Expression and immunolocalisation of TpFABP as a candidate antigen for the serodiagnosis of rabbit Taenia pisiformis cysticercosis

The larval stage of Taenia pisiformis, also known as Cysticercus pisiformis, is the causative agent of cysticercosis and the cause of severe health problems in rabbits that negatively impacts on husbandry production. To date, there is no fast detection method to identify early infections in rabbits. In the present study, a new dot-ELISA-based on an endogenous antigen fatty acid-binding protein (FABP) was developed for the detection of cysticercosis, and its potential was then evaluated using test serum samples. Immunolocalisation showed that T. pisiformis FABP (TpFABP) localised to the parenchyma of the bladder wall of the cysticercus and perinuclear cytoplasm of parenchyma of the adult parasite. After cloning and expression, recombinant TpFABP (rTpFABP) protein was used for serodiagnosis of T. pisiformis infection in rabbits by dot-ELISA. The antibody was detected 14 days post-infection in rabbits experimentally infected with T. pisiformis. Based on the necropsy results, the sensitivity and specificity of 169 serum samples tested by rTpFABP dot-ELISA were found to be 98.2% (54/55) and 92.1% (105/114), respectively. These data suggest that the dot-ELISA developed in this study has potential for detection of T. pisiformis infection in rabbits.


Introduction
Cysticercosis, an infection caused by the larvae of Taenia pisiformis (Bloch, 1780) [27], is one of the most common parasitic disease in rabbits [11]. During the life cycle of T. pisiformis, the cysticerci present in the abdominal cavity of infected rabbit are ingested by a definitive host (canids and felines), following which the adult T. pisiformis individuals parasitises and matures in the host small intestine [2,23]. The gravid proglottids of T. pisiformis released from infected dogs are in turn ingested by rabbit through contaminated food or water. The proglottids discharge oncospheres in the rabbit intestine and penetrate the intestinal mucosa and blood vessels. The oncospheres reach the liver parenchyma, then migrate to liver capsule, greater omentum and mesentery and develop into cysticerci [18,20]. China is the world's largest producer of rabbits [5], and T. pisiformis severely affects rabbit breeding. Rabbits infected with T. pisiformis are emaciated and have weak resistance to other diseases; in particular, it can also cause death especially for breeding rabbit.
The rapid and accurate detection of cysticercosis in rabbits is crucial for arresting its negative impact on husbandry production. In general, as there are no obvious early clinical symptoms in rabbits infected with T. pisiformis, it is a major challenge to control this disease. The presence of T. pisiformis-specific antibodies in serum from infected rabbits can provide the foundation for detection of this parasite [6,29]. Crude antigens from oncospheres or mature metacestodes have been used in previous studies [6,29]. However, due to the limited availability of crude parasite antigens, only a few serologic tests have been used to detect anti-T. pisiformis antibodies, including enzymelinked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) [6,29]. In addition, the standard ELISA and IFAT methods are too complex to be used routinely under field conditions. Keeping these considerations in mind, dot-ELISA is one of the better serodiagnostic strategies due to its sensitivity and convenience.
Fatty acid-binding proteins (FABPs), multigenic cytosolic proteins are found in most animal groups. They are involved in the uptake and transport of hydrophobic ligands to different cellular fates [10,13]. In helminthic parasites, FABPs are proven to be involved in acquisition and utilisation of host-derived hydrophobic substances, as well as in signalling and cellular interactions [16]. In the present study, a new FABP homologue TpFABP, from T. pisiformis was cloned and expressed and its immunolocalisation was then analyzed. Based on these results, a new recombinant FABP (rTpFABP) protein-based dot-ELISA was developed for the serodiagnosis of T. pisiformis infections in the rabbit industry.

