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Fig. 3. Comparative analysis of ELISA with panel of antigen/sera. (A–D) hyper immune serum raised against T. evansi buffalo, dog, lion and leopard isolates respectively, (E and F) rabbit control serum and anti rabbit conjugate control respectively, (G) hyper immune serum from bovine, (H) immune serum from buffalo, (I and J) control serum from bovine and buffalo respectively, (K) antibovine conjugate control.
animals compared to other regions of India. The calculated 2 values were found to be lesser than the critical/table 2 value. The comparative analysis of sero-epidemiology by ELISA and CATT/T. evansi is shown in Table 1.
serum remained non-reactive. Out of 1051 bovine field serum samples 293 samples remained positive, while 33 out of 100 camel serum samples and 2 sera out of 51 horse sera were found to be positive.
3.2.3. CATT/T. evansi In CATT/T. evansi, all the hyperimmune/immune sera were reactive (Table 2). The buffalo immune serum showed highest activity among all the sera. The rabbit hyperimmune serum against lion and leopard isolates showed the minimal reactivity. While the control rabbit/bovine/buffalo
4. Discussion T. evansi an important parasite in the subtropics is covered by a thick uniform coat of VSG. The parasites escape the host defense mechanism by the continuous variation of these antigens. In susceptible animals the VSG is
Fig. 4. Frequency distribution graph to determine the cut-off value of rVSG based ELISA (arrow indicates “cut off PP” value).
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Table 1 Sera samples (positive and negative) collected from different regions of India. State
Species
Antigen/test ELISA (rVSG)
Karnataka Tamil Nadu Maharashtra Odisha Uttarakhand West Bengal Rajasthan
Bovine Horse Bovine Bovine Bovine Bovine Bovine Camel
ELISA (VSG RoTat 1.2)
CATT/T. evansi
P
N
P
N
P
N
115 3 26 35 12 14 106 31
252 48 54 141 30 100 166 69
129 3 28 37 9 21 98 32
238 48 52 139 33 93 174 68
111 3 25 35 8 12 102 30
256 48 55 141 34 102 170 70
P – positive, N – negative.
expressed at early, middle and later stages of infection (Verloo et al., 2001). Moreover, apart from being the major determinant of immuno evasion, as it is remaining at the outer most layer of the parasite, the host immune system elicits sufficient level of antibody production against the parasite (Gadelha et al., 2011). The surface epitopes of living trypanosomes are confirmationally labile (Freymann et al., 1990). Further it is also known that anti-VSG antibody recognizes glycosylated and deglycosylated VSG equally (Reinwald, 1985). The above findings and molecular epidemiological studies on trypanosomosis suggest that VSG can act as a potent antigen in the diagnosis of T. evansi infection. A number of genes of trypanosomes have been heterologously expressed for several purposes. For instance, oligopeptidase B from T. b. brucei (Rae et al., 2006), variable surface glycoprotein (VSG) gene of T. evansi in insect cells (Urakawa et al., 2001), ISG-75 gene of T. b. gambiense (Tran et al., 2008), hypoxanthine guanine phospho ribosyl transferase gene of T.b. brucei (Allen and Ullman, 1993), beta-tubulin gene of T. evansi (Li et al., 2007), actin gene of T. evansi (Li et al., 2009) and N-terminal region of VSG gene from T. evansi (Sengupta et al., 2012) in prokaryotic system. In the present study VSG gene of T. evansi encoding a polypeptide of 402 amino acids has been expressed in E. coli and the expressed protein was characterized for its possible application in the serodiagnosis of surra/T. evansi infection. The prokaryotic expression system such as E. coli is advantageous over other systems for its easy manipulation and expression system.
VSGs are the predominant antigens in antibody detection tests for sleeping sickness and surra (Magnus et al., 1978; Verloo et al., 2001) and are also implicated in the control measures against trypanosomes, including diagnosis and vaccination. The whole cell lysates of T. evansi leads to strong cross reactions with T. vivax, T. congolense and even T. cruzi (OIE, 2010). Earlier reports suggest that VSG expressed in heterologus system can be used as an antigen for the diagnosis of T. evansi infection. For instance ELISA and CATT test using VSG (expressed in insect cells) as a diagnostic antigen (Bajyana Songa and Hamers, 1988; Lejon et al., 2005). In the present study the rVSG based ELISA was not immunoreactive with T. annulata/B. bigemina antibody. The immunoblot and ELISA with hyperimmune/ immune sera revealed that, rVSG is immunoreactive. However the OD values obtained with WCL/RoTat 1.2 antigen were found to be higher than rVSG due to the presence of other paratopes apart from VSG. The comparative study of ELISA revealed that, rVSG developed in the present study exhibits high sensitivity and specificity when compared with the standard reagents procured from OIE reference laboratory on surra. The Cohen’s kappa co-efficient of agreement was found to be 0.93, and 0.86 respectively with rVSG, and VSG RoTat 1.2 antigens. Hence rVSG turns out to be an excellent diagnostic antigen for the serodiagnosis using ELISA and it can be used as a rapid, reliable, potential and promising perspective tool in the serological diagnosis of carrier status which can be used effectively in control programme of surra.
Table 2 Immunoreactivity of different hyperimmune/immune sera with CATT/T. evansi. Serum dilution
Hyper immune rabbit serum raised against recombinant 48.3 kDa protein Hyper immune rabbit serum raised against WCL antigen of buffalo isolate of T. evansi Hyper immune rabbit serum raised against WCL antigen of canine isolate of T. evansi Hyper immune rabbit serum raised against WCL antigen of lion isolate of T. evansi Hyper immune rabbit sera serum raised against WCL antigen of leopard isolate of T. evansi Hyper immune bovine serum raised against WCL antigen of buffalo isolate of T. evansi Immune buffalo serum against the buffalo isolate of T. evansi
1:4
1:8
1:16
1:32
++ ++ + + + ++ ++
+ + + – – ± +
– – – – – – ±
– – – – – – –
+++, Strongly positive (very strong agglutination); ++, positive (strong agglutination); +, positive (moderate agglutination); ±, weakly positive (weak agglutination); −, negative (absence of agglutination).
