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Flagellar antigen based CI-ELISA for sero-surveillance of surra
Veterinary Parasitology 219 (2016) 17–23
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Short communication
Flagellar antigen based CI-ELISA for sero-surveillance of surra M. Ligi, P.P. Sengupta ∗ , G.R. Rudramurthy, H. Rahman National Institute of Veterinary Epidemiology & Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, P.B. No. 6450, Bengaluru 560064, Karnataka, India
a r t i c l e
i n f o
Article history: Received 17 July 2015 Received in revised form 23 January 2016 Accepted 25 January 2016 Keywords: Surra Flagellar antigen Monoclonal antibody ROC analysis CI-ELISA
a b s t r a c t Trypanosoma evansi causes a disease known as ‘surra’ in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive–negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen’s Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma evansi a haemoflagellated parasite causes a chronic wasting disease called surra. It is considered as an important disease in domestic, wild herbivores and carnivores animals in tropical and sub-tropical countries. Cattle, buffaloes, camels and horses are the most susceptible hosts of surra in south–east Asia (Holland et al., 2004; Desquesnes et al., 2013). T. evansi believed to be originated from Trypanosoma brucei (Lai et al., 2008) is mainly transmitted mechanically by the tabanid flies, in carnivores, the transmission of the disease has also been observed after feeding on infected meat (Raina et al., 1985). The clinical symptoms of trypanosomosis include severe anemia, fever, hypoglycemia, weight loss, poor body weight gain, poor draughtability, infertility, abortion and even death. The calving rate in cows and fertility rate in infected bulls is reduced significantly in the high risk areas of infection (Dargantes et al., 2009). The sub clinical stage of the disease in lactating cows can even cause a significant decrease in milk production (Pholpark et al., 1999). In India a high percentage (12.74%) of T. evansi infection has been reported in horses (Laha and Sasmal, 2008), high mortality rate in young female buffaloes (10%), causes a
∗ Corresponding author. Principal Scientist, Parasitology Laboratory, National Institute of Veterinary Epidemiology & Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, P.B. No. 6450, Bengaluru 560064, Karnataka, India. Fax: +91 80 23093222. E-mail address: pinakiprasad [email protected] (P.P. Sengupta). http://dx.doi.org/10.1016/j.vetpar.2016.01.019 0304-4017/© 2016 Elsevier B.V. All rights reserved.
significant loss in agriculture industry (Dargantes et al., 2009). The animals serve as carriers for the disease by exhibiting low levels of fluctuating parasites for years after recovery. The detection of carrier status of animals is very important in controlling the disease. Acute or sub acute infections can be satisfactorily diagnosed by conventional parasitological techniques, but more difficult in latent or chronic infection where parasitaemia becomes very low. The clinical signs of trypanosomosis in general are indicative but the sub-clinical case must be confirmed by highly sensitive laboratory methods. The development of sensitive serological tests helps in the detection of carrier status of animals. The detection of carrier status and subsequent treatment of the animals lead to effective control of the disease, as well as better production. Several serological [indirect fluorescent antibody test (IFAT) or enzyme linked immunosorbent assay (ELISA), card agglutination test (CATT/T. evansi) etc.] and parasitological (thick or wet blood smear) diagnostic tests have also been developed for detection of trypanosomosis. Several sensitive and specific diagnostic tools such as, nucleic acid detection by PCR (Sengupta et al., 2010; Rudramurthy et al., 2013), serological tests etc., have been developed for the detection of carrier status of the infection. The serological test such as, ELISA is likely to correctly identify healthy animals and qualifies as a universal test as it is not strain specific (OIE, 2012). However, only clinical stages of infections can be diagnosed satisfactorily by parasitological test, but not latent or chronic infection (Fernandez et al., 2009).
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Table 1 Age and species wise sero-prevalence of T. evansi infection. Test→
CI-ELISA
CATT/T.evansi
State
Species
Age
P
N
% Positive
P
N
% Positive
Karnataka
Cattle
0–2 years 2–4 years 4–6 years >6 years 0–2 years 2–4 years 4–6 years 6–8 years >8 years 4–6 years 8–10 years 10–12 years >12 years 4–6 years 6–8 years 8–10 years >10 years
3 11 22 19 5 16 11 7 23 1 1 2 1 2 1 1 2
19 52 41 29 7 22 26 18 37 11 9 31 42 16 9 24 32
13.63 17.46 34.92 39.58 41.66 42.1 29.72 28 38.33 8.33 10 6.06 2.32 11.11 10 4 5.88
3 11 22 20 5 16 10 7 23 1 1 2 1 2 1 1 2
19 52 41 28 7 22 27 18 37 11 9 31 42 16 9 24 32
13.63 17.46 34.92 41.66 41.66 42.1 27.02 28 38.33 8.33 10 6.06 2.32 11.11 10 4 5.88
Buffalo
Horse
Donkey
Odisha
Cattle
0–2 years 2–4 years 4–6 years >6 years
9 16 36 31
19 37 48 39
32.14 30.18 42.85 44.28
9 16 36 31
19 37 48 39
32.14 30.18 42.85 44.28
West-Bengal
Cattle
2–4 years 4–6 years >6 years
1 4 5
5 16 11
16.66 20 31.25
1 4 5
5 16 11
16.66 20 31.25
Rajasthan
Camel
0–2 years 2–4 years 4–6 years 6–8 years 8–10 years >10 years
3 12 15 9 13 24
42 29 58 44 83 68
6.66 29.26 20.54 16.98 13.54 26.08
3 12 15 9 13 25
42 29 58 44 83 67
6.66 29.26 20.54 16.98 13.54 27.17
P = positive, N = negative.
T. evansi is longitudinal and fusiform in appearance; flagellum protrudes from the basal body. The attachment of parasite to the host surfaces is mediated by flagellum (Bastin et al., 2000). The flagellar pocket region can be considered as a privileged site for possible immunological intervention (Radwanska et al., 2000). Gadelha et al. (2005) reported that, the paraflagellar rod protein may be the critical organelle mediating attachment to vector cell surface. Several flagellar pocket-associated proteins have been identified and found to contribute to trafficking and virulence (Field and Carrington. 2009). Moreover, these proteins are involved in the binding of macromolecules, interactions with the host and also elicit host antibody response. Further, several genes such as, paraflagellar rod protein gene 1 (PFR1) and paraflagellar rod protein gene 2 (PFR2) have been cloned and expressed in prokaryotic system (Maharana et al., 2011a,b). The, flagellar pocket antigens and paraflagellar rod protein have been explored in the development of vaccine and diagnostic test respectively (Mkunza et al., 1995; Obishakin et al., 2014). Earlier studies report the development of monoclonal antibody against crude cell membrane protein (Rayulu et al., 2007). The present study is aimed at exploring native flagellar antigen (FLA) in the development of diagnostic ELISA. In this study the FLA was isolated from the purified trypanosomes followed by characterization of the protein by SDS-PAGE analysis, immunoblot and indirect ELISA. Further, the monoclonal antibodies (MAbs) were raised against native crude FLA; subsequently the MAbs were characterized and explored in the development of CI-ELISA for diagnosis of surra.
2. Materials and methods 2.1. T. evansi stabilates, antigens preparation, and serum samples The buffalo isolate of T. evansi, maintained in the Parasitology laboratory, NIVEDI, Bengaluru, India was used in the present study. The parasites were propagated and purified (Sengupta et al., 2010, 2012, 2014; Rudramurthy et al., 2013, 2015) to isolate the FLA antigen. The FLA antigen was prepared by following the protocol mentioned earlier (Purohit et al., 1987). The whole cell lysate (WCL) antigen was prepared from the purified trypanosomes as mentioned earlier (Sengupta et al., 2012) for comparative evaluation study along with VSG RoTat 1.2 antigen and CATT/T. evansi test. VSG RoTat 1.2 antigen was obtained from Koning Leopold Institute of Tropical Medicine, Antwerp, Belgium (OIE reference laboratory of surra) and used as per manufacturer’s instructions. The CATT/T. evansi kit uses freeze dried trypanosomes of T. evansi VSG RoTat 1.2 (Bajyana Songa and Hamers, 1988; Verloo et al., 2001). Hyperimmune/immune sera raised in rabbit/bovine/buffalo (Sengupta et al., 2012) and available in our laboratory, were used in the present study. While, hyperimmune serum against purified FLA antigen was raised in rat by following the standard protocol (Sengupta et al., 2012). The experimental animals were handled as per the standards of animal ethics; feed and drinking water were given ad libitum. The sera sample (1230) from the field were collected randomly between the period 2012–2015 across different states of India (Table 1) were screened for trypanosomosis in ELISA, CATT/T. evansi and CI- ELISA.
