Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens

Parasitology International 64 (2015) 288–294

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Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens Bartłomiej Ferra, Lucyna Holec-Gąsior, Józef Kur ⁎ Faculty of Chemistry, Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Narutowicza 11/12 Street, Gdańsk 80-233, Poland

a r t i c l e

i n f o

Article history: Received 30 December 2014 Received in revised form 16 March 2015 Accepted 17 March 2015 Available online 25 March 2015 Keywords: Animals Toxoplasma gondii Recombinant antigens ELISA Serodiagnosis

a b s t r a c t Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1 + rMIC1 + rMAG1, M2: rSAG2 + rGRA1 + rROP1, and M3: rGRA1 + rGRA2 + rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific antiT. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly used TLA. © 2015 Published by Elsevier Ireland Ltd.

1. Introduction Toxoplasmosis is a zoonotic infection with the protozoan parasite Toxoplasma gondii, which can affect most warm-blooded animals from mammals to birds all over the world. Humans and animals can be intermediate hosts and can become infected by ingestion of food and water contaminated with T. gondii oocysts, by consumption of tissue cysts in infected animal tissues, or congenitally [1]. In farm animals, T. gondii infection generally shows no signs or symptoms despite having been infected with the parasite. However, the main result of the infection in farm animals is abortion, which especially causes significant reproductive losses and, as a result, economic losses. The parasite can pass on to the fetus inside pregnant animals which can lead to stillbirths. During early pregnancy, ingestion of oocysts is known to cause reabsorption. During the mid-phase of pregnancy, fetal mummification can occur. Animals infected during later stages of pregnancy can have weaker ⁎ Corresponding author. Tel.: +48 58 3472302; fax: +48 58 3471822. E-mail address: [email protected] (J. Kur).

http://dx.doi.org/10.1016/j.parint.2015.03.004 1383-5769/© 2015 Published by Elsevier Ireland Ltd.

offspring. These young may survive but can also die within a few weeks after birth. Some adult animals after infection during pregnancy may even become barren [2]. T. gondii infection of farm animals also has implications for public health because consumption of infected raw or undercooked meat, milk, contaminated food, or water can facilitate zoonotic transmission [3,4]. In farm animals, tissue cysts of T. gondii are most frequently observed in tissues of infected sheep, goats, cows, pigs, and less frequently in poultry, rabbits, and horses [1]. Under natural conditions, seroprevalence of toxoplasmosis in farm animals depends, inter alia, on the age of animals, the geographical area, even on the hygienic conditions on the farms, and farm management. The most common sources of human infection include ingestion of tissue cysts in raw or undercooked pork, beef, mutton, and in some countries horse meat. In the European Union, zoonotic diseases have to be reported according to their epidemiological situation (Directive 2003/99/EC), but despite this regulation, no surveillance data are available for T. gondii [5]. Based on data obtained in some regions of European countries, it is estimated that in Europe seroprevalence of toxoplasmosis may vary in sheep from 4% up to 92% (average 35.9%) [1], in horses

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from 0% up to 80% (average 25.8%) [6], and in pigs from 0% up to 64% (average 6%) [1]. Diagnosis of T. gondii infection in humans and animals is based on detection of cysts by microscopy, bioassay, polymerase chain reaction (PCR), or detection of antibodies by serology. However, these methods have their limitations. None of these methods are suitable for the analysis of a large number of samples [7]. For this reason, serological techniques play a major role in the diagnosis of toxoplasmosis. Among available serological tests, the enzyme-linked immunosorbent assay (ELISA) has been adapted for the detection of IgM, IgG, and IgA class antibodies, and it is considered the gold standard today. Serological methods for the detection of T. gondii specific antibodies usually involve the preparation of whole Toxoplasma lysate antigens (TLA) from tachyzoites grown in mice or tissue cultures. The production of these native parasitic antigens is inappropriate because it is expensive and laborious; moreover, the quantity of the antigen mixture is difficult to standardize. Heterologous expression of recombinant T. gondii antigens in a prokaryotic or eukaryotic system could be a potential alternative for producing safer diagnostic antigens with lower costs of production and purification. In recent years, the utility of recombinant antigens is demonstrated in the diagnosis of toxoplasmosis in humans [8]. The use of recombinant antigens for the diagnosis of T. gondii infections proves highly beneficial to improving the standardization of the method since the antigen composition of the test is precisely known. Furthermore, the advantages of these proteins include reduced production costs of antigens and the possibility of using more than one defined antigen for the detection of specific antibodies. A completely new approach is the construction of a new generation of recombinant products, the socalled chimeric antigens, which can replace native antigens from a lysed whole parasite. This new kind of diagnostic tool contains different immunoreactive epitopes from various antigens of T. gondii. So far, there have been only a few studies demonstrating a usefulness of the recombinant chimeric antigens in the detection of specific anti-T. gondii antibodies in human sera [9–14]; however, they have not been implemented in diagnostic tests yet. Recombinant chimeric antigens have major advantages over commonly tested individual recombinant antigens and/or mixture of antigens such as (1) higher sensitivity of immunoassay tests; (2) the quality of the chimeric antigen, which is easy to define; and (3) the method can be easily standardized. The use of recombinant chimeric antigens in serological tests of toxoplasmosis in animals has not been reported yet. The results of our previous research on recombinant chimeric antigens in the serodiagnosis of toxoplasmosis in humans, such as MIC1-MAG1 [12], and MIC1-MAG1-SAG1 [13], which are very promising, encouraged us to further work on this topic. In our opinion, it is necessary to develop new diagnostic tools and methods for detection of T. gondii infection in animals. This will allow for reducing the reproductive losses of animals and minimize the risk of parasite transmission to humans through the ingestion of raw or undercooked meat. The aim of this study was to improve the performance of the IgG ELISA on the basis of a new recombinant product, chimeric antigens. This study demonstrated the diagnostic utility of the T. gondii recombinant chimeric antigens composed of different combinations of three well-characterized proteins, including dense granule antigens (GRA1, GRA2, and GRA6), cyst matrix antigen (MAG1), microneme antigen (MIC1), rhoptry antigen (ROP1), and surface antigens (SAG1 and SAG2). The selection of these antigens is based on previous results obtained in the immunoassays, which determine the reactivity of these proteins with specific anti-T. gondii antibodies. The dense granule antigens (GRA) belong to a group of excreted/secreted antigens (ESA), which appear as both a soluble or a membrane-associated form in the vacuole, and play an important role in the structural modification of the parasitophorous vacuole (PV) [15,16]. The ESA antigens have been shown as important components in the process of invasion and replication of T. gondii within the host cell; it has also been observed that ESA proteins are highly immunogenic [17]. The cyst matrix protein MAG1 is an important antigen,