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Seroprevalence study revealed that the disease is more prevalent in central (Madhya Pradesh), eastern (West Bengal) and southern (Karnataka and Tamil Nadu) parts of India. Further, in each state, no significant difference was observed between seropositivity and seronegativity using three different antigens/test (rVSG/VSG RoT at 1.2 and CATT/T. evansi test) (df = 2, p > 0.05). Earlier studies, reports up to 40% of seroprevalence in eastern India (Laha and Sasmal, 2009), and is in agreement with the present study (38.9%). However, with available data, a varied seroprevalence of 12.2–38.9% was observed from different states of India. Hence a larger panel of samples needs to be studied further for seroprevalence. As per our literature search, this is the first report of prokaryotic expression of VSG of T. evansi and its subsequent use in sero-diagnosis. In conclusion, in the present study we have developed a recombinant VSG antigen in prokaryotic system and thereafter the immunoreactivity of the antigen was evaluated and compared with VSG RoTat 1.2 based ELISA and CATT/T. evansi agglutination test. The latter two antigens were obtained from OIE reference laboratory, Belgium. The comparative study showed that immunoreactivity of rVSG is in agreement with other antigens/test, exhibiting high diagnostic value and the rVSG based ELISA can be used a reliable and perspective tool for serosurveillance to control surra. Acknowledgements The authors are thankful to those who extended cooperation and help in collection of samples. The work was done under the financial support by the Department of Biotechnology (DBT), Government of India (Project number: BT/PR3478/ADV/90/122/2011). References Allen, T.E., Ullman, B., 1993. Cloning and expression of the hypoxanthineguanine phosphoribosyltransferase gene from Trypanosoma brucei. Nucleic Acids Res. 21, 5431–5438. Bajyana Songa, E., Hamers, R., 1988. A card agglutination test (CATT) for veterinary use based on an early VAT Ro Tat 1.2 of Trypanosoma evansi. Ann. Soc. Belg. Med. Trop. 68, 233–240. Borst, P., Fase-Fowler, F., Gibson, W.C., 1987. Kinetoplastid DNA of Trypanosoma evansi. Mol. Biochem. Parasitol. 23, 31–38. Cohen, J., 1960. A coefficient of agreement for nominal scales. Educ. Psychol. Meas. 20, 37–46. Davison, H.C., Thrusfield, M.V., Muharsini, S., Husein, A., Partoutomo, S., Masake, R., Luckins, A.G., 1999. Evaluation of antigen- and antibodydetection tests for Trypanosoma evansi infections of buffaloes in Indonesia. Epidemiol. Infect. 123, 149–155. Diall, O., Nantulya, V.M., Luckins, A.G., Diarra, B., Kouyate, B., 1992. Evaluation of mono- and polyclonal antibody-based antigen detection immunoassays for diagnosis of Trypanosoma evansi infection in the dromedary camel. Rev. Elev. Med. Vet. Pays. Trop. 45, 149–153. Field, M.C., Carrington, M., 2009. The trypanosome flagellar pocket. Nat. Rev. Microbiol 7, 775–786. Freymann, D., Down, J., Carrington, M., Roditi, I., Turner, M., Wiley, D., 1990. 2.9 A˚ Resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma evansi. J. Mol. Biol. 216, 141–160. Gadelha, C., Holden, J.M., Allison, H.C., Field, M.C., Jennifer, M., 2011. Specializations in a successful parasite: what makes the blood stream form African trypanosomes so deadly? Mol. Biochem. Parasitol. 179, 51–58. Gibson, W.C., de, C., Marshall, T.F., Godfrey, D.G., 1980. Numerical analysis of enzyme polymorphism: a new approach to epidemiology and
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taxonomy of trypanosomes of the subgenus Trypanozoon. Adv. Parasitol. 18, 175–246. Holland, W.G., Thanh, N.G., My, L.N., Do, T.T., Goddeeris, B.M., Vercruysse, J., 2004. Prevalence of Trypanosoma evansi in water buffaloes in remote areas in northern Vietnam using PCR and serological methods. Trop. Anim. Health Prod. 36, 45–48. Laha, R., Sasmal, N.K., 2009. Detection of Trypanosoma evansi infection in clinically ill cattle, buffaloes and horses using various diagnostic tests. Epidemiol. Infect. 137, 1583–1585. Lanham, S.M., Godfrey, D.G., 1970. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 28, 521–534. Lejon, V., Claes, F., Verloo, D., Maina, M., Urakawa, T., Majiwa, P.A.O., Buscher, P., 2005. Recombinant RoTat 1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary camels. Int. J. Parasitol. 35, 455–460. Li, S.Q., Fung, M.C., Reid, S.A., Inoue, N., Lun, Z.R., 2007. Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protein T. evansi, T. equiperdum and T.b. brucei infection in mice. Parasite Immunol. 29, 191–199. Li, S.Q., Yang, W.B., Lun, Z.R., Ma, L.J., Xi, S.M., Chen, Q.L., Song, X.W., Kang, J., Yang, L.Z., 2009. Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T.b. brucei infection. Parasitol. Res. 104, 429–435. Lowry, O.H., Rosenbruogh, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Magnus, E., Vervoort, T., Van Meirvenne, N., 1978. A card-agglutination test with stained trypanosomes (C.A.T.T.) for the serological diagnosis of T.b. gambiense trypanosomiasis. Ann. Soc. Belg. Med. Trop. 58, 169–176. Mathieu-Daude, F., Tibayrenc, M., 1994. Isoenzyme variability of Trypanosoma brucei sl.: genetic, taxonomic and epidemiological significance. Exp. Parasitol. 78, 1–19. Nantulya, V.M., Bajyana-Songa, E., Hamers, R., 1989a. Detection of circulating trypanosomal antigens in Trypanosoma evansi infected animals using a T. brucei group specific monoclonal antibody. Trop. Med. Parasitol. 40, 263–266. Nantulya, V.M., Lindqvist, K.J., Diall, O., Olaho-Mukani, W., 1989b. Two simple antigen-detection enzyme immunoassays for the diagnosis of Trypanosoma evansi infections in the dromedary camel (Camelus dromedarius). Trop. Med. Parasitol. 