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2.2. SDS PAGE, immunoblot and indirect ELISA FLA antigen (120 g) and WCL antigen (130 g) were loaded into SDS-PAGE and subjected to electrophoresis. The electrophoresed gel was stained with PAGE blue staining solution as per the manufacturer’s instructions (Invitrogen). For immunoblotting, the electrophoresed proteins were transferred onto nitrocellulose membrane by following the standard protocol and then the membrane was treated with primary antibody using hyper immune/immune sera (1:100 dilution) raised in rat/rabbits/bovine/buffalo (Sengupta et al., 2012) for determining the immunoreactivity of FLA antigen. Later the membrane was treated with secondary antibody conjugated with horse radish peroxidase (1:1000 dilution). The substrate solution containing diamino benzidine tetrahydrochloride (DAB) was added finally to develop the immunoblot (Sambrook and Russell, 2001). Further, immunoreactivity of the FLA antigen was determined using ELISA. For ELISA, the micro titer plates (maxisorp® , nunc) were coated overnight at 4 ◦ C with 100 l/well of purified FLA antigen (200 ng/well)/WCL (150 ng/well)/VSG Ro Tat 1.2 (600 ng/well) in PBS (pH 7.2). After overnight incubation the microtitre plates were washed four times with washing buffer [0.25% (v/v) Tween20 in PBS, pH 7.2] and blocked with 150 l/well blocking buffer (3% skimmed milk powder and 0.05% Tween-20 in PBS) for 1 h at 37 ◦ C and washed. The rat/rabbit/bovine hyperimmune sera, buffalo immune sera and field/herd [cattle (25), buffalo (18), camel (23), horse (10) and donkey (5)] serum samples were diluted (1:100) with half strength blocking buffer and added (100 l/well) followed by incubation for 1 h at 37 ◦ C. After washing, the respective secondary antibody such as, anti-rat IgG (for rat sera), anti-rabbit IgG (for rabbit sera), anti-bovine IgG (for buffalo and cattle sera), anti horse IgG (for horse and donkey sera) and protein G (for camel sera) conjugated with horse radish peroxidase [diluted as per manufacturer’s instruction (sigma)] was added (100 l/well) and incubated for 1 h at 37 ◦ C. The microtitre plates were then washed and 100 l/well enzyme substrate (chromogenic) solution (5 mg of O-phenylene diamine dihydrochloride (sigma) and 0.03% (v/v) H2 O2 ) was added to develop the color. The reaction was stopped by adding 1 M H2 SO4 (100 l/well) and O.D was recorded at 492 nm in an ELISA reader (Bench mark microplate reader, BioRad). Further, the specificity of FLA antigen was determined with cattle serum samples clinically infected with Theileria annulata and Babesia bigemina (diagnosed by blood smear examination). The field serum samples were subjected to ELISA in duplicate using FLA antigen, VSG RoTat 1.2 antigen, CATT/T. evansi for comparative evaluation. 2.3. Production of MAbs 2.3.1. Immunization of Balb/c mice and preparation of splenocytes For immunization process, two Balb/c mice (8–12 weeks old) were used and other two Balb/c mice were maintained as healthy control. Primary immunization was given with emulsified FLA antigen (200 g/mice), followed by 1st booster injection and second booster injection (200 g/mice) 21 days after primary injection and first booster injection respectively subcutaneously. The third booster injection (1000 g/mice) was given 3–4 days before the fusion in saline intraperitoneally. The immunized mice were euthanized and washed with 70% alcohol. From it, spleen was taken and transferred aseptically into a sterile petri dish containing 5 ml Iscove’s Modified Dulbecco’s Medium (IMDM) (Hyclone) without supplements. The medium was flushed gently into spleen with the help of 5 ml glass syringe attached with 26G needle. The splenocytes were spun down at 214 g for 5 min at room temperature. The pellet was distributed with 10 ml IMDM by inverting the tubes
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gently and the splenocytes were counted in haemocytometer by diluting the aliquot of suspension in RBC lysis buffer. Simultaneously, for positive control, the blood was collected from the mice and serum was separated and used as the positive control. 2.3.2. Preparation of B-cell myeloma and feeder layer B cell myeloma such as, SP2/0 (Shulman et al., 1978) which are HAT sensitive (HGPRT−/TK− and non-Ig secretor) were used in the present study. The SP2/O cells were grown to log phase in IMDM supplemented with, 15% fetal bovine serum (FBS) (Hyclone) and 20 mM l-glutamate and harvested by centrifugation at 214 × g for 5 min at room temperature. The pellet was washed (twice), resuspended with IMDM (without any supplements) and cells were counted. For macrophage preparation, non-immunized BALB/c mouse was euthanized, and washed with 70% alcohol and was taken into laminar flow hood. Without damaging the abdominal muscle, the skin was cut open and 10 ml of pre-cooled IMDM was injected into abdomen using 10 ml glass syringe fitted with 18G needle. Abdomen was tapped by holding the needle in place to dislodge the cells adhered to the abdominal cavity and then the medium was retracted into the syringe (peritoneal exudates). The obtained peritoneal exudate, enriched with macrophages was kept on ice in a polypropylene tube and the cells were counted. Meanwhile, the blood was also collected from the mice; serum was separated and used as a negative control serum. 2.3.3. Production of hybridoma Twenty million splenocytes and 2 million myeloma (SP2/O) cells (10:1) were taken in a screw capped tube containing 10 ml of IMDM for hybridoma production. These cells were mixed and spun at 214 g for 10 min at room temperature. The residual medium was removed using sterile gauze, and the cell pellet was used for hybridoma production. Five hundred microlitre of polyethelene glycol (PEG) and dimethyl sulphoxide (DMSO) mixture (Sigma–Aldrich) was added within one minute to the cell pellet drop wise (cell pellet was disrupted after each drop) and exposed for another one minute. Five milliliter of IMDM (without any supplementation) was added first 1 ml over minute, drop wise to the tube slowly after one minute incubation. The cells were incubated in the CO2 (5%) incubator for 45 min and were pelleted at 214 g for 10 min at room temperature and spun at 214 g for 10 min at room temperature. The cell pellet obtained was suspended in IMDM, supplemented with 0.1 mM sodium hypoxanthine (H), 0.4 M aminopterine (A) and 0.016 mM thymidine (T) (Gibco® Life technologies), 15% fetal bovine serum (FBS) and 20 mM l-glutamate. The cells were plated out in a 96 well culture plate (0.2 ml per well), containing feeder layer/macrophages (4 × 106 cells/96 well plate). Controls were maintained in two wells containing only SP2/O) and macrophages. The plates were then incubated in CO2 (5%) incubator at 37 ◦ C for 7 days. On the 7th day, the medium was replaced with IMDM, supplemented with 0.1 mM hypoxanthine, 0.016 mM thymidine (Gibco, Life technologies), 15% FBS and 20 mM l-glutamate and were kept in CO2 incubator. 2.3.4. Screening of hybridoma clones and selection of monoclones By indirect ELISA, the clones appeared in the plates were screened on the 10th day for antibody production. The positive clones obtained were expanded by transferring the clones into 500 l cultures, 1 ml cultures, and finally into 5 ml culture flasks. In each expansion step, to screen positive clones, indirect ELISA was carried out in duplicate (as described in Section 2.2). The positive clones obtained were preserved in IMDM containing FBS (40%) and DMSO (10%) at −80 ◦ C and also in liquid nitrogen till further use. By dilution cloning technique from the positive hybridoma, the monoclones were selected and the hybridoma cells were counted and
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Fig. 1. Analysis of crude flagellar antigen by SDS-PAGE and Immunoblot. (a) SDS PAGE analysis: Lane 1 = Purified FLA antigen, Lane 2 = WCL antigen, Lane M = Prestained protein ladder, (b) Immunoblot analysis: Lane M = Prestained protein ladder, lanes 1 and 2 = MAb versus FLA antigen and MAb versus SP2/O cells culture supernatant.