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which is responsible for building the matrix of the cyst and the cyst wall surrounding the bradyzoites [18]. Moreover, this antigen is a very immunogenic protein, which is expressed in both tachyzoite and bradyzoite and indicates that the humoral response occurs very early after infection [19,20]. The MIC1 antigen belongs to a group of microneme proteins (MIC) which are secreted first, after contact with the host cell. All of MIC proteins are involved in host cell recognition and attachment [21]. Moreover, MIC1 has been described as lactosespecific lectin having the ability to bind to the lactose-containing glycoprotein receptors located on the surface of host cells [22]. The soluble rhoptry protein ROP1 also belongs to a group of ESA antigens; this protein is released into the nascent PV during early stages of host cell invasion and then quickly disappears [23–25]. The surface of T. gondii is covered by a family of glycosylphosphatidylinositol (GPI)-anchored antigens (SAGs) and SAG-related sequence (SRS) proteins [26]. The major surface antigen SAG1 and antigen SAG2 (identified as an intrinsically unstructured protein) can interact with many cellular and surface molecules of infected host [27–29]. 2. Materials and methods 2.1. T. gondii recombinant antigens, recombinant chimeric antigens, and TLA Eight T. gondii recombinant antigens, rGRA1 (amino acids 24–190), rGRA2ex2 (amino acids 51–185), rGRA6 (amino acids 30–231), rMAG1 (amino acids 30–222), rMIC1ex2 (amino acids 25–182), rROP1 (amino acids 85–396), rSAG1 (amino acids 49–313), and rSAG2 (amino acids 30–231), containing His6-tags at both ends for the purification by metal-affinity chromatography were obtained as previously described [30–34]. Five T. gondii recombinant chimeric antigens, GRA1-GRA2-GRA6 (amino acids: 31–170 of GRA1, 51–185 of GRA2, and 40–230 of GRA6), MIC1-MAG1-SAG1S (amino acids: 25–182 of MIC1, 30–222 of MAG1, and 49–198 of SAG1), SAG1L-MIC1-MAG1 (amino acids: 49–311 of SAG1, 25–182 of MIC1, and 30–222 of MAG1), SAG2-GRA1ROP1S (amino acids: 31–170 of SAG2, 26–190 of GRA1, and 85–250 of ROP1), and SAG2-GRA1-ROP1L (amino acids: 31–170 of SAG2, 26–190 of GRA1, and 85–396 of ROP1), containing His6-tags at both ends for the purification by metal-affinity chromatography were obtained in analogous manner as the previously described for MIC1-MAG1-SAG1S [13]. All of the recombinant proteins were analyzed by SDS–PAGE on 12% acrylamide gels and stained with Coomassie blue. The calculated molecular masses for GRA1-GRA2-GRA6, MIC1-MAG1-SAG1S, SAG1LMIC1-MAG1, SAG2-GRA1-ROP1S, and SAG2-GRA1-ROP1L were 59.1 kDa, 57.6 kDa, 69.2 kDa, 56.4 kDa, and 72.2 kDa, respectively. The recombinant antigens were purified by means of a one-step chromatography procedure using metal-affinity chromatography with Ni2+ bound to iminodiacetic acid-agarose (Novagen). The concentration of purified proteins was determined by the Bradford method using bovine serum albumin (BSA) as a standard. The yields of purified GRA1GRA2-GRA6, MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1ROP1S, and SAG2-GRA1-ROP1L proteins were 48 mg/L, 30 mg/L, 52 mg/L, 36 mg/L, and 37 mg/L, respectively, with a purity of approximately 95% for each protein. Toxoplasma lysate antigen (TLA) from tachyzoites (strain RH) was prepared according to the method previously described [35]. 2.2. Test sera Three groups of serum samples derived from different animal species: 118 horses sera, 215 ovine sera, and 192 swine sera were received from Veterinary Hygiene Station (Gdańsk, Poland). These serum samples have been obtained from epidemiological studies conducted on a farm animal population from the northern region of Poland. All serum samples were analyzed and divided into seropositive and seronegative