40, 415–418. OIE, 2010. Trypanosoma evansi (Surra). In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organization for Animal Health, Paris, Available from http://www.oie.int Rae, D., Hazell, C., Andrew, N.W., Morty, R.E., Fulop, V., 2006. Expression, purification and preliminary crystallographic analysis of oligopeptidase B from Trypanosoma brucei. Acta Crystallogr. F: Struct. Biol. Cryst. Commun. 62, 808–810. Reinwald, E., 1985. Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense: protection against proteolytic attack. Eur. J. Biochem. 151, 385–391. Roge, S., Van Reeta, N., Odiwuora Tran, T., Schilderman, K., Vandamme, S., Vandenberghe, I., Verveckene, W., Gillingwater, K., Claes, F., Devreese, B., Guisez, Y., Buscher, P., 2013. Recombinant expression of trypanosome surface glycoproteins in Pichia pastoris for the diagnosis of Trypanosoma evansi infection. Vet. Parasitol. 197, 571–579. Rudramurthy, G.R., Sengupta, P.P., Balamurugan, V., Prabhudas, K., Rahman, H., 2013. PCR based diagnosis of trypanosomiasis exploring invariant surface glycoprotein (ISG) 75 gene. Vet. Parasitol. 193, 47–58. Sambrook, J., Russell, D., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Sengupta, P.P., Balumahendiran, M., Suryanarayana, V.V.S., Raghavendra, A.G., Shome, B.R., Ganjendragad, M.R., Prabhudas, K., 2010. PCR-based diagnosis of surra-targeting VSG gene: experimental studies in small laboratory rodents and buffalo. Vet. Parasitol. 171, 22–31. Sengupta, P.P., Balumahendiran, M., Balamurugan, V., Rudramurthy, G.R., Prabhudas, K., 2012. Expressed truncated N-terminal variable surface glycoprotein (VSG) of Trypanosoma evansi in E. coli exhibits immunoreactivity. Vet. Parasitol. 187, 1–8. Snedecor, G.W., Cochran, W.G., 1968. Statistical Methods. Indian Edition. Oxford & IBH Publishing Co., New Delhi, pp. 1–593. Songa, E.B., Paindavoine, P., Wittouck, E., Viseshakul, N., Muldermans, S., Steinert, M., Hammers, R., 1990. Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates. Mol. Biochem. Parasitol. 43, 167–179.
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Verloo, D., Holland, W., My, L.N., Thanh, N.G., Tam, P.T., Goddeeris, B., Vercruysse, J., Buscher, P., 2000. Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam. Vet. Parasitol. 29, 87–96. Verloo, D., Magnus, E., Buscher, P., 2001. General expression of RoTat 1.2 variable antigen type in Trypanosoma evansi isolates from different origin. Vet. Parasitol. 97, 183–189. Wright, P.F., Nilsson, E., Van Rooij, E.M.A., Lelenta, M., Jeggo, M.H., 1993. Standardisation and validation of enzyme linked immunosorbent assay techniques for the detection of antibody in infectious disease diagnosis. Rev. Sci. Technol. 12, 435–450. Zhang, Z.Q., Baltz, T., 1994. Identification of Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma brucei brucei using repetitive DNA probes. Vet. Parasitol. 53, 197–208.
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Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance P.P. Sengupta a,∗ , G.R. Rudramurthy a , M. Ligi a , M. Roy a , V. Balamurugan a , P. Krishnamoorthy a , M. Nagalingam a , L. Singh b , H. Rahman a a
National Institute of Veterinary Epidemiology and Disease Informatics, Hebbal, Bengaluru 560024, Karnataka, India Collaborating Unit, AICRP on ADMAS, State Disease Diagnostic Centre, Old B.P. Lab. Campus, Gopinath Marg, New Colony, Jaipur 302001, Rajasthan, India b
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 20 August 2014 Accepted 22 August 2014 Keywords: Trypanosoma evansi Surra Variable surface glycoprotein ELISA
a b s t r a c t Trypanosoma evansi, a haemoflagellate, causes “surra” an important chronic wasting disease of a wide range of wild and domestic herbivorous and carnivorous animals including cattle, buffaloes, camels, horses, etc. The untreated recovered animal can act as a carrier without exhibiting the disease symptoms and can be a source of infection to healthy animals. The diagnosis and subsequent treatment of the carrier animals is helpful to curb the disease. As the parasitaemia in carrier animals is very scanty, the conventional blood smear examination, which is widely practiced in the field, cannot detect such condition. For this purpose improved diagnostics are very much useful for mass sero-screening test such as ELISA. In the present study, the VSG of T. evansi was expressed in prokaryotic system (E. coli) and thereafter its immunoreactivity has been evaluated in immuno blot and enzyme immuno assay. The expressed protein showed 95.6% sensitivity, 98.0% specificity and 0.93 Cohen’s kappa value, when compared with standard antigens. The developed antigen has also been validated with field serum samples from bovine, camel and horse collected from different states of India. The data showed that the developed recombinant antigen can be a diagnostic tool to detect carrier animals as well as control of the disease. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma (T) evansi, a haemoflagellated parasite and a petite mutant of T. brucei (Field and Carrington, 2009) causes a fatal wasting disease called surra, results in havoc economical loss. Surra has a wide range of hosts such as cattle, buffaloes, camels, horses, etc. The mammalian hosts for surra in south east Asia are mostly cattle, buffaloes
∗ Corresponding author at: Parasitology Laboratory, National Institute of Veterinary Epidemiology and Disease Informatics, IVRI Campus, Hebbal, Bengaluru 560024, Karnataka, India. Tel.: +91 080 23419576; fax: +91 080 23415329. E-mail address: pinakiprasad [email protected] (P.P. Sengupta). http://dx.doi.org/10.1016/j.vetpar.2014.08.012 0304-4017/© 2014 Elsevier B.V. All rights reserved.