100 cells were suspended in 20 ml IMDM (with all supplements) and plated out in 96 wells culture plate (∼one cell/well). The plate was incubated in CO2 (5%) incubator till the appearance of clones. The wells containing the single clone (monoclone) were screened by indirect ELISA (Section 2.2), expanded further and preserved at −80 ◦ C and also in liquid nitrogen in IMDM containing FBS (40%) and DMSO (10%) till further use. Later, the MAbs were produced in larger scale in 250 ml culture flasks and the cells were grown till the IMDM color changes to yellow and further tested by indirect ELISA (>1.2 OD). The supernatant rich in MAbs was collected by centrifugation at 857 × g for 3 min and preserved at −80 ◦ C till further use.
2.4. Characterization of MAbs 2.4.1. Immunoblot To characterize the MAbs, the immunoblot analysis was carried out by following the standard protocol (Sambrook and Russell, 2001). Here, the electrophoresed proteins (FLA antigen and control antigens) were transferred on to nitrocellulose membrane by following the standard protocol. The membrane was then treated with MAbs and SP2/O clones (undiluted culture supernatant). The substrate solution containing diamino benzidine tetrahydrochloride (DAB) was added finally to develop the immunoblot.
2.4.2. Isotyping Mouse MAb was isotyped using mouse isotyping (IgG1, IgG2a, IgG2b, IgG3, IgA and IgGM) antibodies (Sigma–Aldrich) by antigen mediated ELISA as per manufacturer’s instructions. For antigen mediated ELISA, microtitre plates (Maxisorp® , Nunc) were coated with 100 l/well of FLA antigen (200 ng/well) for overnight at 4 ◦ C. After overnight incubation the plate was washed four times with washing buffer (Section 2.2) and the undiluted MAb (culture supernatant from monoclone) was added into each well (100 l/well) and incubated for 2 h at 37 ◦ C. The plate was then washed and isotyping antibodies (100 l/well) were added and incubated at 37 ◦ C for 1 h. After washing anti goat IgG was added in each well (100 l/well) and incubated for 30 min at 37 ◦ C, followed by washing and addition of 100 l/well substrate solution (Section 2.2). The plate was then incubated for 30 min to develop the color and then read in ELISA reader at 492 nm.
2.5. CI-ELISA By checker board titration, the optimum concentration of FLA antigen, serum dilution and MAb dilution was determined. Microtitre plates (Maxisorp® , Nunc were coated with purified FLA 400 ng/well) overnight at 4 ◦ C in PBS (pH 7.2). The microtitre plates were washed and blocked after overnight incubation. The test serum samples (20 l of test serum, 20 l of blocking buffer and 60 l MAb) was added in duplicate and incubated at 37 ◦ C for 1 h on ELISA shaker followed by washing. The test was included with strongly positive (>90% competition), weakly positive (60–70% competition), negative, MAb (0% competition) and conjugate control wells simultaneously. Hundred microlitre of antimouse antibody horseradish peroxidase conjugate (diluted as per manufacturer’s instruction) was added to each well and incubated at 37 ◦ C for 1 h followed by washing and addition of substrate solution (100 l/well). The plates were incubated till the visible color develops in the monoclonal antibody control wells and then the reaction was stopped by the addition of (100 l/well) stopping solution. The plates were then read at 492 nm in an ELISA reader (Section 2.2). The OD values obtained were converted to percentage inhibition (PI) values by using the formula: PI = 100−{(OD in test well/OD in Cm well) × 100} (Singh et al., 2004).
2.5.1. Determination of optimum antigen concentration, MAb and serum dilutions For the optimization of antigen concentration, MAbs and serum dilutions, the buffalo immune serum raised against T. evansi (buffalo isolate) and healthy buffalo serum were selected. The different combination of antigen concentration, serum dilution and MAb dilution was tested and has been established that FLA antigen reacted well with undiluted/neat MAb and 1:100 diluted immune/hyperimmune serum without background activity at 200 ng/well in indirect ELISA. Hence, initial antigen concentration, serum dilution and MAb dilution was chosen at 200 ng/well, 5 l/well and 40 l/well respectively. The concentration/dilution was increased two fold up to 600 ng/well (antigen), 35 l/well (serum) and 80 l/well (MAb). The optimum concentration/dilution of each reagent was found to be the maximum difference in PI value between positive and negative at highest dilution. The optimum dilution of rat hyper immune serum raised against FLA antigen, bovine hyper immune serum raised against
M. Ligi et al. / Veterinary Parasitology 219 (2016) 17–23
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Fig. 2. ROC (receiver operating characteristic) graph depicting diagnostic sensitivity and specificity of CI-ELISA.
T. evansi (buffalo isolate) with respective healthy serum was also determined simultaneously.
2.5.2. Statistical analysis Receiver operating characteristic (ROC) analysis was carried out to determine the cut off PI value and optimum combination of diagnostic specificity and sensitivity of FLA antigen in ELISA using cattle, buffalo, camel, horse and donkey sera by keeping CATT/T. evansi as gold standard test. An agreement of FLA antigen in relation to CATT/T. evansi was determined for detecting antibody against T. evansi. A trial version of software MedCalc (version 12.7.2, USA) was used to generate ROC curve. The Cohen’s kappa (Cohen, 1960) value were determined using on line version of Kappa–VassarStats (cited at: www.vassarstats.net/kappa.html) software. A group of serum samples collected from cattle, buffaloes, horse, donkey and camels from four different states of India were subjected to new CI-ELISA and CATT/T. evansi for comparative analysis. The percentage prevalence of surra in different species (age wise) across different states of India was also carried out. Further, the significance of the test was determined by Chi square (2) analysis (Snedecor and Cochran 1968) of the epidemiological data (species and age wise) obtained from both the tests. The specificity of CI-ELISA was also evaluated with cattle serum samples clinically infected with T. annulata and B. bigemina.
3.2. Production and characterization of MAbs The three clones which showed very high reactivity with FLA antigen by indirect ELISA (OD > 1.4) were selected, while, the control clones (SP2/0, spleen and macrophages) showed lesser O.D values (<0.2). Immunoblot analysis revealed that the MAb was highly specific and reacted with FLA antigen. However, immunoreactivity of MAb was not observed with control proteins (Fig. 1b). Isotyping analysis revealed that MAbs belong to isotype of IgG, subtype IgG3 and is used in the development of CI-ELISA. 3.3. Diagnostic sensitivity and specificity of CI- ELISA versus CATT/T. evansi
3. Results
The optimal concentration of FLA antigen, buffalo immune serum and MAb was found to be 400 ng/well, 35 l/well and 80 l/well respectively. The maximum difference in the PI value between rat/bovine hyperimmune serum versus (v/s) control serum was found to 15 l/well, while between buffalo immune serum v/s control serum was found to 20 l/well. Hence, for the development of CI-ELISA, 20 l/well serum was chosen as an optimal value, since the field serum samples were obtained as immune serum samples. The statistical analysis of CI-ELISA showed positive–negative cut off PI value was found to be >55. The optimum combination of diagnostic sensitivity and specificity was found to be respectively at 95.8 and 94.4 at >55 cut-off PI value (Fig. 2), with a Cohen’s kappa coefficient of agreement value of 0.83.