Ethics statement
All animals were handled in strict accordance with animal protection law of the People's Republic of China (a draft of an animal protection law in China released on September 18, 2009)

TpFABP amplification and structural prediction
Total RNA was isolated from mature metacestodes (provided by the parasitology laboratory at the Sichuan Agricultural University, China) using Trizol reagent (Invitrogen, Shanghai, China) according to the manufacturer's instructions. The cDNA was obtained using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Shanghai, China) following the manufacturer's protocol. Based on the cDNA sequence of T. solium FABP (GenBank: DQ273765), the gene-specific primers for TpFABP were designed as follows (letters in parentheses represent the code of degenerate primers): PCR was performed in a 25 lL final volume, including 12.5 lL of PCR mixture (Invitrogen, Shanghai, China), 0.4 lM of each primer (forward and reverse), 1 lL of cDNA template and 9.5 lL ddH 2 O. The amplification conditions consisted of an initial denaturing step at 94°C for 5 min, followed by 35 cycles of amplification, 94°C for 50 s, 56°C for 45 s, and 72°C for 50 s and a final extension step at 72°C for 10 min. The PCR products were cloned into pMD19-T vector (TaKaRa, Dalian, China), and sequenced using an ABI PRISM TM 377XL DNA Sequencer (ABI, Foster City, USA). The new TpFABP sequence was deposited in GenBank with Accession Number GU205472. BepiPred 1.0 server (http://www.cbsdtu.dk/services/ BepiPred/) was used to predict the location of linear B-cell epitopes [17]. PredictProtein (http://www.predictprotein.org/) was used to infer the secondary structures [22]. The alignment of TpFABP amino acid sequences with those of other Taeniidae cestodes and Oryctolagus cuniculus was performed using ClustalX 1.83 software [25], and the MegAlign program of DNAstar software package [4] was utilised to calculate the percentage identities.

rTpFABP expression and western blotting
The expression sequence of TpFABP was amplified by F2 5 0 -GGGATCCATGGAGGCATTCCTCGGTA-3 0 and R2 5 0 -CGCTCGAGTTACGTCCCTTTAAAGTAGGTTC-3 0 using the same PCR conditions described above. The PCR products were subcloned into the BamH1 and Xhol sites of the expression vector pET32a (Novagen, Darmstadt, Germany) and expressed in Escherichia coli BL21 (DE3) induced by 0.6 M isopropyl-b-D-thiogalactoside (IPTG). The TpFABP fusion proteins (fused with the Trx-Tag TM thioredoxin) were dissolved using 8 M urea, purified on an Ni-IDA sefinoseTM resin (Bio-Rad, California, USA), and the concentration of the purified protein was determined by a Biophotometer (Eppendorf, Hamburg, Germany) using a BCA Protein Assay Kit (Beyotime, Haimen, China) according to the manufacturer's instructions.
The rTpFABP protein was separated on a 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose (NC) filtre membrane (Sigma, San Francisco, USA) by electroblotting. The membrane was washed three times for 5 min with 0.01 M phosphate buffer solution (PBS), and blocked with 5% non-fat milk powder in 0.01 M PBS for 2 h at room temperature. The rabbit antisera was probed with 1:100 dilution, and added directly to the blocking solution (including 5% non-fat milk powder and 0.01 M PBS) at 4°C overnight. The rabbit T. pisiformis antisera were sourced from animals at 50 days post-experimental infection (provided by the laboratory of parasitology in Sichuan Agricultural University). The membrane was then washed three times with PBS for 5 min each, and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000 dilution; Sigma, San Francisco, USA) for 1.5 h at room temperature. Finally, the membranes were washed three times with 0.01 M PBS for 5 min. The membrane was exposed to HRPdiaminobenzidine (DAB) substrate (Tiangen, Beijing, China) following the manufacturer's instructions.

Immunolocalisation
Polyclonal antisera against rTpFABP protein were raised in two 9-week old female parasite-free New Zealand White rabbits (obtained from Laboratory Animal Centre of Sichuan Agricultural University, China) by consecutive subcutaneous inoculation of the rTpFABP protein as described by Hu et al. (2002) [12]. The immune sera were obtained by centrifugation at 4,000· g for 10 min in room temperature. IgG fractions were isolated using a Protein G affinity chromatography column (Bio-Rad, California, USA) and stored at À80°C.
The collection of fresh mature metacestodes and adult of T. pisiformis was previously described [30]. The parasites were freshly fixed in Bouin's solution for 24 h and then embedded in paraffin. The sections were cut serially at 7 lm thickness using a slicer (Leica, Wetzlar, Germany). Immunolocalisation was carried out using a streptavidin biotin complex-peroxidase (SABC-POD) with rabbit IgG kit (Boster, Wuhan, China) according to the manufacturer's protocol. The purified IgG fractions against TpFABP were diluted to 200 times. Peroxidase activity was visualised by incubating sections with a DAB-Plus Kit (Boster, Wuhan, China). Finally, the slides were counterstained with Mayer's haematoxylin, examined with an optical microscope and photographed (Nikon, E800).