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groups in accordance with the results obtained using the agglutination test (Toxo-Screen DA, bioMérieux) and immunofluorescence test, with the use of slides coated with T. gondii antigen (bioMérieux). All of serum samples were also seronegative for specific anti-Neospora caninum antibodies by the use of commercial competitive-inhibition enzyme-linked immunosorbent assay (cELISA) (VMRD, Inc). Moreover, in this study 15 serum samples seronegative for T. gondii and seropositive for N. caninum were used for estimating the recombinant antigens, recombinant chimeric antigens, and TLA cross-reactivity with specific N. caninum antibodies and were used to determine cutoff value. None of these sera was found to score above the cutoff what indicates the absence of cross-reactivity. Horses serum samples were divided into two groups: group I—36 sera from naturally infected horses (IgG anti-T. gondii positive) and group II—82 from healthy animals (IgG anti-T. gondii negative). Thirty-two of the seronegative serum samples were used to determine the cutoff value. Ovine serum samples were divided into two groups: group I—140 sera from naturally infected sheep and group II—75 from healthy animals. Twenty-four of the seronegative serum samples were used to determine the cutoff value. Swine serum samples were divided into two groups: group I—81 sera from naturally infected pigs and group II—111 from healthy animals. 24 of the seronegative serum samples were used to determine the cutoff value. 2.3. IgG ELISA MaxiSorp multiwells plates (Nunc, Denmark) were coated with mixtures of recombinant proteins, recombinant chimeric antigens, or with a TLA at final concentrations of 2.5 μg/ml for each recombinant protein and 1 μg/ml for the TLA in a coating buffer (0.05 M carbonate buffer, pH 9.6). After overnight incubation at 4 °C, the plates were washed three times with PBS–0.1% Triton X-100 and blocked for 1 h at 37 °C in blocking solution (1% bovine serum albumin, 0.5% Triton X-100 in PBS). The cells were then washed three times and incubated for 1 h at 37 °C with the animals serum diluted 1:100 in blocking solution. Next, the plates were washed three times with washing buffer and respectively incubated with: anti-sheep or anti-swine IgG peroxidase-labeled conjugates (Jackson ImmunoResearch) diluted 1:16000, and anti-horse IgG peroxidase-labeled conjugates (Jackson ImmunoResearch) diluted 1:40000 in blocking solution for 1 h at 37 °C, after which o-phenylenediamine dihydrochloride chromogenic substrate (Sigma) was added. After 45 min of incubation at 37 °C in darkness, the reaction was stopped by the addition of 2 M sulfuric acid, and the OD492 was measured using a microtiter plate reader (Multiskan FC; Thermo Scientific). Each serum sample was examined twice. The results were determined for each sample by calculating the mean OD reading of duplicate wells. A positive result was defined as any value higher than the average OD reading plus 2 standard deviations (cutoff value) obtained with

serum samples from the control groups II, which consisted of seronegative serum samples. 3. Result 3.1. Reactivity of IgG antibodies from horses sera in ELISA with mixtures of recombinant proteins and recombinant chimeric antigens Eight independent IgG ELISAs were developed using different five recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2GRA6), and three different mixtures of selected recombinant proteins from which the individual chimeric antigens are composed (M1: rSAG1 + rMIC1 + rMAG1, M2: rSAG2 + rGRA1 + rROP1, and M3: rGRA1 + rGRA2 + rGRA6) as a coating antigen to evaluate the potential of each of these proteins for the serodiagnosis of horses toxoplasmosis. The obtained results were referenced to the results obtained in IgG ELISA using the same pool of positive and negative sera, which was tested with TLA. For all antigens, the same pool of sera from group II (negative control) was used to obtain the relative absorbance of each serum sample and the cutoff value (Table 1). The sensitivity of IgG ELISAs calculated from all 36 positive serum samples tested were diverse for the individual antigens, whereas the specificity obtained for all antigens preparation was 100% (Table 1). The 100% sensitivity was observed only in IgG ELISAs with TLA and chimeric antigen SAG2GRA1-ROP1L. Relatively high reactivity was noticed for mixture M1 (rSAG1 + rMIC1 + rMAG1) and chimeric antigen GRA1-GRA2-GRA6, which amounted to 88.9% and 86.1%, respectively. Definitely lower sensitivity at level 77.8% was observed for IgG ELISAs with chimeric antigen SAG1L-MIC1-MAG1 and mixture M2 (rSAG2 + rGRA1 + rROP1), even lower reactivity was obtained for chimeric antigen MIC1-MAG1-SAG1S (75%). In IgG ELISA with mixture M3 (rGRA1 + rGRA2 + rGRA6), only 66.7% serum samples with specific anti-T. gondii antibodies were identified correctly. The lowest sensitivity was noticed for IgG ELISA with chimeric antigen SAG2-GRA1-ROP1S, and it amounted to 50%. For some chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, and GRA1-GRA2-GRA6) and mixtures (M1: rSAG1 + rMIC1 + rMAG1, and M3: rGRA1 + rGRA2 + rGRA6), the mean and the range of absorbance values were relatively high, but they were also characterized by high cutoff values. Comparable values of absorbance were obtained for the chimeric antigen SAG2-GRA1-ROP1L and TLA. All of the 36 positive serum samples from group I reacted above the cutoff value (range absorbance: 0.439–1.358 and 0.445–0.910 for the chimeric antigen SAG2-GRA1-ROP1L and TLA, respectively), while none of the 50 negative serum samples from group II were found to score above the cutoff value (range absorbance: 0.129–0.433 and 0.202–0.429 for the chimeric antigen SAG2-GRA1-ROP1L and TLA, respectively). Moreover, also the reactivity of chimeric antigen SAG2-GRA1-ROP1L (mean absorbance: 0.714) with specific anti-T.gondii antibodies was comparable to the reactivity of TLA (mean absorbance: 0.578).