3.1. Characterization and immunoreactivity of FLA antigen
3.4. Sero- prevalence of T. evansi
In SDS-PAGE analysis, nine bands were obtained in the flagellar portion between 18 to 130 kDa at 18, 20, 30, 50, 55, 60, 75, 90 and 130 kDa. FLA was analyzed by SDS-PAGE (Fig. 1a) and its immunogenicity was tested by immunoblot (Fig. 1b). The FLA antigen and control antigens remained respectively reactive and non reactive in ELISA. The FLA antigen showed high reactivity by indirect ELISA (O.D > 1.2) with hyperimmune sera, while the control sera showed low O.D values of <0.2. The FLA antigen remained non reactive with serum samples clinically infected with T. annulata and B. bigemina.
The statistical analysis of the CI-ELISA data by keeping variable such as, species and age revealed that, the disease is more prevalent among adult cattle in Karnataka (>4 years, 2 = 0.09, df = 1, p > 0.05) and West Bengal (>6 years, 2 = 0.00, df = 1, p > 0.05). In Karnataka, calves (up to 4 years, 2 = 0.00, df = 1, p > 0.05) and aged buffaloes (>8 years, 2 = 0.00, df = 1, p < 0.05) were found more sero-positive than middle aged cattle. The disease prevalence in horse and donkey was also found to be more or less equal among the different age groups. However, in cattle, all age group were almost equally seropositive in Odisha. Camel samples from Rajasthan revealed that the
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calves (<2 years, 2 = 0.00, df = 1, p > 0.05) are less susceptible to T. evansi infection than adults (>2 years, 2 = 0.009, df = 1, p > 0.05). The comparative analysis of CI-ELISA with CATT/T. evansi showed that the new assay is in agreement with the standard test. The age wise prevalence of disease in different species from different states of India is shown inTable 1.
4. Discussion The flagellar membrane mediates attachment of parasite to host tissues and harbors multiple virulence factors. The flagellum also mediates host parasite interactions (Langousis and Hill, 2014). Earlier, Rayulu et al. (2009) developed one MAb (MAb engineered against cell membrane antigen) based sensitive and rapid latex agglutination test (MAb LAT) to detect the circulating antigens of Indian isolate of T. evansi in domestic animals. In the present study, competitive inhibition ELISA has been developed exploring monoclonal antibodies against purified FLA antigen. SDS PAGE analysis of purified FLA protein showed nine bands in SDS-PAGE ranging from 18 to 130 kDa. Earlier a varying range of 4–12 protein bands were observed in different isolates of which 58.7 (∼60) kDa was also present in some isolates. Earlier study showed that even in some geographical regions also the polypeptide bands were varied in different isolates (Singh et al., 1995). The purified FLA protein remained non- reactive in ELISA with T. annulata and B. bigemina infected serum. Apart from this, the new CI-ELISA test also remained non-reactive with such sera. In ELISA and Western blot analysis, both the MAb showed immunoreactivity against purified flagellar protein. The purified FLA protein remained immunoreactive with the panel of sera samples consisting of experimentally produced/field samples including different host species namely cattle, buffalo, camel, horse and donkey. Besides, CI-ELISA test remained reactive with experimentally produced T .evansi buffalo immune sera, bovine hyperimmune sera, and rat immune sera. On comparison with CATT/T. evansi test, new CI-ELISA test emerged as sensitive (95.8%) and specific (94.4%,) with a Cohen’s kappa value of 0.83. The developed assay detected T. evansi antibodies from different host species such as, cattle, buffalo, camel, horse and donkey. The statistical analysis of the epidemiological data (species and age wise) revealed no significant variation in both the test systems i.e., CI-ELISA and CATT/T. evansi. The cattle samples clinically infected with T. annulata and B. bigemina did not react in CI-ELISA. Thus, CI-ELISA incorporating MAbs turns out to be a potent diagnostic tool for the detection of the antibodies of T. evansi. However, it needs to test with the antibodies of other flagellated protozoa for checking cross-reaction. The seroprevalence study revealed that the prevalence of disease is observed more in adult cattle (up to 44%, 2 = 0.0, df = 1, p > 0.05) compared to calves. Besides, T. evansi infection was found to be more in buffalo calves than adults (Table 1). The incidence of T. evansi infection in camels, horse and donkey was found to be more or less equal among different age groups. However, a large number of horse and donkey serum samples need to be tested for further conclusion. Though cattle and buffalo sera are collected from the same location/state (Karnataka) the variation in the disease incidence among different age groups is observed between cattle and buffalo. The present study provides preliminary information to carry out for further study in determining the risk factors associated with different age groups and species. The comparative analysis revealed that the new assay is comparable with CATT/T. evansi. The authors expect that the new ELISA test in the present study can be employed for a mass level screening for effective control of trypanosomosis. However, further evaluation of the test for its
cross reactivity with other species and organisms is needed before mass screening of animals. Acknowledgements The present study is a part of the Ph.D. work of the senior author registered under Jain University, Bengaluru, India. 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).The authors are thankful to the concerned PIs of RRUs of AICRP on ADMAS and those who help in collection of samples. The help in statistical analysis by Dr. K.P. Suresh, Scientist, NIVEDI, is thankfully acknowledged. References 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. Bastin, P., Pullen, T.J., Moreira-Leita, F.F., Gull, K., 2000. Inside and outside of the trypanosome flagellum: a multifunctional organelle. Microbes Infect. 2, 1865–1874. Cohen, J., 1960. A coefficient of agreement for nominal scales. Educ. Psychol. Meas. 20, 37–46. Dargantes, A.P., Mercado, R.T., Dobson, R.J., Reid, S.A., 2009. Estimating the impact of Trypanosoma evansi infection (surra) on buffalo population dynamics in southern Philippines using data from cross-sectional surveys. Int. J. Parasitol. 39, 1109–1114. Desquesnes, M., Holzmuller, P., Lai, D.H., Dargantes, A., Lun, Z.R., Jittaplapong, S., 2013. Trypanosoma evansi and Surra: a review and perspectives on origin, history, distribution, taxonomy, morphology, hosts, and pathogenic effects. BioMed Res. Int. 2013, 22 Article ID 194176. Fernandez, D., Gonzalez-Baradat, B., Eleizalde, M., Gonzalez-Marcano, E., Perrone, T., Mendoza, M., 2009. Trypanosoma evansi: a comparison of PCR and parasitological diagnostic tests in experimentally infected mice. Exp. Parasitol. 121, 1–7. Field, M.C., Carrington, M., 2009. The trypanosome flagellar pocket. Nat. Rev. Microbiol. 7, 775–786. Gadelha, C., Wickstead, B., de Souza, W., Gull, K., Cunha-e-Silva, N., 2005. Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa. Eukaryot. Cell 4, 516–525. 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., 2008. Endemic status of Trypanosoma evansi infection in a horse stable of eastern region of India- a field investigation. Trop. Anim. Health Prod. 40, 357–361. Lai, D.H., Hashimi, H., Lun, Z.R., Ayalag, F.J., Lukes, J., 2008. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc. Natl. Acad. Sci. U. S. A. 105, 1999–2004. Langousis, G., Hill, K.L., 2014. Motility and more: the flagellum of Trypanosoma brucei. Nat. Rev. Microbiol. 12, 505–518. Maharana, B.R., Rao, J.R., Tewari, A.K., Singh, Harkirat, 2011a. Isolation and characterization of paraflagellar rod protein gene (PFR 1) in Trypanosoma evansi and its conservation among their kinetoplastid parasites. Indian J. Anim. Res. 45, 283–288. Maharana, B.R., Rao, J.R., Tewari, A.K., Singh, Harkirat, 2011b. Cloning and expression of paraflagellar ROD protein 2 (PFR 2) gene of Trypanosoma evansi. J. Vet. Parasitol. 25, 118–123. Mkunza, F., Olaho, W.M., Powell, C.N., 1995. Partial protection against natural trypanosomiasis after vaccination with a flagellar pocket antigen from Trypanosoma brucei rhodesiense. Vaccine 13, 151–154. Obishakin, E., Stijlemans, B., Santi-Rocca, J., Vandenberghe, I., Devreese, B., Muldermans, S., Bastin, P., Magez, S., 2014. Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes. PLoS One 9 (12), http://dx.doi. org/10.1371/journal.pone.0115893, e115893. OIE, 2012. Trypanosoma evansi infection (surra). In: OIE Terrestrial Manual 2012. World Organization for Animal Health, Paris, cited at: http://www.oie. inthttp://www.oie.int. Pholpark, S., Pholpark, M., Polsar, C., Paengpassa, Y., Kashiwazaki, Y., 1999. Influence of Trypanosoma evansi on milk yield of dairy cattle in northeast Thailand. Prev. Vet. Med. 42, 39–44. Purohit, S.K., Jatkar, P.R., Lodha, K.R., 1987. Isolation of flagella and cell membrane of Trypanosoma evansi. Indian Vet. J. 64, 18–20. Radwanska, M., Magez, S., Dumont, N., Pays, A., Nolan, D., Pays, E., 2000. Antibodies raised against the flagellar pocket fraction of Trypanosoma brucei preferentially recognizer HSP60 in c DNA expression library. Parasite Immunol. 22, 639–650. Raina, A.K., Kumar, R., Rajora, V.S., Sridhar, S.R.P., 1985. Oral transmission of Trypanosoma evansi infection in dogs and mice. Vet. Parasitol. 18, 67–69.