Collection of sera
Three healthy rabbits were used as the negative control. Positive polyclonal antisera against rTpFABP protein were as described above.
Experimental sera were collected from seven 90-day old healthy white female New Zealand White rabbits (Oryctolagus cuniculus) sourced from the Laboratory Animal Centre of Sichuan Agricultural University, China. The rabbits were orally infected with 5,000 mature viable T. pisiformis eggs. Serum samples were collected every 7 days. The rabbits were humanely sacrificed (50 mg/kg ketamine and 100 mg/kg sodium pentobarbital [Sigma, San Francisco, USA]) at 49 days post-infection. Necropsies were as performed as previously described [3].
Test serum samples (n = 169) were collected from rabbits from a local slaughterhouse, and the serum was separated and stored at À20°C. The abdominal cavity of rabbits was examined for the presence of T. pisiformis cysticerci, as previously described.

Dot-ELISA
Total T. pisiformis-specific IgG antibodies of rabbits were detected by dot-ELISA using rTpFABP as the test antigen following the methodology described by Piña et al. (2011) [21] with some modifications. Briefly, 50, 100 and 200 ng of the purified antigen (10 lg/mL) were dotted on marked circular regions at the centre of each NC strip. The positive polyclonal sera (1:50, 1:100, 1:200) and goat anti-rabbit IgG-HRP conjugate (1:7000) (Sigma, San Francisco, USA) were diluted using PBS Tween-20 and 5% (w/v) non-fat milk. ELISA dots were detected by HRP-DAB (Invitrogen, Shanghai, China). The visual reading by two independent observers was the same for all tests, and no difference in colour intensity was observed. In addition to the background colour, a tan-yellow reaction was designated as a positive result. The best dilution of rTpFABP antigen and rabbit sera was determined by the colour reaction intensity in the positive dot-ELISA.
The remainder of the experimental and test sera were detected by dot-ELISA and described as above. The percentage sensitivity was calculated as dot-ELISA positive · 100/true positive, and the percentage specificity was calculated as dot-ELISA negative · 100/true negative [28].
rTpFABP expression and western blotting rTpFABP was successfully expressed in E. coli strain BL21 (DE3). The molecular weight of the recombinant protein was about 36 kDa, and the solubility of rTpFABP protein was identified as inclusion bodies. rTpFABP was recognised by rabbit T. pisiformis cysticercosis antisera in western blotting analysis ( Figure 2).

Immunolocalisation of TpFABP
TpFABP was localised in perinuclear cytoplasm (PC) of adult T. pisiformis proglottids (Figure 3). Furthermore, the positive signal was observed in the parenchyma of the bladder wall of the cysticercus, and intensely localised in outer layer of cystic wall (OCW) and middle layer of cystic wall (MCW).

Dot-ELISA
Combinations of various amount of antigens tested with various dilutions of positive polyclonal sera did not show any differences (data not shown). Therefore, 200 ng of rTpFABP antigen and 1:100 of sera in each strip were determined to be the optimal combination for the full set of sample tests. The experimental sera were found to be positive at 14 days postinfection, and remained through to 49 days post-infection ( Figure 4) when the rabbits were sacrificed.
Of the 169 rabbits sampled from the slaughterhouse, T. pisiformis cysticerci were found in 55 rabbits by necropsy, but it was not present in 114 rabbits. Fifty-four rabbits tested positive and 105 were negative by dot-ELISA. Thus, the sensitivity and specificity of dot-ELISA using rTpFABP antigen to detect T. pisiformis cysticercus were 98.2% (54/55) and 92.1% (105/114), respectively. There was no cross-reaction between rTpFABP and the positive sera of Sarcoptes scabiei, Psoroptes cuniculi, Eimeria spp. and Passalurus ambiguus.