Table 1 Comparison of the immunoreactivities of the mixture of antigens (M1: rMIC1 + rMAG1 + rSAG1, M2: rSAG2 + rGRA1 + rROP1, M3: rGRA1 + rGRA2 + rGRA6), the MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, GRA1-GRA2-GRA6 chimeric antigens, and the TLA in the IgG ELISA using a pool of 86 horse sera. Recombinant antigens/mixtures of recombinant antigens

MIC1-MAG1-SAG1S SAG1 L-MIC1-MAG1 M1 (rSAG1 + rMIC1 + rMAG1) SAG2-GRA1-ROP1S SAG2-GRA1-ROP1L M2 (rSAG2 + rGRA1 + rROP1) GRA1-GRA2-GRA6 M3 (rGRA1 + rGRA2 + rGRA6) TLA

Pool of seropositive sera (n = 36)

Pool of seronegative sera (n = 50)

No. (%) of positive sera

Mean absorbance value

Range absorbance values

No. (%) of positive sera

Mean absorbance value

Range absorbance values

27 (75) 28 (77.8) 32 (88.9) 18 (50) 36 (100) 28 (77.8) 31 (86.1) 24 (66.7) 36 (100)

1.059 1.088 1.293 0.596 0.714 0.889 1.416 1.348 0.578

0.401–2.321 0.397–2.435 0.470–2.637 0.236–1.956 0.439–1.358 0.238–1.871 0.669–2.518 0.409–2.412 0.445–0.910

0 0 0 0 0 0 0 0 0

0.561 0.573 0.654 0.314 0.332 0.388 0.730 0.720 0.346

0.299–0.797 0.283–0.790 0.329–0.788 0.154–0.458 0.129–0.433 0.156–0.511 0.313–0.986 0.375–1.002 0.202–0.429

Cutoff value

0.828 0.793 0.799 0.517 0.437 0.512 1.002 1.021 0.433

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Table 2 Comparison of the immunoreactivities of the mixture of antigens (M1: rMIC1 + rMAG1 + rSAG1, M2: rSAG2 + rGRA1 + rROP1, M3: rGRA1 + rGRA2 + rGRA6), the MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, GRA1-GRA2-GRA6 chimeric antigens, and the TLA in the IgG ELISA using a pool of 191 ovine sera. Recombinant antigens/mixtures of recombinant antigens

Pool of seropositive sera (n = 140) No. (%) of positive sera

Mean absorbance value

Range absorbance values

Pool of seronegative sera (n = 51) No. (%) of positive sera

Mean absorbance value

Range absorbance values

MIC1-MAG1-SAG1S SAG1 L-MIC1-MAG1 M1 (rSAG1 + rMIC1 + rMAG1) SAG2-GRA1-ROP1S SAG2-GRA1-ROP1L M2 (rSAG2 + rGRA1 + rROP1) GRA1-GRA2-GRA6 M3 (rGRA1 + rGRA2 + rGRA6) TLA

137 (97.9) 140 (100) 109 (77.9) 140 (100) 140 (100) 140 (100) 134 (95.7) 129 (92.1) 140 (100)

0.658 1.650 0.724 1.079 1.479 1.291 0.762 0.847 0.544

0.294–1.223 0.612–2.655 0.410–1.633 0.419–3.066 0.447–3.530 0.530–3.753 0.273–1.673 0.193–1.798 0.316–1.066

0 0 4 (7.8) 0 0 0 0 0 0

0.271 0.380 0.433 0.273 0.258 0.345 0.262 0.280 0.266

0.119–0.372 0.182–0.485 0.120–0.834 0.109–0.397 0.111–0.401 0.106–0.500 0.060–0.432 0.061–0.502 0.129–0.313

3.2. Reactivity of IgG antibodies from ovine sera in ELISA with mixtures of recombinant proteins, and recombinant chimeric antigens In order to assess the potential utility of recombinant chimeric antigens and mixtures of recombinant proteins to the serodiagnosis of ovine toxoplasmosis, the same procedure was used as in the case of horses. The sensitivity of IgG ELISAs calculated from all 140 positive serum samples tested varied for the individual antigens, whereas the specificity obtained for almost all antigens preparation was 100%, only mixture M1 (rSAG1 + rMIC1 + rMAG1) had a lower specificity at level 92.2% (Table 2). The 100% sensitivity was observed in IgG ELISAs with TLA, chimeric antigens SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and mixture M3 (rSAG2 + rGRA1 + rROP1). The high sensitivity more than 90% was noticed for IgG ELISAs with chimeric antigens MIC1-MAG1-SAG1S, GRA1-GRA2-GRA6, and mixture M3 (rGRA1 + rGRA2 + rGRA6), which amounted to 97.9%, 95.7%, and 92.1%, respectively. The lowest sensitivity at level 77.9% was obtained for IgG ELISA with mixture M1 (rSAG1 + rMIC1 + rMAG1). The highest mean and range of absorbance values were noticed for chimeric antigens SAG1L-MIC1-MAG1 (mean absorbance value 1.650), SAG2GRA1-ROP1L (mean absorbance value 1.479), SAG2-GRA1-ROP1S (mean absorbance value 1.079), and mixture M2 (mean absorbance value 1.291). Moreover, the mean and the range of absorbance values for this recombinant protein preparation were definitely higher that values obtained for TLA (mean absorbance value 0.544, and range absorbance value 0.316–1.066).