M. Ligi et al. / Veterinary Parasitology 219 (2016) 17–23 Rayulu, V.C., Singh, A., Chaudhri, S.S., 2007. Monoclonal antibody based immunoassays for the detection of circulating antigens of Trypanosoma evansi in buffaloes. Ital. J. Anim. Sci. 6, 907–910. Rayulu, V.C., Chaudhri, S.S., Singh, A., 2009. Evaluation of parasitological and monoclonal antibody assays in detection of Trypanosoma evansi. Indian J. Anim. Sci. 7, 978–981. 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. Rudramurthy, G.R., Sengupta, P.P., Metilda, B., Balamurugan, V., Prabhudas, K., Rahman, H., 2015. Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis. Indian J. Exp. Biol. 53, 7–15. 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 immuno-reactivity. Vet. Parasitol. 187, 1–8.
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Sengupta, P.P., Rudramurthy, G.R., Ligi, M., Roy, M., Balamurugan, V., Krishnamoorthy, P., Nagalingam, M., Singh, L., Rahman, H., 2014. Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance. Vet. Parasitol. 205, 490–498. Shulman, M., Wilde, C.D., Köhler, G., 1978. A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269–270. Singh, V., Singh, A., Chhabra, M.B., 1995. Polypeptide profiles and antigenic characterization of cell membrane and flagellar preperations of different stocks of Trypanosoma evansi. Vet. Parasitol. 56, 269–279. Singh, R.P., Sreenivasa, B.P., Dhar, P., Shah, L.C., Bandyopadhyay, S.K., 2004. Development of a monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus. Vet. Microbiol. 98, 3–15. Snedecor, G.W., Cochran, W.G., 1968. Statistical Methods Indian Edition. Oxford & IBH Publishing Co., New Delhi, pp. 1–593. 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.
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Short communication
Flagellar antigen based CI-ELISA for sero-surveillance of surra M. Ligi, P.P. Sengupta ∗ , G.R. Rudramurthy, H. Rahman National Institute of Veterinary Epidemiology & Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, P.B. No. 6450, Bengaluru 560064, Karnataka, India
a r t i c l e
i n f o
Article history: Received 17 July 2015 Received in revised form 23 January 2016 Accepted 25 January 2016 Keywords: Surra Flagellar antigen Monoclonal antibody ROC analysis CI-ELISA
a b s t r a c t Trypanosoma evansi causes a disease known as ‘surra’ in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive–negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen’s Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma evansi a haemoflagellated parasite causes a chronic wasting disease called surra. It is considered as an important disease in domestic, wild herbivores and carnivores animals in tropical and sub-tropical countries. Cattle, buffaloes, camels and horses are the most susceptible hosts of surra in south–east Asia (Holland et al., 2004; Desquesnes et al., 2013). T. evansi believed to be originated from Trypanosoma brucei (Lai et al., 2008) is mainly transmitted mechanically by the tabanid flies, in carnivores, the transmission of the disease has also been observed after feeding on infected meat (Raina et al., 1985). The clinical symptoms of trypanosomosis include severe anemia, fever, hypoglycemia, weight loss, poor body weight gain, poor draughtability, infertility, abortion and even death. The calving rate in cows and fertility rate in infected bulls is reduced significantly in the high risk areas of infection (Dargantes et al., 2009). The sub clinical stage of the disease in lactating cows can even cause a significant decrease in milk production (Pholpark et al., 1999). In India a high percentage (12.74%) of T. evansi infection has been reported in horses (Laha and Sasmal, 2008), high mortality rate in young female buffaloes (10%), causes a
∗ Corresponding author. Principal Scientist, Parasitology Laboratory, National Institute of Veterinary Epidemiology & Disease Informatics (NIVEDI), Ramagondanahalli, Yelahanka, P.B. No. 6450, Bengaluru 560064, Karnataka, India. Fax: +91 80 23093222. E-mail address: pinakiprasad [email protected] (P.P. Sengupta). http://dx.doi.org/10.1016/j.vetpar.2016.01.019 0304-4017/© 2016 Elsevier B.V. All rights reserved.
significant loss in agriculture industry (Dargantes et al., 2009). The animals serve as carriers for the disease by exhibiting low levels of fluctuating parasites for years after recovery. The detection of carrier status of animals is very important in controlling the disease. Acute or sub acute infections can be satisfactorily diagnosed by conventional parasitological techniques, but more difficult in latent or chronic infection where parasitaemia becomes very low. The clinical signs of trypanosomosis in general are indicative but the sub-clinical case must be confirmed by highly sensitive laboratory methods. The development of sensitive serological tests helps in the detection of carrier status of animals. The detection of carrier status and subsequent treatment of the animals lead to effective control of the disease, as well as better production. Several serological [indirect fluorescent antibody test (IFAT) or enzyme linked immunosorbent assay (ELISA), card agglutination test (CATT/T. evansi) etc.] and parasitological (thick or wet blood smear) diagnostic tests have also been developed for detection of trypanosomosis. Several sensitive and specific diagnostic tools such as, nucleic acid detection by PCR (Sengupta et al., 2010; Rudramurthy et al., 2013), serological tests etc., have been developed for the detection of carrier status of the infection. The serological test such as, ELISA is likely to correctly identify healthy animals and qualifies as a universal test as it is not strain specific (OIE, 2012). However, only clinical stages of infections can be diagnosed satisfactorily by parasitological test, but not latent or chronic infection (Fernandez et al., 2009).
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M. Ligi et al. / Veterinary Parasitology 219 (2016) 17–23
Table 1 Age and species wise sero-prevalence of T. evansi infection. Test→
CI-ELISA
CATT/T.evansi
State
Species
Age
P
N
% Positive
P
N
% Positive
Karnataka
Cattle
0–2 years 2–4 years 4–6 years >6 years 0–2 years 2–4 years 4–6 years 6–8 years >8 years 4–6 years 8–10 years 10–12 years >12 years 4–6 years 6–8 years 8–10 years >10 years
3 11 22 19 5 16 11 7 23 1 1 2 1 2 1 1 2
19 52 41 29 7 22 26 18 37 11 9 31 42 16 9 24 32
13.63 17.46 34.92 39.58 41.66 42.1 29.72 28 38.33 8.33 10 6.06 2.32 11.11 10 4 5.88
3 11 22 20 5 16 10 7 23 1 1 2 1 2 1 1 2
19 52 41 28 7 22 27 18 37 11 9 31 42 16 9 24 32
13.63 17.46 34.92 41.66 41.66 42.1 27.02 28 38.33 8.33 10 6.06 2.32 11.11 10 4 5.88
Buffalo
Horse
Donkey
Odisha
Cattle
0–2 years 2–4 years 4–6 years >6 years
9 16 36 31
19 37 48 39
32.14 30.18 42.85 44.28
9 16 36 31
19 37 48 39
32.14 30.18 42.85 44.28
West-Bengal
Cattle
2–4 years 4–6 years >6 years
1 4 5
5 16 11
16.66 20 31.25
1 4 5
5 16 11
16.66 20 31.25
Rajasthan
Camel
0–2 years 2–4 years 4–6 years 6–8 years 8–10 years >10 years
3 12 15 9 13 24
42 29 58 44 83 68
6.66 29.26 20.54 16.98 13.54 26.08
3 12 15 9 13 25
42 29 58 44 83 67
6.66 29.26 20.54 16.98 13.54 27.17
P = positive, N = negative.