and horses (Holland et al., 2004). The disease is clinically characterized by anemia, recurrent fever, oedema, muscular weakness, loss of appetite and abortion, with a morbidity and mortality of 50–70%. The animals serve as carriers for the disease by exhibiting low levels of fluctuating parasites for years after recovery. The detection of carrier status in animal is very important in controlling the disease. Acute or subacute infection can be satisfactorily diagnosed by conventional parasitological techniques, but it becomes difficult in latent or chronic infection where parasitaemia is very low. The application of advanced laboratory techniques such as DNA amplification and sensitive serological tests can be helpful in the detection of carrier status of animal. Several genes have been analyzed/targeted for diagnosis of trypanosomosis, such as
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variable surface glycoprotein (VSG) (Sengupta et al., 2010), invariant surface glycoprotein (ISG) (Rudramurthy et al., 2013), kinetoplastid DNA, nuclear DNA and multilocus isoenzymes analysis (Gibson et al., 1980; Borst et al., 1987; Songa et al., 1990; Stevens and Godfrey, 1992; MathieuDaude and Tibayrenc, 1994; Zhang and Baltz, 1994). VSG is the primary immunogen of T. evansi in eliciting the antibody response in host. Potential serological tests have been developed for T. evansi based on the detection of either antigens or antibodies such as antigen detection ELISA for camels (Nantulya et al., 1989b; Diall et al., 1992; Verloo et al., 1998), buffaloes (Nantulya et al., 1989a; Davison et al., 1999; Verloo et al., 2000), antibody detection based on the predominant variant antigen type (VAT) RoTat 1.2 (Verloo et al., 2000), CATT/T. evansi (Bajyana Songa and Hamers, 1988; Verloo et al., 2000), LATEX/T. evansi (Verloo et al., 1998, 2000) and ELISA/T. evansi (Verloo et al., 2000). Earlier the VSG was expressed in different host cells, such as, insect cell line (Urakawa et al., 2001), yeast (Roge et al., 2013) and E. coli (Sengupta et al., 2012), etc. Further, it has also been reported that the immunoreactivity of the glycosylated and deglycosylated VSG remained same against anti-VSG antibody (Reinwald, 1985). In our earlier studies also, we showed that expressed N-terminal portion of VSG exhibits immunoreactivity. In the present study the whole VSG gene of T. evansi has been expressed in E. coli and assessed its immunoreactivity and further the rVSG has been evaluated for its potentiality to explore as a diagnostic antigen in ELISA for the serodiagnosis of surra using field sera samples. 2. Materials and methods 2.1. T. evansi stabilates and whole cell lysate (WCL) antigen preparation Different isolates of T. evansi such as buffalo, dog, lion and leopard, maintained in the Parasitology Laboratory, NIVEDI, Bengaluru, India, were used in the present study. The different isolates of T. evansi were propagated in rats as mentioned earlier (Sengupta et al., 2010) and purified using diethyl amino ethyl (DEAE) cellulose (DE.52-Whatman) column (Lanham and Godfrey, 1970). The WCL antigens from different isolates were prepared as mentioned earlier (Sengupta et al., 2012), the protein concentration of the supernatant was estimated (Lowry et al., 1951) and kept in aliquots at −80 ◦ C till further use. 2.2. VSG RoTat 1.2 antigen The VSG RoTat 1.2 antigen was obtained from the Koning Leopold Institute of Tropical Medicine, Antwerp, Belgium (OIE reference laboratory of surra) and used as per manufacturer’s instruction. The antigen was used in ELISA at 600 ng/well to compare the immunoreactivity with expressed protein. 2.3. Preparation of hyper immune/immune sera in animals The hyper immune sera against T. evansi were raised in rabbits (buffalo, dog, lion and leopard isolates) and also
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in cattle (buffalo isolate); while immune serum was raised in buffalo (buffalo isolate) (Sengupta et al., 2012) and sera available in the parasitology laboratory were used in the present study. 2.4. Sera collection from field The bovine field sera (n = 1051) were collected randomly from different states of India, such as Karnataka, Tamil Nadu, Maharashtra, Odisha, West Bengal and Uttarakhand, which covers southern, northern, eastern and western regions of India, between 2010 and 2013. The sera from camel (n = 100) were collected from Rajasthan (India) from the field, while horse sera (n = 51) were collected from Karnataka state in India from an organized herd in 2014. All the animals were apparently healthy. However, the disease status of the animals was not available. The sera separated after the sampling was preserved at −80 ◦ C for further use. The sera included in the present study were screened for trypanosomosis by serological tests such as CATT/T. evansi and ELISA in duplicate. The ELISA was carried out using rVSG and VSG RoTat 1.2 antigens for comparative evaluation. 2.5. Amplification of VSG gene by RT-PCR A set of expression primers TEVSG-F (5 -CATGAATTCCAAGGCGCTCGTTGG-3 ) corresponding to 10–24 bp and TEVSG-R (5 -GCCTGTAAGCTTTTTGTTTTTTGCATCTGATTC3 ) to 1216–1196 bp were designed from our published sequence EF495337. The forward and reverse primers were respectively introduced with EcoRI and HindIII restriction site for cloning into expression vector. The histidine (His) sequences were introduced in the forward as well as the reverse primers to purify the expressed protein. The total RNA was isolated from purified trypanosomes (buffalo isolate) and preserved at −80 ◦ C, till further use. RT-PCR was carried out from the total RNA to synthesize VSG specific cDNA using random nanomer by following the standard protocol described earlier (Sengupta et al., 2010). The cDNA obtained was subjected to PCR for the amplification of the VSG gene. The 25 l PCR reaction mixture contained Taq buffer, 10 mm dNTPS, 20 Pmol each of TEVSG-F/R primers and 3 units (U) Taq DNA polymerase (MBI fermentas). The PCR cycle was carried out with initial denaturation at 94 ◦ C for 3 min, followed by 35 cycles of one min, denaturation at 94 ◦ C for 1 min., primer annealing at 55 ◦ C for 1 min, and extension at 72 ◦ C for 1.20 min with a final extension at 72 ◦ C for 10 min. The amplified product was analyzed in 1% agarose gel and documented in gel doc analyzer (BioRad). 2.6. Construction of expression cassette in pET33 (b) vector The amplicon run on the agarose gel was extracted using Qiagen gel extraction kit as per manufacturer’s instruction. The purified product was initially ligated into pGEMT@ Easy Cloning vector (Promega) and transformed into E. coli Top10 competent cells. The transformed cells were plated on Luria Bertini (LB) agar containing ampicillin (50 g/ml),
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isoprophyl -thiogalacto pyranoside (IPTG) (1 mM) and X-.gal (20 l/ml) for the selection of recombinant clones. The white positive clones were selected and confirmed by colony lysis, colony touch PCR, restriction enzyme digestion (EcoRI single digestion) and DNA sequencing using T7 and Sp6 universal primers. The VSG gene from pGEMT@ Easy vector was released using EcoRI and HindIII digestion and cloned into EcoRI and HindIII site of pET33(b) expression vector system (Novagen) and the recombinant plasmid was named as pET33(b)VSG. The plasmid pET33 (b) VSG was transformed into E. coli (Top 10) competent cells by following the standard protocol (Sambrook and Russell, 2001). The transformed cells were plated on the LB agar medium containing kanamycin (50 g/ml) and incubated at 37 ◦ C for overnight. The clones appeared on the plate were screened as mentioned earlier. The recombinant plasmid DNA isolated from the positive clones was sequenced using vector specific (T7 promoter/terminator primers) and gene specific (TEVSGF/R) primers to determine the orientation of the insert in the vector backbone.