3.3. Reactivity of IgG antibodies from swine sera in ELISA with mixtures of recombinant proteins, and recombinant chimeric antigens In the next part of the study, the diagnostic utility of the same recombinant proteins in the serodiagnosis of swine toxoplasmosis was assessed. The sensitivity of IgG ELISAs calculated from all 81 positive

Cutoff value

0.373 0.487 0.574 0.400 0.406 0.525 0.433 0.505 0.314

serum samples tested varied for the individual antigens, whereas the specificity obtained for all antigens preparation was 100% (Table 3). All IgG-positive pig serum samples were detected only by the TLA. The slightly lower sensitivity (above 90%) was observed in IgG ELISAs based on a recombinant chimeric antigen such as GRA1-GRA2-GRA6 (96.3%), SAG2-GRA1-ROP1L (93.8%), and SAG2L-MIC1-MAG1 (90.1%). Relatively high sensitivity was noticed for mixture of antigens M1 (rSAG1 + rMIC1 + rMAG1), and M2 (rSAG2-rGRA1-rROP1), which amounted to 88.9% and 81.5%, respectively. Definitely lower sensitivity was observed for IgG ELISAs with cocktail M3 (rGRA1 + rGRA2 + rGRA6), and chimeric antigen MIC1-MAG1-SAG1S, at level 54.3% and 45.7%, respectively. The recombinant chimeric antigen SAG2-GRA1ROP1S can detect specific anti-T. gondii antibodies only in 28.4% of serum samples. The mean and the range of absorbance values observed for the most reactive chimeric antigens GRA1-GRA2-GRA6 (mean absorbance value 0.694), SAG2-GRA1-ROP1L (mean absorbance value 0.550), and SAG1L-MIC1-MAG1 (mean absorbance value 0.691) were comparable to the results obtained for TLA (mean absorbance value 0.734). Moreover, the difference between absorbance values for the positive and negative sera was the greatest for these chimeric antigens. 4. Discussion In this study, we developed IgG ELISAs assays based on recombinant T. gondii chimeric antigens (composed of three different T. gondii proteins), with reference to the assays based on a mixture of three recombinant T. gondii antigens or TLA. All these chimeric antigens gave 100% specificity with sheep, pig, and horse sera, some of them also have very high sensitivity between 90% and 100%. Over the past 20 years, numerous recombinant antigens of T. gondii have been evaluated for their potential as diagnostic antigens for the detection of specific antibodies in human serum samples [8]. However, recombinant antigens are not frequently used in the serodiagnosis of toxoplasmosis in farm animals. To date, only a few studies

Table 3 Comparison of the immunoreactivities of the mixture of antigens (M1: rMIC1 + rMAG1 + rSAG1, M2: rSAG2 + rGRA1 + rROP1, M3: rGRA1 + rGRA2 + rGRA6), the MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, GRA1-GRA2-GRA6 chimeric antigens, and the TLA in the IgG ELISA using a pool of 168 swine sera. Recombinant antigens/mixtures of recombinant antigens

Pool of seropositive sera (n = 81)

Pool of seronegative sera (n = 87)

Cutoff value

No. (%) of positive sera

Mean absorbance value

Range absorbance values

No. (%) of positive sera

Mean absorbance value

Range absorbance values

MIC1-MAG1-SAG1S SAG1 L-MIC1-MAG1 M1 (rSAG1 + rMIC1 + rMAG1) SAG2-GRA1-ROP1S SAG2-GRA1-ROP1L M2 (rSAG2 + rGRA1 + rROP1) GRA1-GRA2-GRA6 M3 (rGRA1 + rGRA2 + rGRA6) TLA

37 (45.7) 73 (90.1) 72 (88.9) 23 (28.4) 76 (93.8) 66 (81.5) 78 (96.3) 44 (54.3) 81 (100)

0.433 0.691 0.664 0.252 0.550 0.436 0.694 0.705 0.734

0.184–1.159 0.248–1.304 0.182–1.308 0.104–1.116 0.174–1.488 0.114–1.112 0.300–1.909 0.169–1.738 0.414–1.337

0 0 0 0 0 0 0 0 0

0.271 0.321 0.284 0.179 0.273 0.224 0.329 0.432 0.297

0.063–0.393 0.059–0.391 0.058–0.407 0.066–0.261 0.062–0.375 0.062–0.274 0.096–0.382 0.112–0.676 0.059–0.364

0.404 0.393 0.415 0.262 0.384 0.275 0.387 0.677 0.369

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demonstrated the usefulness of T. gondii recombinant antigens in ELISA, enzyme-linked immunoassay (EIA), latex agglutination test (LAT), or rapid immunochromatographic test (ICT) for detection of specific antibodies in the ovine and swine sera [35–41]. Furthermore, until now, there has been no report of using recombinant antigens for the serodiagnosis of horses. Previously described research shows the possibilities of using single recombinant antigens in the serodiagnosis of toxoplasmosis in sheep and pigs. Unfortunately, this approach is not always satisfactory. Immunoassays such as IgG ELISAs based on single proteins can give variable results, and the detection of all positive sera which contain specific antibodies is impossible. The main explanation for this state of affairs is the fact that the humoral immune response of the host is strictly correlated with the stage of infection. Most of the T. gondii antigens are characteristic of a particular developmental form of the parasite and appear in specific stages of the infection. For these reasons, also specific IgG antibodies may be present at one stage of infection but absent at another stage. The solution to this problem is the use for IgG ELISA a mixture of two or three recombinant antigens, which are characteristic of different stage of T. gondii infection. The higher sensitivity of immunoassay based on a combination of proteins results from the fact that several different epitopes of various antigens may be recognized for specific antibodies present in the serum from acute and/or chronic T. gondii infection. Unfortunately, this approach also has its disadvantages such as (1) production and purification of few antigens and (2) need for the standardization of the mixture and diagnostic test. The construction of recombinant chimeric antigens allows for the elimination of these problems. The advantages of their application are (1) lower costs of the production and purification, (2) the possibility of obtaining a well-characterized single antigen which contains several different immunodominant regions from various T. gondii proteins, (3) better standardization of the diagnostic test, (4) higher sensitivity and specificity of the test, and (5) reproducibility of the test within and between laboratories. For all these reasons, recombinant chimeric antigens can replace the preparation of TLA in routine the serodiagnosis of toxoplasmosis in animals. In this study, we evaluated the usefulness of T. gondii recombinant chimeric antigens composed of three different well-characterized parasite proteins for the detection of specific IgG antibodies in sera from naturally infected sheep, pigs, and horses for the first time. The used recombinant chimeric antigens showed varying sensitivity in IgG ELISA tests with different serum samples, from high (e.g. 100% for SAG2-GRA1-ROP1L with sera from horses and sheep) to low (28.4% for SAG2-GRA1-ROP1S with sera from swine). Similar regularity can be observed in the case of a mixture of different recombinant antigens, which were able to detect specific antibodies only in 54.3% (M3: rGRA1 + rGRA2 + rGRA6) in the sera of pigs, but also 100% (M2: rSAG2 + rGRA1 + rROP1) in the sera from sheep. The explanation for these varying results is strictly associated with the immune response to infection, which may be differentiated between species. It is commonly known that different species of animals have varying ability to recognize and fight against various pathogens. For these reasons, it is often difficult to develop a diagnostic test that could be used for the diagnosis of a disease in all species. During the analysis of the results, it is easy to see that the chimeric antigens in IgG ELISA tests have a higher sensitivity than a mixture of recombinant antigens. In our opinion, the higher reactivity of the chimeric antigens than the mixtures of recombinant proteins may be explained by the fact that the epitopes of the chimeric antigens are probably more exposed to the antiT. gondii antibodies. At the same time, a steric obstruction between molecules may occur in the case of a combination of recombinant proteins. The exception is only the IgG ELISA based on mixture M1 (rMIC1 + rMAG1 + rSAG1) with horses sera. The sensitivity of this assay amounted to 88.9%, and it is definitely higher than the sensitivity of IgG ELISAs based on chimeric antigens MIC1-MAG1-SAG1S (75%) and SAG1L-MIC1-MAG1 (77.8%). It is commonly known that recombinant proteins produced in a bacterial expression system, such as E. coli,

