Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Simple Summary The accurate and early diagnosis of Haemonchus contortus infection is crucial for effective control. Early stage detection of H. contortus infection has not been attempted in goat, even though both immature worm and fourth larval stage are blood sucking. This study was carried out to detect the H. contortus infection during early stage in goat. The results of this study assessed that rHc-HCA59 could detect the antibody in H. contortus infected goats’ sera during early period with good sensitivity and specificity using immunodiagnostic techniques. Our findings illustrated that combined use of ELISA and western blotting based on rHc-HCA59 is a powerful tool for early detection of H. contortus infection in goats. Abstract Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.


Introduction
Small ruminant farming industry contributes one of the major means to develop living standards in numerous developing countries and plays an important role in the national economy [1]. Among small ruminant species, goats are particularly vulnerable to gastrointestinal nematodes in humid, tropical, and subtropical regions and are particularly overwhelmed by Haemonchus contortus (H. contortus) [2]. It is an important blood sucking parasite found in abomasum that may remove about 0.05 mL of blood per day by seepage from lesions [3]. H. contortus infection leads to significant economic losses to small ruminant farming industry as a consequence of high mortality and morbidity [1].
The accurate and prepatent diagnosis of H. contortus infection is crucial for epidemiological surveillance and control. Prepatent stage detection of H. contortus infection has not been attempted in goats, even though both immature worm and fourth larval stage are blood sucking. The current diagnosis of hemonchosis mainly relies on the use of traditional fecal egg counts and serological-based methods, such as FAMACHA (eye-lid coloration) and blood packed cell volume [4]. It is difficult to detect H. contortus eggs in feces before the third week of post infection (21-25 days) [5]. H. contortus starts blood feeding on 11th day post infection (PI) [4]. Until the infection shows clinical signs, young animals suffer from anemia, diarrhea, edema, weight loss, severe frailty, and ultimately death [6]. These methods are often time-consuming, inaccurate, unreliable, and laborious to perform [7].
Immunodiagnosis provides possible avenues to overcome current limitations and develop improved diagnostic assay for detection of H. contortus. The immunodiagnosis of H. contortus is dependent on direct and indirect techniques. To detect specific antibodies, indirect methods are used. ELISA or western blotting can be used to detect anti-Haemonchus antibodies. These techniques are based on target antigens (secreted, purified native, whole parasite extract, or recombinant proteins) being immobilized on a solid support, followed by incubation with serum containing antigen-specific antibodies. The use of recombinant antigens in the design of a specific diagnostic technique facilitates the development of highly sensitive and specific assays that display a high antigen concentration and reduce or eliminate background reactions. Easy production of antigens in expression systems leads to simple and efficient antigen development which can reduce the production costs associated with diagnosis [8].
Excretory and secretory products (ESPs) are produced and released by the parasites during infection [9]. H. contortus ESPs (HcESPs) contain many proteins that can perform various functions including host immune response [10]. In previous study, ESPs have been reported as diagnostic antigen for prepatent detection of H. contortus infection in sheep [11]. Hepatocellular carcinoma-associated antigen 59 (HCA59) was identified as one of HcESPs and can be isolated from different larval stages of this parasite [12]; however, its diagnosis potency is still unknown. HCA59 belongs to TLS1 (Telomere length and silencing protein 1) family which is potential candidate for immunological applications and provides the molecular features for understanding tumorigenicity [13].
The aim of the current study was to evaluate the combined use of ELISA and western blotting to detect specific antibodies against the antigen of H. contortus during prepatent stage of infection. Early diagnosis of this infection will assist in treating carrier animals quickly with suitable anthelmintics.

Recombinant Protein Purification
The recombinant plasmid expression, rHc-HCA59 was provided by Ministry of Education (MOE) joint international Research Laboratory, Preventive Veterinary Medicine, Nanjing Agricultural University (NAU) (GenBank: CDJ80864.1) and purified by standard protocol described previously [14]. Briefly, transformation of recombinant plasmids into E.coli BL21 (DE3) was performed and cultured in ampicillin (100 µg/mL) containing LB medium (Luria Bertini). After that, induction of protein expression was done at 37 • C using IPTG (Isopropyl β-D-thiogalactopyranoside; Sigma-Aldrich) to make OD 600 0.6. Culture was centrifuged at 4500 rpm for 15 min and supernatant was discarded. Pallet was lysed by using lysozyme (10 µg/mL Sigma-Aldrich) followed by sonication. 12% (w/v) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to examine the sonication products. Manufacturer's instructions were practiced to purify recombinant protein using Ni 2+ nitrilotriacetic acid column (GE Healthcare, Chicago, IL, USA). The rHc-CS protein purity and quantification were performed by SDS-PAGE with Coomassie blue stain and Bradford method respectively [15]. Protein toxins were removed utilizing Toxin Eraser TM Endotoxin Removal kit (GenScript, Piscataway, NJ, USA).