2.6.1. Expression of VSG in E. coli host [BL-21(DE3) pLys S] The recombinant plasmid pET33(b)VSG isolated was transformed into E. coli BL21(DE3)PLysS (Invitrogen) expression host system by following the standard protocol (Sambrook and Russell, 2001). The transformed cells were plated on the LB agar medium containing chloramphenicol (34 g/ml) and kanamycin (50 g/ml) and incubated at 37 ◦ C for overnight. The positive BL21 recombinant clones appeared in the plate were selected as mentioned earlier to express the protein.
2.7. Expression and purification of rVSG Eight recombinant positive clones were analysed for the expression of VSG. The inoculum for the protein expression was prepared by inoculating a single positive colony into 5 ml LB broth containing chloramphenicol (34 g/ml) and kanamycin (30 g/ml) and incubated in a shaker incubator at 37 ◦ C for overnight. Two hundred and fifty microlitre of the overnight culture was further inoculated into 25 ml LB broth containing respective antibiotics and incubated at 37 ◦ C in a shaker incubator for 3 h. After 3 h incubation the gene expression was induced by adding IPTG to a final concentration of 1 mM and incubated at 37 ◦ C in a shaker incubator. The optimal post induction time (PIT) was determined by the SDS PAGE analysis of culture collected (1 ml) at every 1 h interval (0–8 h). The protein was expressed in bulk after the determination of optimal PIT and the His tagged recombinant VSG protein (rVSG) was purified using NiNTA agarose column (Qiagen, USA) by following the manufacturer’s instructions. The purified protein was dialyzed against phosphate buffered saline (PBS) pH 7.2 and stored in aliquots at −20 ◦ C after estimating the protein concentration (Lowry et al., 1951) till further use.
2.8. Characterization of rVSG 2.8.1. SDS PAGE and immunoblot SDS PAGE was carried out by loading 40 g and 60 g of purified and cell lysate samples respectively into the 10% polyacrylamide gel as described by Sambrook and Russell (2001). Prior to loading, the protein samples were boiled in electrophoresis sample buffer (50 mM Tris pH 6.8, 10% glycerol, 5% -mercaptoethanol, 2% SDS and 0.1% bromophenol blue) for 5 min. The gel was stained for overnight on a shaker with page blue staining solution (Fermentas USA) followed by destaining. For immunoblot assay the electrophoresed protein was transferred on to nitrocellulose membrane following the standard protocol and then it was developed by treating the membrane with anti Histagged antibodies (1:1000 dilutions). The membrane was later treated with substrate solution containing diamino benzidine tetrahydrochloride (DAB) as a substrate. Further, the immuno reactivity of the expressed protein was determined using hyper immune/immune sera. Later the membrane was treated with respective secondary antibody conjugated with horse radish peroxidase and then treated with substrate solution to develop the blot.
2.8.2. Enzyme linked immunosorbent assay (ELISA) The antigen concentration, sera dilution and conjugate dilution for ELISA were optimized by checker board titration. ELISA was carried out by overnight coating of ELISA plates (Maxisorp, Nunc) at 4 ◦ C with purified recombinant VSG, WCL of T. evansi (Buffalo isolate), and RoTat 1.2 antigens respectively at a concentration of 12.5 g/well, 600 ng/well, and 600 ng/well in PBS (pH 7.2) respectively, whereas antigen free control wells received 100 l/well of PBS. After overnight incubation and in subsequent incubation steps the plates were washed 4 times with washing buffer [0.25% (v/v) Tween-20 in PBS, pH 7.2]. One hundred micro liters of blocking buffer [3% w/v lactalbumin enzymatic hydrolysate, 5% w/v skimmed milk powder & 0.05% v/v tween 20 in PBS (pH 7.2)] was added to each well and plates were incubated for 1 h at 37 ◦ C on a plate shaker (Heidoltch titramax 101). Hyperimmune/immune sera from rabbit/bovine/buffalo and field sera were diluted in dilution buffer (1:1 diluted blocking buffer in PBS, pH 7.2) and added (100 l/well) followed by incubation for 1 h at 37 ◦ C in a shaker incubator. Secondary antibody, anti bovine (for bovine sera)/anti rabbit (for rabbit sera)/anti horse (for horse sera)/protein G (for camel sera) conjugated with horseradish peroxidase (1:10,000) was added to each well (100 l/well) and further incubated for 1 h on a plate shaker. Hundred micro liters of substrate-chromogen (ortho phenylene di-amine) solution containing 0.03% H2 O2 was added to each well after four washes, and incubated for 15–20 min for the development of color. The reaction was stopped after the development of color by adding 100 l/well stopping solution (1 M H2 SO4 ) and plate was shaken for 10 s to read OD at 492 nm in ELISA reader (Biorad micro plate reader, bench mark). The specificity/cross reactivity of rVSG was evaluated with cattle serum samples clinically infected with Theileria annulata and Babesia bigemina in ELISA.