often lose their antigenic properties due to incorrect folding. Furthermore, recombinant chimeric antigens are not naturally occurring proteins; it is possible that some fragments of immunodominant regions interact with each other and/or the amino acid sequence of linked antigens does not allow to create all functional epitopes. For this reason, some of the epitopes characteristic of native proteins are not present in the recombinant antigen and therefore cannot be recognized by specific antibodies. On the other hand, during the construction of chimeric antigens, we should pay attention to not create additional hydrophobic domains which can be recognized by specific antibodies. This possibility was excluded in the case of presented chimeric antigens by performing analysis using bioinformatics software. The results of IgG ELISAs using sera from different groups of animals (horses, sheep, and pigs) confirmed a high diagnostic potential of the chimeric recombinant antigens of T. gondii. The reactivity of the different recombinant chimeric antigens largely depends on the type of proteins of T. gondii and the size of immunodominant regions of proteins. The chimeric proteins comprising a larger immunodominant fragment of individual parasite antigens were characterized by higher reactivity. This regularity can be particularly seen in the case of chimeric antigens composed of fragments of SAG2, GRA1 proteins, and different-sized fragments of ROP1. The SAG2-GRA-ROP1L chimeric antigen having a longer fragment of ROP1 protein (amino acid residues from 85 to 396) is characterized by 100% sensitivity with both ovine and equine sera and by 93.8% with swine sera. While the chimeric antigen SAG2GRA1-ROP1S, which has a short fragment of the ROP1 protein (amino acid residues 85–250), has a much lower sensitivity at level 50% with equine sera and 28.4% with pig sera, specific anti-T. gondii antibodies were correctly detected only in sheep serum samples. A similar pattern was observed for the chimeric antigens composed of SAG1, MIC1, and MAG1 proteins. However, in this case, the reactivity of the chimeric protein MIC1-MAG1-SAG1S containing shorter fragment of SAG1 (amino acid residues 49–198) was only slightly lower than the reactivity of the chimeric antigen SAG1L-MIC1-MAG1 containing a larger fragment of the SAG1 protein (amino acid residues 49–311). Exceptions are the results of IgG ELISAs with swine sera used, where a significant decrease in sensitivity of the assay of 90.1% to 45.7% was noted. The analysis of the results of the IgG ELISAs for different groups of animals shows that usually both the mean and the range values of the measured absorbance are much higher for the chimeric antigens containing longer fragments of immunodominant regions of T. gondii proteins. The explanation for this might be that a greater number of specific anti-T. gondii antibodies are able to recognize the chimeric antigen having a longer immunodominant fragment of individual proteins. Another reason may be the higher structural stability, which is particularly evident in the case of antigens containing fragments of varying length of the ROP1 protein. It is possible that the shorter fragment of the ROP1 antigen is not at all recognized by specific IgG antibodies, and the measured absorbance value derived from the antibodies specifically bound to the fragments of SAG2 and GRA1 antigens. This hypothesis would also explain why the mean absorbance values obtained from the mixture of antigens and SAG2-GRA1-ROP1L chimeric antigen were comparable. The study of the ROP1 antigen showed that the amino acid sequence of these proteins contains an interesting charge asymmetry, suggesting a role of different proteins in the heterotypic binding [25,42,43]. The mature ROP1 protein has a series of tandem octapeptide repeats rich in proline and glutamic acid (gives the acidic character to the N-terminal region and the center of protein), the antigen also contains a region rich in arginine (alkaline region in the C-terminal), which probably stabilizes the structure of protein. The SAG2-GRA1-ROP1L chimeric antigen with a fragment of ROP1 corresponding to 85–396 amino acid residues, contains a part of the alkaline C-terminal region rich in arginine, which is likely to be crucial for antigen recognition by the specific antibodies. The high reactivity of the SAG2-GRA1-ROP1L chimeric antigen can be also explained by another hypothesis, the C-terminal region of ROP1 could contain at least one immunodominant B-cell epitope recognized