Study Populations
Local crossbred goats aged 4 to 6 months (n = 38) were housed indoors and provided with whole shelled corn, hay and water ad libitum. All goats were dewormed with Levamisole (8 mg/kg body weight) to eliminate the naturally attained infection with helminthes at 2-week intervals. Microscopic analysis of fecal samples was performed twice per week for helminth eggs and helminth-free goats were used in further experiments. All goats were experimentally infected orally with 8000 L3 of H. contortus. Blood samples were collected from all goats prior to challenging infection (negative) and at 14th post infection day (prepatent stage). Sera were prepared and stored at −20 • C. Serum samples from goats with a positive diagnosis for Toxoplasma gondii (T. gondii), Fasciola hepatica (F. hepatica), and Trichinella spiralis (T. spiralis), provided by MOE joint international Research Laboratory, Preventive Veterinary Medicine NAU, were also used for the evaluation of the ELISA specificity.
Feces samples (n = 38) from all goats were collected at 7th and 14th post infection day (prepatent stage) and conventional fecal egg counting was performed with the McMaster method as described previously [16]. McMaster egg counting was based on detection of H. contortus eggs in 3 g of feces dispersed in saturated NaCl (42 mL), providing a diagnostic sensitivity of 50 epg (eggs per gram). At the end of the experiment, all goats were confirmed for the presence of H. contortus eggs in feces then dewormed to use in another experiment.

Western Blotting Assay
Western blotting plays preliminary role in selection of target protein to diagnose the infection of H. contortus and to evaluate the immunogenicity and immunoreactivity of antigen [17]. Western blotting analysis was performed to detect H. contortus-specific antibody responses in sera of experimentally infected goats using recombinant H. contortus HCA59 (rHc-HCA59). The purified rHc-HCA59 was first deposited in SDS-PAGE wells individually, then transferred onto PVDF (polyvinyl difluoride membrane, Millipore, Billerica, USA) with the help of semi dry system (Novablot Hoefer, USA) and transfer solution (Tris 48 mM, glycine 39 mM, SDS 0.0375%, methanol 20%). The membrane was blocked with 5% skimmed milk diluted in TBS-T (Tris-buffered saline containing 0.05% Tween 20) for 2 h at 37 • C. The PVDF membrane was cut, washed (three times) and incubated with anti-H. cortortus 1:100 diluted goat serum (primary antibody) for 2 h at 37 • C. Following three washes with TBS-T the strips were incubated with 1:5,000 diluted secondary antibodies, horseradish peroxidase (HRP) conjugated rabbit anti-goat IgG (Sigma, Hilden, Germany). Subsequently, the strips were washed five times and immunoreactions were observed by virtue of substrate (Tanon™ High-sig ECL Western Blotting, Shanghai, China). Western blotting also helped to check the specificity of rHc-HCA59 against infected sera of T. gondii, F. hepatica, and T. spiralis as primary antibody.

Development of Indirect ELISA
To standardize the indirect ELISA development, different concentrations of rHc-HCA59 (65 to 1.01 µg/mL) and serum dilutions (1:25; 1:50; 1:100; 1:200) were optimized. Negative and positive sera of H. contortus were used as controls. The highest P/N (OD 450 of infected serum/OD 450 of non-infected serum) value (>2.1) was used to optimize the experimental conditions. Indirect ELISA with optimal conditions of was performed as discussed previously [18]. Briefly, the reaction was performed in 96-well flat bottom without lid high binding plates (Costar, Cambridge, MA, USA). The plate wells were coated with 100 µL of optimum concentration diluted antigen in 0.05 M carbonate buffer solution (CBS; pH 9.6) and incubated overnight at 4 • C. The next day, all wells were blocked with 100 µL BSA (bovine serum albumin; 5%) for 2h at 37 • C. Serum samples diluted 1:50 in BSA were added in triplicate and the plates were again incubated for 2 h at 37 • C. After this duration, diluted secondary antibody in 5% BSA (HRP conjugated rabbit anti-goat IgG; 1:6000) was added and incubated again at 37 • C for 1 h. After each incubation period, plates were washed three times with TBS-T. Finally, plates were washed five times and 100 µL of tetramethylbenzidine (TMB) was added in each well and incubated for 10 min in the dark at room temperature. The color reaction was stopped by adding 100 µL/well of 2M H 2 SO 4 . The optical density (OD) of wells was read at a wavelength of 450 nm using micro plate reader (Thermo Fischer Scientific, Waltham, MA, USA).