T. evansi is longitudinal and fusiform in appearance; flagellum protrudes from the basal body. The attachment of parasite to the host surfaces is mediated by flagellum (Bastin et al., 2000). The flagellar pocket region can be considered as a privileged site for possible immunological intervention (Radwanska et al., 2000). Gadelha et al. (2005) reported that, the paraflagellar rod protein may be the critical organelle mediating attachment to vector cell surface. Several flagellar pocket-associated proteins have been identified and found to contribute to trafficking and virulence (Field and Carrington. 2009). Moreover, these proteins are involved in the binding of macromolecules, interactions with the host and also elicit host antibody response. Further, several genes such as, paraflagellar rod protein gene 1 (PFR1) and paraflagellar rod protein gene 2 (PFR2) have been cloned and expressed in prokaryotic system (Maharana et al., 2011a,b). The, flagellar pocket antigens and paraflagellar rod protein have been explored in the development of vaccine and diagnostic test respectively (Mkunza et al., 1995; Obishakin et al., 2014). Earlier studies report the development of monoclonal antibody against crude cell membrane protein (Rayulu et al., 2007). The present study is aimed at exploring native flagellar antigen (FLA) in the development of diagnostic ELISA. In this study the FLA was isolated from the purified trypanosomes followed by characterization of the protein by SDS-PAGE analysis, immunoblot and indirect ELISA. Further, the monoclonal antibodies (MAbs) were raised against native crude FLA; subsequently the MAbs were characterized and explored in the development of CI-ELISA for diagnosis of surra.
2. Materials and methods 2.1. T. evansi stabilates, antigens preparation, and serum samples The buffalo isolate of T. evansi, maintained in the Parasitology laboratory, NIVEDI, Bengaluru, India was used in the present study. The parasites were propagated and purified (Sengupta et al., 2010, 2012, 2014; Rudramurthy et al., 2013, 2015) to isolate the FLA antigen. The FLA antigen was prepared by following the protocol mentioned earlier (Purohit et al., 1987). The whole cell lysate (WCL) antigen was prepared from the purified trypanosomes as mentioned earlier (Sengupta et al., 2012) for comparative evaluation study along with VSG RoTat 1.2 antigen and CATT/T. evansi test. VSG RoTat 1.2 antigen was obtained from Koning Leopold Institute of Tropical Medicine, Antwerp, Belgium (OIE reference laboratory of surra) and used as per manufacturer’s instructions. The CATT/T. evansi kit uses freeze dried trypanosomes of T. evansi VSG RoTat 1.2 (Bajyana Songa and Hamers, 1988; Verloo et al., 2001). Hyperimmune/immune sera raised in rabbit/bovine/buffalo (Sengupta et al., 2012) and available in our laboratory, were used in the present study. While, hyperimmune serum against purified FLA antigen was raised in rat by following the standard protocol (Sengupta et al., 2012). The experimental animals were handled as per the standards of animal ethics; feed and drinking water were given ad libitum. The sera sample (1230) from the field were collected randomly between the period 2012–2015 across different states of India (Table 1) were screened for trypanosomosis in ELISA, CATT/T. evansi and CI- ELISA.
M. Ligi et al. / Veterinary Parasitology 219 (2016) 17–23
2.2. SDS PAGE, immunoblot and indirect ELISA FLA antigen (120 g) and WCL antigen (130 g) were loaded into SDS-PAGE and subjected to electrophoresis. The electrophoresed gel was stained with PAGE blue staining solution as per the manufacturer’s instructions (Invitrogen). For immunoblotting, the electrophoresed proteins were transferred onto nitrocellulose membrane by following the standard protocol and then the membrane was treated with primary antibody using hyper immune/immune sera (1:100 dilution) raised in rat/rabbits/bovine/buffalo (Sengupta et al., 2012) for determining the immunoreactivity of FLA antigen. Later the membrane was treated with secondary antibody conjugated with horse radish peroxidase (1:1000 dilution). The substrate solution containing diamino benzidine tetrahydrochloride (DAB) was added finally to develop the immunoblot (Sambrook and Russell, 2001). Further, immunoreactivity of the FLA antigen was determined using ELISA. For ELISA, the micro titer plates (maxisorp® , nunc) were coated overnight at 4 ◦ C with 100 l/well of purified FLA antigen (200 ng/well)/WCL (150 ng/well)/VSG Ro Tat 1.2 (600 ng/well) in PBS (pH 7.2). After overnight incubation the microtitre plates were washed four times with washing buffer [0.25% (v/v) Tween20 in PBS, pH 7.2] and blocked with 150 l/well blocking buffer (3% skimmed milk powder and 0.05% Tween-20 in PBS) for 1 h at 37 ◦ C and washed. The rat/rabbit/bovine hyperimmune sera, buffalo immune sera and field/herd [cattle (25), buffalo (18), camel (23), horse (10) and donkey (5)] serum samples were diluted (1:100) with half strength blocking buffer and added (100 l/well) followed by incubation for 1 h at 37 ◦ C. After washing, the respective secondary antibody such as, anti-rat IgG (for rat sera), anti-rabbit IgG (for rabbit sera), anti-bovine IgG (for buffalo and cattle sera), anti horse IgG (for horse and donkey sera) and protein G (for camel sera) conjugated with horse radish peroxidase [diluted as per manufacturer’s instruction (sigma)] was added (100 l/well) and incubated for 1 h at 37 ◦ C. The microtitre plates were then washed and 100 l/well enzyme substrate (chromogenic) solution (5 mg of O-phenylene diamine dihydrochloride (sigma) and 0.03% (v/v) H2 O2 ) was added to develop the color. The reaction was stopped by adding 1 M H2 SO4 (100 l/well) and O.D was recorded at 492 nm in an ELISA reader (Bench mark microplate reader, BioRad). Further, the specificity of FLA antigen was determined with cattle serum samples clinically infected with Theileria annulata and Babesia bigemina (diagnosed by blood smear examination). The field serum samples were subjected to ELISA in duplicate using FLA antigen, VSG RoTat 1.2 antigen, CATT/T. evansi for comparative evaluation. 2.3. Production of MAbs 2.3.1. Immunization of Balb/c mice and preparation of splenocytes For immunization process, two Balb/c mice (8–12 weeks old) were used and other two Balb/c mice were maintained as healthy control. Primary immunization was given with emulsified FLA antigen (200 g/mice), followed by 1st booster injection and second booster injection (200 g/mice) 21 days after primary injection and first booster injection respectively subcutaneously. The third booster injection (1000 g/mice) was given 3–4 days before the fusion in saline intraperitoneally. The immunized mice were euthanized and washed with 70% alcohol. From it, spleen was taken and transferred aseptically into a sterile petri dish containing 5 ml Iscove’s Modified Dulbecco’s Medium (IMDM) (Hyclone) without supplements. The medium was flushed gently into spleen with the help of 5 ml glass syringe attached with 26G needle. The splenocytes were spun down at 214 g for 5 min at room temperature. The pellet was distributed with 10 ml IMDM by inverting the tubes