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by the majority of individuals, whereas responses to the N-terminal region of ROP1 would be much more variable. The same situation probably occurs in the case of the SAG1 antigen. Amino acid sequence analyses show that the C-terminal region of SAG1 may contain potential epitopes for B-cell recognition. This hypothesis is confirmed by studies carried out on recombinant antigens using human sera. In 2000, Lecordier et al. [44] showed that only the N-terminal hydrophilic part of GRA6 protein was recognized by a pool of positive human sera in an immunoblot, while the same serum samples yielded no positive results with the C-terminal part of GRA6. In 2008, Holec et al. [33] observed the different reactivity between fragments of the MIC1 antigen. In the IgG ELISA with the MIC1ex2 (N-terminal part of MIC1), the MIC1ex34 (C-terminal part of MIC1), and the MIC1, the reactivity of serum samples from patients with acute and chronic infections was examined. The result also showed that only the N-terminal part of MIC1 (rMIC1ex2) reacts strongly with both groups of sera from the acute (96.1%) and chronic (75%) phase of toxoplasmosis. These results demonstrated that for some antigens, the reactivity of specific IgG antibodies was correlated with particular parts of the antigen, and probably the same situation occurred for the ROP1 and SAG1 proteins. In conclusion, our results indicate that from all tested protein preparations in the IgG ELISA assay, the best antigen for the serodiagnosis of toxoplasmosis regardless of the test group of animals is a recombinant chimeric antigen SAG2-GRA1-ROP1L. The high diagnostic potential has also chimeric antigens GRA1-GRA2-GRA6 and SAG1L-MIC1-MAG1. The obtained results confirmed that recombinant chimeric antigens are better than commonly tested mixtures of proteins (particularly in the case of human toxoplasmosis) and can be an alternative to the native polyvalent antigen TLA. In our opinion, in the near future, it will be possible to commercialize a rapid and inexpensive immunoassay method based on chimeric antigens for routine diagnosis of toxoplasmosis in both humans and animals.

[12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20] [21] [22]

[23] [24]

[25]

[26] [27]

Acknowledgments This study was financed from State Resources for Science in the years 2012–2014 (grant no. IP2011 017571). This research work was supported by the European Social Fund, the State Budget and the Pomorskie Voivodeship Budget according to the Operational Programme Human Capital, Priority VIII, Measure 8.2, Sub-measure 8.2.2: ‘Regional Innovation Strategy’ within the system project of the Pomorskie Voivodeship “InnoDoktorant–Scholarships for PhD students, VIth edition.”

[28] [29]

[30]

[31]

[32]

References [33] [1] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217–58. [2] Buxton D. Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in sheep and goats: recent advances. Vet Res 1998;29:289. [3] Hill DE, Dubey JP. Toxoplasma gondii prevalence in farm animals in the United States. Int J Parasitol 2013;43:107–13. [4] Jones JL, Dubey JP. Foodborne toxoplasmosis. Clin Infect Dis 2012;55:845–51. [5] Kijlstra A, Jongert E. Toxoplasma-safe meat: close to reality? Trends Parasitol 2009; 25:18–22. [6] Tassi P. Toxoplasma gondii infection in horses. A review. Parassitologia 2007;49: 7–15. [7] Montoya JG. Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J Infect Dis 2002;185(Suppl. 1):73–82. [8] Holec-Gąsior L. Toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis: current status of studies. Clin Vaccine Immunol 2013;20: 1343–51. [9] Beghetto E, Spadoni A, Bruno L, Buffolano W, Gargano N. Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. J Clin Microbiol 2006;44:2133–40. [10] Dai J, Jiang M, Wang Y, Qu L, Gong R, Si J. Evaluation of a recombinant multiepitope peptide for serodiagnosis of Toxoplasma gondii infection. Clin Vaccine Immunol 2012;19:338–42. [11] Dai JF, Jiang M, Qu LL, Sun L, Wang YY, Gong LL, et al. Toxoplasma gondii: enzymelinked immunosorbent assay based on a recombinant multi-epitope peptide for

[34]

[35]

[36]

[37]

[38]

[39]

[40]