Determination of Cut-Off Value
A total 38 serum samples from non-infected goats were evaluated to determine the positive cutoff value from mean OD 450 of known negative sera + 3 standard deviation [19]. While, negative cut-off value was assessed from mean OD 450 of known negative sera + 2 standard deviation [20]. The experiment was repeated twice and for the interpretation, any goat sera that had an OD 450 value greater than the positive cut off value was considered as seropositive while OD 450 value lower than negative cut-off value was considered as sero-negative [21]. Furthermore, any goat sera which had OD 450 value between positive and negative cut-off values was considered as false negative/positive.

Calculation of Sensitivity and Specificity
To investigate the further feasibility of indirect ELISA, serum samples (n = 38) positive with H. contortus were evaluated and diagnostic sensitivity was calculated as "ELISA positive × 100/true H. contortus-positive" and serum samples (n = 38) from non-infected goats were evaluated to calculate specificity. Additionally, to evaluate the cross-reactivity, 12 serum samples (4 samples for each parasite) positive with T. gondii, F. hepatica, and T. spiralis were used. This experiment was performed in duplicate. To ratify the rHc-HCA59-based indirect ELISA results, all positive, negative, false positive/negative, and other organisms' sera, were subjected to western blot analyses based on same antigen.

Development of the Western blot-rHc-HCA59
The western blot assay was performed almost as described above. The rHc-HCA59 was deposited in all wells of the SDS-PAGE gel and, after transfer to the nitrocellulose membrane; it was cut to obtain five strips. Each strip was incubated with a 1:100 serum dilution after blocking with 5% skimmed milk for 2 h. The secondary antibody horseradish peroxidase (HRP) conjugated rabbit anti-goat IgG (Sigma, Hilden, Germany) (1:5000) was added to all membranes and incubate for 1 h. Immunoreactions was revealed using substrate (Tanon™ High-sig ECL Western Blotting). All incubation steps were performed at 37 • C and were followed by three washes with TBS-T.

Repeatability of ELISA-rHc-HCA59
In order to evaluate the repeatability of the ELISA, three separate batches of recombinant HCA59 antigen were produced and purified following the methodology described above with distinct purification times, to demonstrate the reproducibility of the recombinant protein expression procedure. Serum samples were selected for testing against each batch of antigen and the averages, and standard deviations, of the O.D. at 450 nm were calculated. Of the samples used for this test, one was strongly reactive positive, three were moderately reactive positive, and four were negative serum samples.

Purification and Western Blotting
The SDS-PAGE results demonstrated the presence of distinct bands, rHc-HCA59 showed a molecular mass of 33 kDa ( Figure 1A). The same band was observed in the Western blotting and confirmed the presence of band that corresponded to the protein of interest ( Figure 1B). Previous study has described the cloning of the 426 bp corresponding to the rHc-HCA59 protein gene from H. contortus and reported that the recombinant protein presented as a distinct molecular mass, 33 kDa [14]. This corroborates with the results observed through SDS-PAGE and western blot in the present study ( Figure 1). Furthermore, the rHc-HCA59 antigen did not show any cross-reaction with goat anti-sera of T. gondii, F. hepatica, and T. spiralis (Figure 2). distinct purification times, to demonstrate the reproducibility of the recombinant protein expression procedure. Serum samples were selected for testing against each batch of antigen and the averages, and standard deviations, of the O.D. at 450 nm were calculated. Of the samples used for this test, one was strongly reactive positive, three were moderately reactive positive, and four were negative serum samples.

Purification and Western Blotting
The SDS-PAGE results demonstrated the presence of distinct bands, rHc-HCA59 showed a molecular mass of 33 kDa ( Figure 1A). The same band was observed in the Western blotting and confirmed the presence of band that corresponded to the protein of interest ( Figure 1B). Previous study has described the cloning of the 426 bp corresponding to the rHc-HCA59 protein gene from H. contortus and reported that the recombinant protein presented as a distinct molecular mass, 33 kDa [14]. This corroborates with the results observed through SDS-PAGE and western blot in the present study ( Figure 1). Furthermore, the rHc-HCA59 antigen did not show any cross-reaction with goat anti-sera of T. gondii, F. hepatica, and T. spiralis ( Figure 2).

Microscopic Examination
McMaster fecal egg counts (FECs) could not detect the eggs of H. contortus in fecal samples of all infected goats (n = 38) collected at 7th and 14th day of infection. While, H. contortus eggs were detected at the 21st day of post infection in feces.