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gently and the splenocytes were counted in haemocytometer by diluting the aliquot of suspension in RBC lysis buffer. Simultaneously, for positive control, the blood was collected from the mice and serum was separated and used as the positive control. 2.3.2. Preparation of B-cell myeloma and feeder layer B cell myeloma such as, SP2/0 (Shulman et al., 1978) which are HAT sensitive (HGPRT−/TK− and non-Ig secretor) were used in the present study. The SP2/O cells were grown to log phase in IMDM supplemented with, 15% fetal bovine serum (FBS) (Hyclone) and 20 mM l-glutamate and harvested by centrifugation at 214 × g for 5 min at room temperature. The pellet was washed (twice), resuspended with IMDM (without any supplements) and cells were counted. For macrophage preparation, non-immunized BALB/c mouse was euthanized, and washed with 70% alcohol and was taken into laminar flow hood. Without damaging the abdominal muscle, the skin was cut open and 10 ml of pre-cooled IMDM was injected into abdomen using 10 ml glass syringe fitted with 18G needle. Abdomen was tapped by holding the needle in place to dislodge the cells adhered to the abdominal cavity and then the medium was retracted into the syringe (peritoneal exudates). The obtained peritoneal exudate, enriched with macrophages was kept on ice in a polypropylene tube and the cells were counted. Meanwhile, the blood was also collected from the mice; serum was separated and used as a negative control serum. 2.3.3. Production of hybridoma Twenty million splenocytes and 2 million myeloma (SP2/O) cells (10:1) were taken in a screw capped tube containing 10 ml of IMDM for hybridoma production. These cells were mixed and spun at 214 g for 10 min at room temperature. The residual medium was removed using sterile gauze, and the cell pellet was used for hybridoma production. Five hundred microlitre of polyethelene glycol (PEG) and dimethyl sulphoxide (DMSO) mixture (Sigma–Aldrich) was added within one minute to the cell pellet drop wise (cell pellet was disrupted after each drop) and exposed for another one minute. Five milliliter of IMDM (without any supplementation) was added first 1 ml over minute, drop wise to the tube slowly after one minute incubation. The cells were incubated in the CO2 (5%) incubator for 45 min and were pelleted at 214 g for 10 min at room temperature and spun at 214 g for 10 min at room temperature. The cell pellet obtained was suspended in IMDM, supplemented with 0.1 mM sodium hypoxanthine (H), 0.4 M aminopterine (A) and 0.016 mM thymidine (T) (Gibco® Life technologies), 15% fetal bovine serum (FBS) and 20 mM l-glutamate. The cells were plated out in a 96 well culture plate (0.2 ml per well), containing feeder layer/macrophages (4 × 106 cells/96 well plate). Controls were maintained in two wells containing only SP2/O) and macrophages. The plates were then incubated in CO2 (5%) incubator at 37 ◦ C for 7 days. On the 7th day, the medium was replaced with IMDM, supplemented with 0.1 mM hypoxanthine, 0.016 mM thymidine (Gibco, Life technologies), 15% FBS and 20 mM l-glutamate and were kept in CO2 incubator. 2.3.4. Screening of hybridoma clones and selection of monoclones By indirect ELISA, the clones appeared in the plates were screened on the 10th day for antibody production. The positive clones obtained were expanded by transferring the clones into 500 l cultures, 1 ml cultures, and finally into 5 ml culture flasks. In each expansion step, to screen positive clones, indirect ELISA was carried out in duplicate (as described in Section 2.2). The positive clones obtained were preserved in IMDM containing FBS (40%) and DMSO (10%) at −80 ◦ C and also in liquid nitrogen till further use. By dilution cloning technique from the positive hybridoma, the monoclones were selected and the hybridoma cells were counted and
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M. Ligi et al. / Veterinary Parasitology 219 (2016) 17–23
Fig. 1. Analysis of crude flagellar antigen by SDS-PAGE and Immunoblot. (a) SDS PAGE analysis: Lane 1 = Purified FLA antigen, Lane 2 = WCL antigen, Lane M = Prestained protein ladder, (b) Immunoblot analysis: Lane M = Prestained protein ladder, lanes 1 and 2 = MAb versus FLA antigen and MAb versus SP2/O cells culture supernatant.
100 cells were suspended in 20 ml IMDM (with all supplements) and plated out in 96 wells culture plate (∼one cell/well). The plate was incubated in CO2 (5%) incubator till the appearance of clones. The wells containing the single clone (monoclone) were screened by indirect ELISA (Section 2.2), expanded further and preserved at −80 ◦ C and also in liquid nitrogen in IMDM containing FBS (40%) and DMSO (10%) till further use. Later, the MAbs were produced in larger scale in 250 ml culture flasks and the cells were grown till the IMDM color changes to yellow and further tested by indirect ELISA (>1.2 OD). The supernatant rich in MAbs was collected by centrifugation at 857 × g for 3 min and preserved at −80 ◦ C till further use.
2.4. Characterization of MAbs 2.4.1. Immunoblot To characterize the MAbs, the immunoblot analysis was carried out by following the standard protocol (Sambrook and Russell, 2001). Here, the electrophoresed proteins (FLA antigen and control antigens) were transferred on to nitrocellulose membrane by following the standard protocol. The membrane was then treated with MAbs and SP2/O clones (undiluted culture supernatant). The substrate solution containing diamino benzidine tetrahydrochloride (DAB) was added finally to develop the immunoblot.
2.4.2. Isotyping Mouse MAb was isotyped using mouse isotyping (IgG1, IgG2a, IgG2b, IgG3, IgA and IgGM) antibodies (Sigma–Aldrich) by antigen mediated ELISA as per manufacturer’s instructions. For antigen mediated ELISA, microtitre plates (Maxisorp® , Nunc) were coated with 100 l/well of FLA antigen (200 ng/well) for overnight at 4 ◦ C. After overnight incubation the plate was washed four times with washing buffer (Section 2.2) and the undiluted MAb (culture supernatant from monoclone) was added into each well (100 l/well) and incubated for 2 h at 37 ◦ C. The plate was then washed and isotyping antibodies (100 l/well) were added and incubated at 37 ◦ C for 1 h. After washing anti goat IgG was added in each well (100 l/well) and incubated for 30 min at 37 ◦ C, followed by washing and addition of 100 l/well substrate solution (Section 2.2). The plate was then incubated for 30 min to develop the color and then read in ELISA reader at 492 nm.
2.5. CI-ELISA By checker board titration, the optimum concentration of FLA antigen, serum dilution and MAb dilution was determined. Microtitre plates (Maxisorp® , Nunc were coated with purified FLA 400 ng/well) overnight at 4 ◦ C in PBS (pH 7.2). The microtitre plates were washed and blocked after overnight incubation. The test serum samples (20 l of test serum, 20 l of blocking buffer and 60 l MAb) was added in duplicate and incubated at 37 ◦ C for 1 h on ELISA shaker followed by washing. The test was included with strongly positive (>90% competition), weakly positive (60–70% competition), negative, MAb (0% competition) and conjugate control wells simultaneously. Hundred microlitre of antimouse antibody horseradish peroxidase conjugate (diluted as per manufacturer’s instruction) was added to each well and incubated at 37 ◦ C for 1 h followed by washing and addition of substrate solution (100 l/well). The plates were incubated till the visible color develops in the monoclonal antibody control wells and then the reaction was stopped by the addition of (100 l/well) stopping solution. The plates were then read at 492 nm in an ELISA reader (Section 2.2). The OD values obtained were converted to percentage inhibition (PI) values by using the formula: PI = 100−{(OD in test well/OD in Cm well) × 100} (Singh et al., 2004).