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Fig. 1. (A) PCR, (B); colony PCR (C) restriction enzyme digestion (RED) of plasmid (pGEMT-Easy+VSG) with EcoRI, (D) RED of plasmid (pET 33b+VSG) with EcoRI and NotI: Lane M: 1 kb DNA ladder, A; lane 1 – PCR control, lane 2 – PCR product, B; lane 1 – PCR control, lanes 2, 3 and 4 – clones 1, 2 and 3 respectively with plasmid PGEMT® Easy+VSG, lanes 4, 5 and 6 – clones 1, 2 and 3 respectively with plasmid pET 33b+VSG, C: lane 1 – undigested plasmid DNA, lanes 2, 3 and 4 – RED of plasmid DNA from clone 1 and 2 respectively, D; lane 1 and 2 – undigested plasmid DNA from clone 1 and 2 respectively, lanes 3 and 4 – RED plasmid DNA from clones 1 and 2 respectively.
The field/herd serum samples were subjected to ELISA in duplicate using rVSG and VSG RoTat 1.2 antigens. 2.8.3. CATT/T. evansi CATT/T. evansi, the rapid direct agglutination test, uses freeze dried trypanosomes of T. evansi VAT RoTat 1.2 (Bajyana Songa and Hamers, 1988; Verloo et al., 2001). All the reagents were used as per instruction of manufacturer. The hyperimmune/immune sera samples were diluted 1:4 dilutions up to 1:32 (double dilutions) with CATT buffer. The test card was rotated for 5 min at 70 rpm and later the card was observed for agglutination to read the results. The field/herd serum samples were tested in duplicate at 1:4 dilutions. 2.8.4. Statistical analysis The optimum combination of diagnostic specificity/sensitivity (Thrushfield, 2005) and cut off positive percentivity (PP) value were determined by frequency analysis graph (Wright et al., 1993). The raw data (OD values) obtained were converted into PP using the formula PP = (test sample OD value − negative control OD value)/(positive control OD value − negative control OD value) × 100. The cut off PP was determined from frequency distribution graph plotted with PP values obtained from 255 known positive and 259 known negative sera (confirmed with CATT/T. evansi) using rVSG. The sensitivity, specificity and cut-off PP value of VSG RoTat 1.2 antigen was determined by keeping CATT/T. evansi as gold standard. Thereafter, sensitivity, specificity and cut-off
PP value of rVSG was determined by keeping VSG RoTat 1.2 antigen as gold standard. Cohen’s kappa test (Cohen, 1960) was used to determine a correlation/agreement between the different diagnostic tests. The Cohen’s kappa was determined using on-line free soft ware KappaVassarStats (cited at: www.vassarstats.net/kappa.html). The Chi-square (2 ) test of independence was done as per Snedecor and Cochran (1968). 3. Results 3.1. Amplification and cloning of VSG gene of T. evansi The expression primer set amplified 1.3 kb of VSG by PCR (Fig. 1A). The recombinant clones were identified by colony touch PCR (Fig. 1B) and restriction enzyme digestion (Fig. 1C and D). The cloned VSG sequence is of 1206 nucleotides (nt) encoding a polypeptide of 402 amino acids (aa), with an apparent molecular weight of 42.7 kDa. The sequencing results showed that VSG in pET 33(b) is in correct orientation with respect to promoter sequence and also in frame with the ATG codon. 3.2. Expression, purification and characterization of recombinant VSG Out of the six positive recombinant clones, five clones showed high expression level. The optimal post induction time (PIT) was found to be 6 h. The concentration of recombinant protein remained same even after 6 h of
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Fig. 2. (A and B) SDS PAGE analysis of unpurified cell lysate samples and purified protein samples respectively, (C) immunoblot analysis: Lane M – prestained protein ladder, A; lane 1 – induced BL 21 DE3 pLys S, lane 2 – induced BL 21 DE3 pLys S with pET33b, lane 3 – uninduced BL 21 DE3 pLys S with pET33b+VSG, lanes 4, 5, 6, 7, 8, 9, 10, 11 – induced pET33b+VSG at 0, 1, 2, 3, 4, 5, 6, 7 h post induction respectively, B; lanes 1, 2 and 4 – induced clones, BL 21 DE3 pLys S, BL21 DE3 pLys S with pET33b, BL 21 DE3 pLys S with pET33b+VSG respectively, lane 3 – uninduced BL21 DE3 pLys S with pET33b+VSG. C; lanes 1, 2, 3, 4 – induced rVSG v/s rabbit hyperimmune sera of T. evansi buffalo, dog, lion and leopard isolates respectively, (lanes 5–8) uninduced rVSG v/s rabbit hyperimmune sera of T. evansi buffalo, dog, lion and leopard isolates respectively, (lane 9 – induced rVSG vs rabbit control serum.