Cut-Off Value of Indirect ELISA
The positive cut-off value of indirect ELISA was 0.371 as determined by mean value (0.256 ± 0.004) plus three multiplied by standard deviations (3 × 0.038) of the OD 450 values obtained from 38 known negative goat sera. While, the negative cut-off value of indirect ELISA was 0.332 as determined by mean value (0.256 ± 0.004) plus two multiplied by standard deviations (2 × 0.038) of the OD 450 values.

Development and Serodiagnostic Potential of Indirect ELISA
The optimum concentration of rHc-HCA59 antigen and optimum dilution of serum were 0.81 µg/well and 1:50, respectively ( Table 1). The rHc-HCA59 has definite potential for serological diagnosis through the established indirect ELISA. The indirect ELISA showed 87% sensitivity against well-characterized positive sera and 100% specificity against helminth free sera. Total 88 serum samples (38 positive for H. contortus collected at prepatent stage of infection; 38 negative; 12 positive for other co-infection parasites) were used to evaluate the diagnostic potential of indirect ELISA. Of which, OD 450 of 28 sera samples were observed above positive cut-off value and scored positive (OD 450 > 0.371). OD 450 value of 38 negative sera samples was observed below the negative cut-off line and scored negative (OD 450 < 0.332) in ELISA-rHc-HCA59. Moreover, five sera samples collected at the prepatent stage from H. contortus positive goats, which were negative in microscopic examination, showed OD 450 value between the positive and negative cut-off line and considered as false negative. While all T. gondii, F. hepatica, and T. spiralis-positive sera tested negative (OD 450 < 0.332) in the developed rHc-HCA59 ELISA, providing evidence of test specificity. Significant differences (p < 0.05) in mean OD values between H. contortus infected goats' sera (0.681 ± 0.012) and uninfected sera (0.268 ± 0.004) were observed by t-test. While, no significant difference between negative and other parasites' serum samples were observed (Figure 3). The results indicated that five serum samples showed false negative OD 450 .

Western Blotting for Ratification of Indirect ELISA Results
To ratify the indirect ELISA results, all positive (n = 33), negative (n = 38), false negative (n = 5) and other organisms' sera (n = 12), were subjected to rHc-HCA59-based western blot. The results of western blot analysis confirmed those samples as positive having OD450 value greater than positive cut-off value and recognised the samples as negative which had OD450 lower than negative cut-off

Western Blotting for Ratification of Indirect ELISA Results
To ratify the indirect ELISA results, all positive (n = 33), negative (n = 38), false negative (n = 5) and other organisms' sera (n = 12), were subjected to rHc-HCA59-based western blot. The results of western blot analysis confirmed those samples as positive having OD 450 value greater than positive cut-off value and recognised the samples as negative which had OD 450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera that had OD 450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot (Figure 4). While no cross-reactivity was observed in sera positive for T. gondii, F. hepatica, and T. spiralis.

Test Repeatability
Three different protein batches were used as coating antigens for testing the rHc-HCA59-ELISA and the results from all three were similar (p > 0.05) and thus, indicate antigen stability. The optical density (OD) values derived from the checkerboard were calculated and showed as the positive to negative (P/N) ratio [22]. The high P/N (OD450 of infected serum/OD450 of non-infected serum) value (>2.1) was observed ( Figure 5).

Test Repeatability
Three different protein batches were used as coating antigens for testing the rHc-HCA59-ELISA and the results from all three were similar (p > 0.05) and thus, indicate antigen stability. The optical density (OD) values derived from the checkerboard were calculated and showed as the positive to negative (P/N) ratio [22]. The high P/N (OD 450 of infected serum/OD 450 of non-infected serum) value (>2.1) was observed ( Figure 5).

Test Repeatability
Three different protein batches were used as coating antigens for testing the rHc-HCA59-ELISA and the results from all three were similar (p > 0.05) and thus, indicate antigen stability. The optical density (OD) values derived from the checkerboard were calculated and showed as the positive to negative (P/N) ratio [22]. The high P/N (OD450 of infected serum/OD450 of non-infected serum) value (>2.1) was observed ( Figure 5).