293

distinguishing recent from past infection in human sera. Exp Parasitol 2013;133: 95–100. Holec-Gąsior L, Ferra B, Drapała D, Lautenbach D, Kur J. A new MIC1-MAG1 recombinant chimeric antigen can be used instead of the Toxoplasma gondii lysate antigen in serodiagnosis of human toxoplasmosis. Clin Vaccine Immunol 2012;19:57–63. Holec-Gąsior L, Ferra B, Drapała D. MIC1-MAG1-SAG1 chimeric protein, a most effective antigen for detection of human toxoplasmosis. Clin Vaccine Immunol 2012;19: 1977–9. Lau YL, Thiruvengadam G, Lee WW, Fong MF. Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii expressed in the yeast Pichia pastoris. Parasitol Res 2011;109:871–8. Coppens I, Andries M, Liu JL, Cesbron-Delauw MF. Intracellular trafficking of dense granule proteins in Toxoplasma gondii and experimental evidences for a regulated exocystosis. Eur J Cell Biol 1999;78:463–72. Lecordier L, Mercier C, Sibley LD, Cesbron-Delauw MF. Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell. Mol Biol Cell 1999;10:1277–87. Carruthers VB. Host cell invasion by the opportunistic pathogen Toxoplasma gondii. Acta Trop 2002;81:111–22. Parmley SF, Yang S, Harth G, Sibley LD, Sucharczuk A, Remington JS. Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in the matrix of tissue cysts. Mol Biochem Parasitol 1994;66:283–96. Di Cristina M, Del Porto P, Buffolano W, Beghetto E, Spadoni A, Guglietta S, et al. The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection. Microbes Infect 2004;6: 164–71. Ferguson DJ, Parmley SF. Toxoplasma gondii MAG1 protein expression. Trends Parasitol 2002;18:482. Carruthers VB, Giddings OK, Sibley LD. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cell. Cell Microbiol 1999;1:225–35. Lourenco EV, Pereira SR, Faca VM, Coelho-Castelo AA, Minero JR, Roque-Barreira MC, et al. Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin. Glycobiology 2001;11:541–7. Bradley PJ, Boothroyd JC. The pro region of Toxoplasma ROP1 is a rhoptry-targeting signal. Int J Parasitol 2001;31:1177–86. Bradley PJ, Hsieh CL, Boothroyd JC. Unprocessed Toxoplasma ROP1 is effectively targeted and secreted into nascent parasitophorous vacuole. Mol Biochem Parasitol 2002;125:189–93. Soldati D, Kim K, Kampmeier J, Dubremetz JF, Boothrooyd JC. Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection. Mol Biochem Parasitol 1995;74:87–97. Lekutis C, Ferguson DJP, Grigg ME, Camps M, Boothroyd JC. Surface antigens of Toxoplasma gondii: variations on a theme. Int J Parasitol 2001;31:1285–92. Graille M, Stura EA, Bossus M, Muller BH, Letourneur O, Battail-Poirot N, et al. Crystal structure of the complex between the monomeric form of Toxoplasma gondii surface antigen 1 (SAG1) and a monoclonal antibody that mimics the human immune response. J Mol Biol 2005;354:447–58. He XL, Grigg ME, Boothroyd JC, Garcia KC. Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily. Nat Struct Biol 2002;9:606–11. Macedo AG, Cunha JP, Cardoso THS, Silva MV, Santiago FM, Silva JS, et al. SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts. Parasit Vectors 2013;6:163. Hiszczyńska-Sawicka E, Brillowska-Dąbrowska A, Dąbrowski S, Pietkiewicz H, Myjak P, Kur J. High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli. Protein Expr Purif 2003;27:150–7. Hiszczyńska-Sawicka E, Kur J, Pietkiewicz H, Holec L, Gąsior A, Myjak P. Efficient production of the Toxoplasma gondii GRA6, p35 and SAG2 recombinant antigens and their applications in the serodiagnosis of toxoplasmosis. Acta Parasitol 2005;50:249–54. Holec L, Hiszczyńska-Sawicka E, Gąsior A, Brillowska-Dąbrowska A, Kur J. Use of MAG1 recombinant antigen for detection of Toxoplasma gondii infection in humans. Clin Vaccine Immunol 2007;14:220–5. Holec L, Gąsior A, Brillowska-Dąbrowska A, Kur J. Toxoplasma gondii: enzyme-linked immunosorbent assay using different fragments of recombinant microneme protein 1 (MIC1) for detection of immunoglobulin G antibodies. Exp Parasitol 2008;119:1–6. Holec-Gąsior L, Kur J, Hiszczyńska-Sawicka E. GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in humans with acute toxoplasmosis. Clin Vaccine Immunol 2009;16:510–4. Holec-Gąsior L, Kur J, Hiszczyńska-Sawicka E, Drapała D, Dominiak-Górski B, Pejsak Z. Application of recombinant antigens in serodiagnosis of swine toxoplasmosis and prevalence of Toxoplasma gondii infection among pigs in Poland. Pol J Vet Sci 2010; 13:457–64. Tenter AM, Vietmeyer C, Johnson AM. Development of ELISAs based on recombinant antigens for the detection of Toxoplasma gondii-specific antibodies in sheep and cats. Vet Parasitol 1992;43:189–201. Andrews CD, Dubey JP, Tenter AM, Webert DW. Toxoplasma gondii recombinant antigens H4 and H11: Use in ELISAs for detection of toxoplasmosis in swine. Vet Parasitol 1997;70:1–11. Jiang T, Gong D, Ma LA, Nie H, Zhou Y, Yao B, et al. Evaluation of a recombinant MIC3 based latex agglutination test for the rapid serodiagnosis of Toxoplasma gondii infection in swines. Vet Parasitol 2008;158:51–6. Tumurjav B, Terkawi MA, Zhang H, Zhang G, Jia H, Goo YK, et al. Serodiagnosis of ovine toxoplasmosis in Mongolis by an enzyme-linked immunosorbent assay with recombinant Toxoplasma gondii matrix antygen 1. Jpn J Vet Res 2010;58:111–9. Terkawi MA, Kameyama K, Rasul NH, Xuan X, Nishikawa Y. Development of an immunochromatographic assay based on dense granule protein 7 for serological detection of Toxoplasma gondii infection. Clin Vaccine Immunol 2013;20:596–601.

294

B. Ferra et al. / Parasitology International 64 (2015) 288–294

[41] Holec-Gąsior L, Ferra B, Hiszczycńska-Sawicka E, Kur J. The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis. Vet Parasitol 2014. http://dx.doi.org/10.1016/j.vetpar.2014. 09.018. [42] Bradley PJ, Boothroyd JC. Identification of the pro-mature processing site of Toxoplasma ROP1 by mass spectrometry. Mol Biochem Parasitol 1999;100: 103–9.

[43] Ossorio PN, Schwartzman JD, Boothroyd JC. A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry. Mol Biochem Parasitol 1992;50:1–15. [44] Lecordier L, Fourmaux MP, Mercier C, Dehecq E, Masy E, Cesbron-Delauw MF. Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies. Clin Diagn Lab Immunol 2000;7:607–11.