PIT (Fig. 2A). The yield of the purified rVSG ranged from 30 to 50 mg/l of bacterial culture. The SDS PAGE analysis of the induced cell lysates and purified products revealed the presence of rVSG at 48.3 kDa [rVSG (42.7 kDa) + vector protein fusion (5.6 kDa) = 48.3 kDa]. However the protein band corresponding to 48.3 kDa was not found in the cell lysates/purified products from controls such as induced BL21(DE-3) pLys S, induced BL-21(DE-3) pLys S with pET 33(b) DNA and uninduced BL-21(DE-3) pLys S with pET 33(b) VSG (Fig. 2B). 3.2.1. Immunoblot and ELISA Western blot analysis showed that the recombinant VSG is highly immunoreactive. The recombinant protein reacted with bovine hyper immune serum and buffalo immune serum raised against T. evansi (buffalo isolate). The reactivity of buffalo immune serum was from 21 days onwards till 60 days. Rabbit hyper immune serum raised against the buffalo, dog, lion and leopard isolates of T. evansi also recognized the recombinant protein (Fig. 2C). The recombinant protein was found to be immunoreactive against different hyper immune and immune sera raised in experimental animals, while the recombinant protein was not reactive with sera from non-infected control animals. The hyper immune sera raised against WCL antigens showed higher OD values against WCL/RoTat 1.2
antigens than rVSG. Moreover the cattle samples infected with infected with T. annulata and B. bigemina did not react with rVSG/VSG RoTat 1.2 antigens in ELISA and CATT/T. evansi test. The comparative performance of the expressed and WCL/VSG RoTat 1.2 antigens in ELISA has been demonstrated in Fig. 3. 3.2.2. Serodiagnosis of surra The optimum combination of diagnostic sensitivity and specificity of rVSG was found to be 95.6% (95% CI, 93.2–97.9) and 98.0% (95% CI, 97–99) respectively in ELISA at >25 PP value, keeping VSG Ro Tat 1.2 as gold standard antigen (Fig. 4). While the sensitivity and specificity of VSG RoTat 1.2 was found to be respectively 95.2% (95% CI, 92.8–97.7) and 94.38% (95% CI, 92.7–96.0) respectively at >30 PP value by keeping CATT/T. evansi as gold standard (fig not shown). The epidemiology revealed that the disease is more prevalent in West Bengal (eastern India) with seropositivity (SP) up to 38.9% (2 = 0.502, df = 2, p > 0.05) followed by, Tamil Nadu (SP = 32.5%, 2 = 0.264, df = 2, p > 0.05), Karnataka (SP = 31.3%, 2 = 2.228, df = 2, p > 0.05), Odisha (SP = 28.5%, 2 = 1.165, df = 2, p > 0.05) and Maharashtra (SP = 19.8%, 2 = 0.094, df = 2, p > 0.05). Uttarakhand experienced very low prevalence of 12.2% seropositivity (2 = 3.305, df = 2, p > 0.05). The central, eastern and southern parts of India experiences high prevalence of surra in
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Fig. 3. Comparative analysis of ELISA with panel of antigen/sera. (A–D) hyper immune serum raised against T. evansi buffalo, dog, lion and leopard isolates respectively, (E and F) rabbit control serum and anti rabbit conjugate control respectively, (G) hyper immune serum from bovine, (H) immune serum from buffalo, (I and J) control serum from bovine and buffalo respectively, (K) antibovine conjugate control.
animals compared to other regions of India. The calculated 2 values were found to be lesser than the critical/table 2 value. The comparative analysis of sero-epidemiology by ELISA and CATT/T. evansi is shown in Table 1.
serum remained non-reactive. Out of 1051 bovine field serum samples 293 samples remained positive, while 33 out of 100 camel serum samples and 2 sera out of 51 horse sera were found to be positive.
3.2.3. CATT/T. evansi In CATT/T. evansi, all the hyperimmune/immune sera were reactive (Table 2). The buffalo immune serum showed highest activity among all the sera. The rabbit hyperimmune serum against lion and leopard isolates showed the minimal reactivity. While the control rabbit/bovine/buffalo
4. Discussion T. evansi an important parasite in the subtropics is covered by a thick uniform coat of VSG. The parasites escape the host defense mechanism by the continuous variation of these antigens. In susceptible animals the VSG is
Fig. 4. Frequency distribution graph to determine the cut-off value of rVSG based ELISA (arrow indicates “cut off PP” value).
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Table 1 Sera samples (positive and negative) collected from different regions of India. State
Species
Antigen/test ELISA (rVSG)
Karnataka Tamil Nadu Maharashtra Odisha Uttarakhand West Bengal Rajasthan
Bovine Horse Bovine Bovine Bovine Bovine Bovine Camel
ELISA (VSG RoTat 1.2)
CATT/T. evansi
P
N
P
N
P
N
115 3 26 35 12 14 106 31
252 48 54 141 30 100 166 69
129 3 28 37 9 21 98 32
238 48 52 139 33 93 174 68
111 3 25 35 8 12 102 30
256 48 55 141 34 102 170 70
P – positive, N – negative.
expressed at early, middle and later stages of infection (Verloo et al., 2001). Moreover, apart from being the major determinant of immuno evasion, as it is remaining at the outer most layer of the parasite, the host immune system elicits sufficient level of antibody production against the parasite (Gadelha et al., 2011). The surface epitopes of living trypanosomes are confirmationally labile (Freymann et al., 1990). Further it is also known that anti-VSG antibody recognizes glycosylated and deglycosylated VSG equally (Reinwald, 1985). The above findings and molecular epidemiological studies on trypanosomosis suggest that VSG can act as a potent antigen in the diagnosis of T. evansi infection. A number of genes of trypanosomes have been heterologously expressed for several purposes. For instance, oligopeptidase B from T. b. brucei (Rae et al., 2006), variable surface glycoprotein (VSG) gene of T. evansi in insect cells (Urakawa et al., 2001), ISG-75 gene of T. b. gambiense (Tran et al., 2008), hypoxanthine guanine phospho ribosyl transferase gene of T.b. brucei (Allen and Ullman, 1993), beta-tubulin gene of T. evansi (Li et al., 2007), actin gene of T. evansi (Li et al., 2009) and N-terminal region of VSG gene from T. evansi (Sengupta et al., 2012) in prokaryotic system. In the present study VSG gene of T. evansi encoding a polypeptide of 402 amino acids has been expressed in E. coli and the expressed protein was characterized for its possible application in the serodiagnosis of surra/T. evansi infection. The prokaryotic expression system such as E. coli is advantageous over other systems for its easy manipulation and expression system.