Discussion
H. contortus is prevalent wherever small ruminants are raised, but the greatest economic losses occur in warm and tropical areas. Detection of H. contortus infection in small ruminants during the 15-day prepatent period is essential for effective control of this disease on farms and field [23]. The current diagnosis of haemonchosis mainly relies on the use of traditional fecal egg counts [4]. After ingestion of infective larva (L3) by host, HCI and pepsin triggered exsheathment for L3 migration to abomasum where L3 develops into L4 stage, which feeds on blood followed by final adult development. It takes approximately 3 weeks post-infection. It is not possible to detect H. contortus eggs in feces before week 3 post-infection (21-25 days) in small ruminants [24]. That is why in this study, no eggs of H. contortus were detected in fecal samples of all infected goats collected at the 7th and 14th days of infection.
Previous study that focused on the development of potential diagnostic assays was not limited to H. contortus detection, but also focused on development of vaccine against haemonchosis and interaction between parasite molecules and host cells [25][26][27]. Different antigens-such as somatic antigens, larval antigen, and crude antigen-were evaluated for immunodiagnosis against H. contortus infection [5,28,29]. However the cross reactivity among different helminthes and low sensitivity are the limiting factors of these antigens.
Recently, modulatory effects of rHc-HCA59 on PBMCs (peripheral blood mononuclear cells ) and maturation of monocyte-derived dendritic cells were evaluated in goat and reported that rHc-HCA59 could be recognized by H. contortus infected goat sera [14]. These features make this antigen an interesting candidate for use in diagnostic assays, such as ELISA and western blotting. In this study, rHc-HCA59 was used as diagnostic antigen for development of indirect ELISA as well as for detection of specific antibody during prepatent period. Previously, different ELISA based methods have been reported to detect anti-H. contortus antibodies in naturally and/or experimentally infected sheep sera [23,30,31]. The ELISA-rHc-HCA59 developed in this study showed 87% sensitivity and 100% specificity results and thus proves that rHc-HCA59 can be used as diagnostic antigen to powerfully detect specific antibodies during prepatent period of H. contortus infection in goat. In contrast to ELISA of this study, DAS-ELISA (double antibody sandwich ELISA) and dot-ELISA were developed to detect Babesia gibsoni [21] using truncated recombinant BgSA1, achieved sensitivity of 100% and 91.9% respectively, and specificity of 97.4% and 84.2% respectively. Secretion time of antigen may influence antigenicity, specificity and sensitivity during prepatent stage of infection. It has been reported that rHc-HCA59 releases at 7th and 14th day of infection [12]. Therefore, this feature gives this antigen potential to detect specific antibody during prepatent stage of infection. Furthermore, rHc-HCA59 did not show any cross reactivity against co-infecting parasites, that is why this antigen showed high specificity. In contrast to this study, previous study showed cross-reactivity against H. contortus infected sheep sera using recombinant protein recIgE1-2 [32].
In the present study, western blot assay was used to complement the results achieved through ELISA and to support the diagnosis of H. contortus infection further. The combination of indirect ELISA and western blotting can be an effective tool to increase sensitivity of serodiagnosis of infection [33]. In this study, rHc-HCA59-based western blotting was used to confirm the results of screening indirect ELISA assay and results confirmed the immunoreactivity in all positive sera that had higher OD value than the positive cut-off value (OD 450 > 0.371). While, this confirmatory test did not show any cross-reactivity against rHc-HCA59 in positive sera of other organisms and negative sera (control) during the ratifying process of indirect ELISA.
Furthermore, five samples with OD 450 between positive and negative cut off values were negative in ELISA but in western blotting, specific antibodies were detected in these samples. One possible reason for this incongruity may be the blocking buffer used in the assay. The results of antibody assay significantly depend on the buffer system used [34]. The blocking buffer used in indirect ELISA was 5% BSA while, western blot analysis used 5% skimmed milk. Another possible cause for this discordance may be the concentration of antibody used in both assays. Lastly, false negative reactions possibly occurred due to the lower dilutions of secondary antibody selected in attempt to make lower OD values of normal goats. The concentration of secondary antibody used in indirect ELISA was 1:6000, while 1:5000 dilutions were used in western blot assay. False negative results are really undesirable, especially if a lot of animals are misdiagnosed. In this study, rHc-HCA59-based western blot assay provided effective support to eliminate the level of false negatives. Hence, the indirect ELISA applied together with the western blotting assay may avoid or decrease false negative results. Further study for the investigation of H. contortus prevalence is needed on a large scale for application of this assay in the field. The rHc-HCA59 produced in current study was expressed in the soluble fraction and was recoverable from culture easily. Different productions of rHc-HCA59 showed almost same yield and the same ELISA test results and proved the stability of antigen.

Conclusions
The indirect ELISA developed here with rHc-HCA59 as an antigen which can detect anti H. contortus antibodies in goat serum at good sensitivity and specificity. The western blot assay developed with rHc-HCA59 was able to detect antibodies that were false negatives in the ELISA test and showed increased sensitivity. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool to detect H. contortus infection during the prepatent period in goats.