2.5.1. Determination of optimum antigen concentration, MAb and serum dilutions For the optimization of antigen concentration, MAbs and serum dilutions, the buffalo immune serum raised against T. evansi (buffalo isolate) and healthy buffalo serum were selected. The different combination of antigen concentration, serum dilution and MAb dilution was tested and has been established that FLA antigen reacted well with undiluted/neat MAb and 1:100 diluted immune/hyperimmune serum without background activity at 200 ng/well in indirect ELISA. Hence, initial antigen concentration, serum dilution and MAb dilution was chosen at 200 ng/well, 5 l/well and 40 l/well respectively. The concentration/dilution was increased two fold up to 600 ng/well (antigen), 35 l/well (serum) and 80 l/well (MAb). The optimum concentration/dilution of each reagent was found to be the maximum difference in PI value between positive and negative at highest dilution. The optimum dilution of rat hyper immune serum raised against FLA antigen, bovine hyper immune serum raised against
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Fig. 2. ROC (receiver operating characteristic) graph depicting diagnostic sensitivity and specificity of CI-ELISA.
T. evansi (buffalo isolate) with respective healthy serum was also determined simultaneously.
2.5.2. Statistical analysis Receiver operating characteristic (ROC) analysis was carried out to determine the cut off PI value and optimum combination of diagnostic specificity and sensitivity of FLA antigen in ELISA using cattle, buffalo, camel, horse and donkey sera by keeping CATT/T. evansi as gold standard test. An agreement of FLA antigen in relation to CATT/T. evansi was determined for detecting antibody against T. evansi. A trial version of software MedCalc (version 12.7.2, USA) was used to generate ROC curve. The Cohen’s kappa (Cohen, 1960) value were determined using on line version of Kappa–VassarStats (cited at: www.vassarstats.net/kappa.html) software. A group of serum samples collected from cattle, buffaloes, horse, donkey and camels from four different states of India were subjected to new CI-ELISA and CATT/T. evansi for comparative analysis. The percentage prevalence of surra in different species (age wise) across different states of India was also carried out. Further, the significance of the test was determined by Chi square (2) analysis (Snedecor and Cochran 1968) of the epidemiological data (species and age wise) obtained from both the tests. The specificity of CI-ELISA was also evaluated with cattle serum samples clinically infected with T. annulata and B. bigemina.
3.2. Production and characterization of MAbs The three clones which showed very high reactivity with FLA antigen by indirect ELISA (OD > 1.4) were selected, while, the control clones (SP2/0, spleen and macrophages) showed lesser O.D values (<0.2). Immunoblot analysis revealed that the MAb was highly specific and reacted with FLA antigen. However, immunoreactivity of MAb was not observed with control proteins (Fig. 1b). Isotyping analysis revealed that MAbs belong to isotype of IgG, subtype IgG3 and is used in the development of CI-ELISA. 3.3. Diagnostic sensitivity and specificity of CI- ELISA versus CATT/T. evansi
3. Results
The optimal concentration of FLA antigen, buffalo immune serum and MAb was found to be 400 ng/well, 35 l/well and 80 l/well respectively. The maximum difference in the PI value between rat/bovine hyperimmune serum versus (v/s) control serum was found to 15 l/well, while between buffalo immune serum v/s control serum was found to 20 l/well. Hence, for the development of CI-ELISA, 20 l/well serum was chosen as an optimal value, since the field serum samples were obtained as immune serum samples. The statistical analysis of CI-ELISA showed positive–negative cut off PI value was found to be >55. The optimum combination of diagnostic sensitivity and specificity was found to be respectively at 95.8 and 94.4 at >55 cut-off PI value (Fig. 2), with a Cohen’s kappa coefficient of agreement value of 0.83.
3.1. Characterization and immunoreactivity of FLA antigen
3.4. Sero- prevalence of T. evansi
In SDS-PAGE analysis, nine bands were obtained in the flagellar portion between 18 to 130 kDa at 18, 20, 30, 50, 55, 60, 75, 90 and 130 kDa. FLA was analyzed by SDS-PAGE (Fig. 1a) and its immunogenicity was tested by immunoblot (Fig. 1b). The FLA antigen and control antigens remained respectively reactive and non reactive in ELISA. The FLA antigen showed high reactivity by indirect ELISA (O.D > 1.2) with hyperimmune sera, while the control sera showed low O.D values of <0.2. The FLA antigen remained non reactive with serum samples clinically infected with T. annulata and B. bigemina.
The statistical analysis of the CI-ELISA data by keeping variable such as, species and age revealed that, the disease is more prevalent among adult cattle in Karnataka (>4 years, 2 = 0.09, df = 1, p > 0.05) and West Bengal (>6 years, 2 = 0.00, df = 1, p > 0.05). In Karnataka, calves (up to 4 years, 2 = 0.00, df = 1, p > 0.05) and aged buffaloes (>8 years, 2 = 0.00, df = 1, p < 0.05) were found more sero-positive than middle aged cattle. The disease prevalence in horse and donkey was also found to be more or less equal among the different age groups. However, in cattle, all age group were almost equally seropositive in Odisha. Camel samples from Rajasthan revealed that the
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calves (<2 years, 2 = 0.00, df = 1, p > 0.05) are less susceptible to T. evansi infection than adults (>2 years, 2 = 0.009, df = 1, p > 0.05). The comparative analysis of CI-ELISA with CATT/T. evansi showed that the new assay is in agreement with the standard test. The age wise prevalence of disease in different species from different states of India is shown inTable 1.
4. Discussion The flagellar membrane mediates attachment of parasite to host tissues and harbors multiple virulence factors. The flagellum also mediates host parasite interactions (Langousis and Hill, 2014). Earlier, Rayulu et al. (2009) developed one MAb (MAb engineered against cell membrane antigen) based sensitive and rapid latex agglutination test (MAb LAT) to detect the circulating antigens of Indian isolate of T. evansi in domestic animals. In the present study, competitive inhibition ELISA has been developed exploring monoclonal antibodies against purified FLA antigen. SDS PAGE analysis of purified FLA protein showed nine bands in SDS-PAGE ranging from 18 to 130 kDa. Earlier a varying range of 4–12 protein bands were observed in different isolates of which 58.7 (∼60) kDa was also present in some isolates. Earlier study showed that even in some geographical regions also the polypeptide bands were varied in different isolates (Singh et al., 1995). The purified FLA protein remained non- reactive in ELISA with T. annulata and B. bigemina infected serum. Apart from this, the new CI-ELISA test also remained non-reactive with such sera. In ELISA and Western blot analysis, both the MAb showed immunoreactivity against purified flagellar protein. The purified FLA protein remained immunoreactive with the panel of sera samples consisting of experimentally produced/field samples including different host species namely cattle, buffalo, camel, horse and donkey. Besides, CI-ELISA test remained reactive with experimentally produced T .evansi buffalo immune sera, bovine hyperimmune sera, and rat immune sera. On comparison with CATT/T. evansi test, new CI-ELISA test emerged as sensitive (95.8%) and specific (94.4%,) with a Cohen’s kappa value of 0.83. The developed assay detected T. evansi antibodies from different host species such as, cattle, buffalo, camel, horse and donkey. The statistical analysis of the epidemiological data (species and age wise) revealed no significant variation in both the test systems i.e., CI-ELISA and CATT/T. evansi. The cattle samples clinically infected with T. annulata and B. bigemina did not react in CI-ELISA. Thus, CI-ELISA incorporating MAbs turns out to be a potent diagnostic tool for the detection of the antibodies of T. evansi. However, it needs to test with the antibodies of other flagellated protozoa for checking cross-reaction. The seroprevalence study revealed that the prevalence of disease is observed more in adult cattle (up to 44%, 2 = 0.0, df = 1, p > 0.05) compared to calves. Besides, T. evansi infection was found to be more in buffalo calves than adults (Table 1). The incidence of T. evansi infection in camels, horse and donkey was found to be more or less equal among different age groups. However, a large number of horse and donkey serum samples need to be tested for further conclusion. Though cattle and buffalo sera are collected from the same location/state (Karnataka) the variation in the disease incidence among different age groups is observed between cattle and buffalo. The present study provides preliminary information to carry out for further study in determining the risk factors associated with different age groups and species. The comparative analysis revealed that the new assay is comparable with CATT/T. evansi. The authors expect that the new ELISA test in the present study can be employed for a mass level screening for effective control of trypanosomosis. However, further evaluation of the test for its
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