VSGs are the predominant antigens in antibody detection tests for sleeping sickness and surra (Magnus et al., 1978; Verloo et al., 2001) and are also implicated in the control measures against trypanosomes, including diagnosis and vaccination. The whole cell lysates of T. evansi leads to strong cross reactions with T. vivax, T. congolense and even T. cruzi (OIE, 2010). Earlier reports suggest that VSG expressed in heterologus system can be used as an antigen for the diagnosis of T. evansi infection. For instance ELISA and CATT test using VSG (expressed in insect cells) as a diagnostic antigen (Bajyana Songa and Hamers, 1988; Lejon et al., 2005). In the present study the rVSG based ELISA was not immunoreactive with T. annulata/B. bigemina antibody. The immunoblot and ELISA with hyperimmune/ immune sera revealed that, rVSG is immunoreactive. However the OD values obtained with WCL/RoTat 1.2 antigen were found to be higher than rVSG due to the presence of other paratopes apart from VSG. The comparative study of ELISA revealed that, rVSG developed in the present study exhibits high sensitivity and specificity when compared with the standard reagents procured from OIE reference laboratory on surra. The Cohen’s kappa co-efficient of agreement was found to be 0.93, and 0.86 respectively with rVSG, and VSG RoTat 1.2 antigens. Hence rVSG turns out to be an excellent diagnostic antigen for the serodiagnosis using ELISA and it can be used as a rapid, reliable, potential and promising perspective tool in the serological diagnosis of carrier status which can be used effectively in control programme of surra.
Table 2 Immunoreactivity of different hyperimmune/immune sera with CATT/T. evansi. Serum dilution
Hyper immune rabbit serum raised against recombinant 48.3 kDa protein Hyper immune rabbit serum raised against WCL antigen of buffalo isolate of T. evansi Hyper immune rabbit serum raised against WCL antigen of canine isolate of T. evansi Hyper immune rabbit serum raised against WCL antigen of lion isolate of T. evansi Hyper immune rabbit sera serum raised against WCL antigen of leopard isolate of T. evansi Hyper immune bovine serum raised against WCL antigen of buffalo isolate of T. evansi Immune buffalo serum against the buffalo isolate of T. evansi
1:4
1:8
1:16
1:32
++ ++ + + + ++ ++
+ + + – – ± +
– – – – – – ±
– – – – – – –
+++, Strongly positive (very strong agglutination); ++, positive (strong agglutination); +, positive (moderate agglutination); ±, weakly positive (weak agglutination); −, negative (absence of agglutination).
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Seroprevalence study revealed that the disease is more prevalent in central (Madhya Pradesh), eastern (West Bengal) and southern (Karnataka and Tamil Nadu) parts of India. Further, in each state, no significant difference was observed between seropositivity and seronegativity using three different antigens/test (rVSG/VSG RoT at 1.2 and CATT/T. evansi test) (df = 2, p > 0.05). Earlier studies, reports up to 40% of seroprevalence in eastern India (Laha and Sasmal, 2009), and is in agreement with the present study (38.9%). However, with available data, a varied seroprevalence of 12.2–38.9% was observed from different states of India. Hence a larger panel of samples needs to be studied further for seroprevalence. As per our literature search, this is the first report of prokaryotic expression of VSG of T. evansi and its subsequent use in sero-diagnosis. In conclusion, in the present study we have developed a recombinant VSG antigen in prokaryotic system and thereafter the immunoreactivity of the antigen was evaluated and compared with VSG RoTat 1.2 based ELISA and CATT/T. evansi agglutination test. The latter two antigens were obtained from OIE reference laboratory, Belgium. The comparative study showed that immunoreactivity of rVSG is in agreement with other antigens/test, exhibiting high diagnostic value and the rVSG based ELISA can be used a reliable and perspective tool for serosurveillance to control surra. Acknowledgements The authors are thankful to those who extended cooperation and help in collection of samples. The work was done under the financial support by the Department of Biotechnology (DBT), Government of India (Project number: BT/PR3478/ADV/90/122/2011). References Allen, T.E., Ullman, B., 1993. Cloning and expression of the hypoxanthineguanine phosphoribosyltransferase gene from Trypanosoma brucei. Nucleic Acids Res. 21, 5431–5438. Bajyana Songa, E., Hamers, R., 1988. A card agglutination test (CATT) for veterinary use based on an early VAT Ro Tat 1.2 of Trypanosoma evansi. Ann. Soc. Belg. Med. Trop. 68, 233–240. Borst, P., Fase-Fowler, F., Gibson, W.C., 1987. Kinetoplastid DNA of Trypanosoma evansi. Mol. Biochem. Parasitol. 23, 31–38. Cohen, J., 1960. A coefficient of agreement for nominal scales. Educ. Psychol. Meas. 20, 37–46. Davison, H.C., Thrusfield, M.V., Muharsini, S., Husein, A., Partoutomo, S., Masake, R., Luckins, A.G., 1999. Evaluation of antigen- and antibodydetection tests for Trypanosoma evansi infections of buffaloes in Indonesia. Epidemiol. Infect. 123, 149–155. Diall, O., Nantulya, V.M., Luckins, A.G., Diarra, B., Kouyate, B., 1992. Evaluation of mono- and polyclonal antibody-based antigen detection immunoassays for diagnosis of Trypanosoma evansi infection in the dromedary camel. Rev. Elev. Med. Vet. Pays. Trop. 45, 149–153. Field, M.C., Carrington, M., 2009. The trypanosome flagellar pocket. Nat. Rev. Microbiol 7, 775–786. Freymann, D., Down, J., Carrington, M., Roditi, I., Turner, M., Wiley, D., 1990. 2.9 A˚ Resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma evansi. J. Mol. Biol. 216, 141–160. Gadelha, C., Holden, J.M., Allison, H.C., Field, M.C., Jennifer, M., 2011. Specializations in a successful parasite: what makes the blood stream form African trypanosomes so deadly? Mol. Biochem. Parasitol. 179, 51–58. Gibson, W.C., de, C., Marshall, T.F., Godfrey, D.G., 1980. Numerical analysis of enzyme polymorphism: a new approach to epidemiology and
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