Ethical statement
All experimental protocols were approved by the Science and Technology Agency of Jiangsu Province (Approval ID: SYXK (SU) 2010-0005).
Study population
Local crossbred goats (n=35, age≃6 months) were bought from a farm in Xuyu city, Jiangsu, China and kept under controlled conditions in animal house of Nanjing Agricultural University (NAU). All goats were orally dewormed twice with Levamisole (8 mg/kg) at two weeks interval to remove natural parasitic infections. Fecal samples were collected from all goats twice a week and analyzed microscopically for helminth eggs. Helminthes free goats were utilized for further experiment, After 25 days of first deworming, goats were divided randomly into group 1 (n=5), group 2 (n=28) and group 3 (n=2).
Female SD rats of 150 g body weight (n=6) were obtained from the Experimental Animal Center of Jiangsu, PR China (Certified: SCXK 2008-0004). Rats were separated randomly into two groups, group 1 (n=3) and group 2 (n=3) to collect polyclonal antibodies. Rats were kept in sterilized room with free access of food and water. Anti-rHc-CS polyclonal antibodies were collected following the methods as described early [47]. Briefly, complete Freund’s adjuvant was equally mixed with rHc-CS protein (300μg) and injected subcutaneously in SD rats of group 1. After 14 days, incomplete Freund’s adjuvant mixed equally with rHc-CS protein was injected twice with one week interval. Finally, SD rats were anesthetized with 25% isoflurane (inhaling anesthesia) after one week of the last dose by open drop method [48]. Group 2 was kept untreated as control. Rats were euthanized by head dislocation after collecting blood from eye to prepare sera.
H. contortus infective larva (L3)
The H. contortus strain was maintained by serial passages in helminth-free goats, at MOE laboratory NAU. H. contortus (L3) used in these experiments were obtained from feces of infected goats using conventional method [49]. Briefly, feces from H. contortus infected-goat were collected, crushed, mixed with water and combined with vermiculite to keep mixture moist at room temperature. The pan was covered with aluminum foil having several holes to allow air flow. After ten days, mixture was filtered through cheesecloth to collect larvae to examine microscopically that were preserved at 4°C in penicillin G mixed with water until use.
Experiment 1
First experiment was performed to assess early diagnostic potential of rHc-CS protein during different stages of H. contortus infection using immunoblotting assay. For this purpose, group 1 (n=5) was artificially infected with 8000 infected larva of H. contortus (L3) orally and group 3 (n=2) was kept uninfected as control. Serum samples were collected from group 1 (infected) and group 3 (uninfected) at specified days; one day prior challenging infection and 7 to 103 days post challenging infection with week interval for antibody detection.
Moreover, McMaster egg count technique was performed to examine the fecal samples collected at 7, 14, 21, 28 and 35 D.P.I as described previously [50].
Experiment 2
Experiment 2 was performed to develop and optimize indirect ELISA based on rHc-CS. In this regard, Group 2 (n=28) was orally infected with 8000 H. contortus L3 and fecal examination was performed to confirm H. contortus infection before collecting the sera. Moreover, at 30 D.P.I the goats of group 2 were euthanized by injecting sodium pentobarbital (>150 mg/kg) intravenously [48]. Furthermore, necropsy was performed to confirm the presence of adult worms embedded in the mucosa.
Purification of recombinant Hc-CS
Recombinant plasmid (pET-32a+rHc-CS, GenBank: CDJ84294.1) was provided by MOE joint international Research Laboratory, Preventive Veterinary Medicine, NAU and protein was purified by following standard protocol [47]. After transformation of recombinant plasmid into E.coli BL21 (DE3), product was cultured in ampicillin containing LB (Luria Bertani) medium. Ni2+ nitrilotriacetic acid column (GE Healthcare, USA) was used to purify recombinant protein following the manufacturer’s instructions. The quantification of rHc-CS protein was analyzed by Bradford method [51] and protein was detoxified using Toxin EraserTM Endotoxin Removal kit (GeneScript, USA).
Immunoblotting assay
Immunoblotting is a method of choice for H. contortus infection. It helps to select target protein for the diagnostic purposes as well as for the antigenic immunogenicity and immunoreactivity evaluation [52]. Immunoblotting was performed to evaluate the immunoreactivity of rHc-CS using rat sera following the methods as described in our previous study [41]. Moreover, immunoblotting analysis was also performed to evaluate the antigenic characteristics of rHc-CS at early stage as well as late stage of H. contortus infection in sera of experimentally infected goats (Group 1). Upon each blood sampling day, the whole immunoblotting practice was repeated whereas the primary antibody differed. All anti H. contortus sera taken from infected (Group 1) and uninfected goats (Group 3), on those sampling days were taken as the primary antibody. Moreover, the specificity of rHc-CS against infected sera of Fasciola hepatica (F. hepatica), Trichinella spiralis (T. spiralis) and Toxoplasma gondii (T. gondii) was also checked by immunoblotting.
Optimization and development of indirect-ELISA
Indirect-ELISA was performed to assess the immunodiagnostic potential of rHc-CS [53]. Indirect-ELISA based on rHc‑CS was optimized by evaluation of numerous format variables including working dilution of rHc-CS antigen (4.5 to 0.07 μg/well), serum samples (1:25 to 1:200 dilutions), secondary antibody HRP conjugated rabbit anti-goat IgG (1:2000 to 1:16000) and working time of antigen coating (37℃ 2h-4℃ overnight, 37℃ 1h- 4℃ overnight), blocking (30-120 min), serum incubation (30-120 min) and TMB reaction (5-15 min). Furthermore, best blocking buffer (2, 3, 4, 5% BSA and 2, 3, 4, 5% Milk) was also determined using same method. Hence, checkerboard titration was used to derive OD values and calculated as the positive to negative (P/N) ratio [54].
In order to evaluate the diagnostic potential and to complement the immunoblotting results, standardized indirect-ELISA was developed as described previously [41, 55]. Moreover, serum samples (n=33) from infected goats were used to calculate diagnostic sensitivity and serum samples of all goats before artificial infection (n=35) were used to calculate diagnostic specificity of rHc-CS based indirect ELISA using following formula [56]. (see Formula 1 in the Supplementary Files)
Additionally, to confirm the cross-reactivity against commonly found parasites, a total of 12 goat serum samples (4 samples for each parasite, Kept in the MOE laboratory of NAU) against F. hepatica, T. spiralis and T. gondii were used. All the experiments were performed in duplicate.
Determination of cut-off value
Sera (n=35) collected from all goats before H. contortus infection were used to determine cut-off value. The cut-off value was calculated by taking “mean absorbance values of known negative sera + (3× standard deviation)” [57]. OD450 value of sera greater than cut-off value was considered as sero-positive and OD450 value below cut-off value was considered as sero-negative [34].
Statistical analysis
ROC analysis was used to simulate the influence of different cut-off values on sensitivity and specificity of the test [38]. ROC curves were obtained using statistical software MedCalc (version 15; http://www.medcalc.be). Statistical analysis of data was assessed by using software (Graph Pad Prism™ v6 07).