Discussion
Nine groups of FABPs have been identified in mammals with variable primary structures (identity, 20-70%), but all the members of this family share a superimposable tertiary structure [1]. In our study, TpFABP amino acid sequence shared the highest identity (95%) with TsFABP1 in primary structure, and had the 10-stranded b-barrel fold, typical for the family of intracellular lipid-binding proteins [15]. Six locations of linear B-cell epitopes between TpFABP, TsFABP1, TsFABP2, EgFABP1 and EgFABP2 had a similar distribution. Together, these suggest a common conservation of this family of genes within cestode parasites as well as a possible common ancestral gene. However, low identities (23.88-41.04%) between the available FABP amino acid sequences from rabbit and the TpFABP from T. pisiformis indicated that they would not share a common ancestral gene.
The adult stages of parasitic platyhelminths are dependent on carbohydrates for their energy metabolism [26], but a  functional b-oxidation pathway has not been demonstrated in cestodes [24]. FABPs synthesise most of their own lipids de novo by combining hydrophobic groups to help platyhelminths, especially long-chain fatty acids and cholesterol [19].
Meanwhile, FABPs have been described as intracellular carriers of fatty acid (FA) [7]. EgFABP1 is specifically expressed in the protoscolex larval stage and associated with protoscolex larval development [8]. Abundant expressions of TsFABP1 and TsFABP2 were found in the canal region of adult T. solium [16], and TsFABP1 also was positive in subtegumental cytons of tissue sections from cysticerci from T. pisiformis [14]. TsFABP1 has been demonstrated to be involved in the transport of several fatty acids required for T. solium nourishment. It is plausible that TsFABP1 is involved in the mechanism by which FAs are mobilised from the translocation site on the tegument membrane, to other cellular compartments in the syncytium. In this study, the positive distribution of TpFABP was similar to T. crassiceps FABP in cysticerci [14], and was widely distributed in the parenchyma of the bladder wall in the cysticerci. The localisation of TpFABP in the cysticerci suggested that the cystic wall layer might be a primary location in the scolex where FA uptake occurs. In tissue sections of adult T. pisiformis, the positive staining distribution was the same as T. solium FABP1 [14]. TpFABP is probably involved in the uptake and transport of fatty acid molecules in the perinuclear cytoplasm to maintain the survival of adult T. pisiformis. Thus, the biological role of TpFABP in T. pisiformis may be similar to that of FABP1 in T. solium [9]. With guaranteed supply of fatty acids for survival by abundant fatty acid-binding proteins, parasites can adjust their biological mechanism to adapt to a changing environment.
Because T. pisiformis infection in rabbits is not associated with specific clinical symptoms, it is difficult to detect it in the early infective stage (up to 7 weeks post-infection). This stage involved adherence of the oncosphere to and migration  across the intestinal wall, followed by transport to the liver parenchyma via the circulatory system. The oncosphere finally migrates to the abdominal cavity of rabbits [18]. Detection of the antibody against T. pisiformis cysticerci could be useful for early detection and treatment of this infection. Circular antibodies in experimental T. pisiformis infections of rabbits have been previously investigated [6], which indicated that antibodies were detected in rabbit sera by 2 weeks post-infection using the in vitro-derived T. pisiformis metacestode antigen. In addition, Wang et al. (2009) [20] investigated the dynamic profile of antibodies in rabbits experimentally infected with T. pisiformis using crude antigen from mature metacestodes, and found that the antibody levels started to increase at week three postinfection and were up to the highest level at week eight post-infection. In our study, dot-ELISA of rTpFABP was successfully established to detect rabbit T. pisiformis cysticercosis under the optimum conditions. The antibodies in experimental sera could be detected by dot-ELISA in the early stage of infection (14 days), and lasted 7 weeks (49 days) post-infection. The dot-ELISA was developed in this study with a high sensitivity (98.2%) and specificity (92.1%) for 169 tested sera samples when compared with the results of necropsy. The cross-reactivity of several parasites of rabbit, a potential probability of false positives, was also carried out, and no cross-reactivity was observed with our panel of positive sera of other parasites. Thus, rTpFABP antigen can detect specifically T. pisiformis cysticercosis in tested rabbits.
Together, the data shows that rTpFABP is a suitable diagnostic antigen, and the results of study demonstrate the efficacy of the FABP-based dot-ELISA for potential detection of T. pisiformis cysticercosis in rabbit.