Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle

Research in Veterinary Science 88 (2010) 446–451

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Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle Hailing Zhao 1, Huifang Liu 1, Yanfen Du, Siguo Liu *, Hongbo Ni, Yong Wang, Chunlai Wang, Wei SI, Jinguo Yang, Jingkai Ling Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 15001, PR China

a r t i c l e

i n f o

Article history: Accepted 23 November 2009

Keywords: Campylobacter fetus Enzyme-linked immunosorbent assay Prokaryotic expression Purification SapA gene

a b s t r a c t Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398 bp) and SapA-C (1422 bp) fragments were amplified from the C. fetus SapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P < 0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P P 0.45: positive; S/P < 0.4: negative; 0.45 > S/P P 0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Campylobacter fetus (formerly Vibrio fetus) belongs to the genus Campylobacter and contains 2 subspecies, namely, C. fetus subsp. fetus and C. fetus subsp. venerealis (Skirrow and Benjamin, 1980; Taylor and Lior, 1985; Kienesberger et al., 2007). Although C. fetus subsp. fetus normally resides in the intestinal tracts of cattle and sheep, its ingestion can result in systemic infection and abortion in pregnant cows and ewes. In contrast, the only known ecological niche for C. fetus subsp. venerealis is the reproductive tract of cattle where it can cause bovine genital campylobacteriosis (BGC). In cows, this disease is characterized by infertility, early embryonic death, and abortion. It arises from venereal transfer from carrier bulls. BGC is an economically important disease in the cattle industry worldwide (Garcia et al., 1983; Salama et al., 1992; Skirrow, 1994; Vargas et al., 2003; Devenish et al., 2005). Moreover, infection with C. fetus in humans can lead to premature birth, sepsis, * Corresponding author. Fax: +86 451 82733132. E-mail address: [email protected] (S. Liu). 1 Both authors contributed equally to this work. 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.11.013

and symptoms similar to those of brucellosis. In patients with immunodeficiency disorders, C. fetus infection is associated with several conditions, including meningitis, bacteremia, arthritis, and diarrhea (Rettig, 1979; Rennie et al., 1994). C. fetus subsp. venerealis can cause cattle abortion and infertility. Therefore, C. fetus is an important pathogen of livestock and humans. Several testing procedures have been employed for the detection of C. fetus in animals. Culture and identification have been well described for the diagnosis of this pathogen (Lander, 1990). However, C. fetus isolation and identification is very difficult since this organism requires strict environmental and biochemical conditions for growth, and the collection of pathological material related to the disease must be carried out under special conditions. Presently, polymerase chain reaction (PCR), DNA fingerprinting, and pulsed-field gel electrophoresis (PFGE) are used to identify C. fetus and enzyme-linked immunosorbent assay (ELISA) is used to detect antibodies against it. C. fetus has a layer of macromolecular surface proteins (molecular weight, approximately 97–149 kDa) that plays a key role in its toxicity (Dubreuil et al., 1988; Dworkin et al., 1995). The presence of many epitopes with relatively high pathogenicity has been pre-

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viously reported (Wang et al., 1993). In particular, the conserved N-terminal, these surface proteins have anti-serum and anti-phagocytic properties (Blaser et al., 1988). A previous study had shown that the presence of the SapA gene without a promoter could lead to a deficiency in the macromolecular surface protein (Murali et al., 1992). There have been numerous reports on the surface proteins of C. fetus. Wang et al. used these surface proteins to immunize BALB/C mice and produce monoclonal antibodies. Grogono-Thomas et al. (2000) studied the roles of the surface layer proteins of C. fetus subsp. fetus in ovine abortion. In this study, the focus was on the SapA gene of C. fetus that encodes surface array proteins (McCoy et al., 1975; Blaser et al., 1988) and plays an important role in the virulence of C. fetus. The SapA-N (1398 bp) and SapA-C (1422 bp) gene fragments from the SapA gene were amplified using PCR. The recombinant proteins were expressed in E. coli and then purified, and their immunological activities were compared. Finally, an indirect ELISA using rSapA-N as the antigen was established.

pended in 40 lL of ultrapure milli-Q water. The DNA concentration was measured in a biophotometer spectrophotometer (Eppendorf). The DNA solution was diluted to the appropriate concentration for PCR amplification. The SapA-N and SapA-C DNA fragments were amplified from the C. fetus gDNA by PCR. The sequences of the employed primer pairs (i.e., SapA-N forward, SapA-N reverse, SapA-C forward, and SapA-C reverse) were listed in Table 1. The antigenicity of SapA and expression of SapA-N and SapA-C fragments were predicted (Fig. 1). The PCR reaction mixture contained 200 nmol/l of each primer, 0.2 mmol/l of each deoxyribonucleotide triphosphate, 6.3 ng template DNA, 0.5 U Taq DNA polymerase (Fermentas), and enough sterile deionized water to make up the final volume to 25 lL. The PCR thermal cycling conditions were as follows: 35 cycles were carried out with initial denaturation at 95 °C for 10 min, 94 °C for 1.5 min, 50 °C (SapA-N) for 1.5 min or 55 °C (SapA-C) for 1.5 min, and 72 °C for 1.5 min, with a final 10-min extension carried out at 72 °C. The amplified DNA fragments were purified using a commercial gel extraction kit (Watson Biotechnologies, Inc.).

2. Materials and methods 2.3. Construction of recombinant plasmids 2.1. Serum samples Serum samples were collected from dairy farms nationwide and were stored in our laboratory. All experiments were in compliance with the guidelines for the welfare of animals and those of the concerned ethical authorities. The normal and negative bovine samples were collected from cattle belonging to a region that had no clinical history of C. fetus infection. The processed vaginal mucus and bull semen through the bacterial culture and biochemical test confirmed that all serum samples were negative. Meanwhile, the bull semen and vaginal mucus were confirmed to be negative using PCR. The positive and subclinical bovine serum samples infected with C. fetus were taken from cattle abortion and aborted fetus. Placental tissue from cattle that underwent abortion, fetal stomach contents, bull semen, as well as abortion and fetal placenta were identified by PCR, bacterial cultures and biochemical test and confirmed to be infected with C. fetus. 2.2. Design of PCR primers, DNA extraction, and amplification of target genes Genomic DNA (gDNA) from C. fetus strain 553 (Jilin Entry-Exit Inspection and Quarantine Bureau, Jilin Province, China) was isolated according to a procedure previously described (Sambrook and Russell, 2001) with the following modifications. A culture loop of each isolate was washed twice in 10 mmol Tris–HCl (pH 8.0) containing 1 mmol ethylenediamine tetra-acetic acid and 0.1 M NaCl. After centrifugation, the cell pellet was resuspended in deionized water and incubated with 20 mmol NaOH and 3.4% sodium dodecyl sulfate (SDS) for 3 h at 37 °C. The reaction mixture was then incubated with 0.2 mg/ml of proteinase K (Sigma) for 2 h at 50 °C. Finally, the DNA was purified from the aqueous phase by phenol–chloroform extraction and precipitated in 0.6  volume of isopropanol. The pellet was washed with 70% ethanol and sus-

The SapA-N and SapA-C DNA fragments were inserted into the pMD18-T vector (Takara) and transformed into E. coli strain DH5a (from stock from the authors’ laboratory). Positive clones were screened, and the recombinant plasmids (named T-SapA-N and T-SapA-C, respectively) were purified for sequencing which was carried out by a commercial laboratory (Bioasia Shanghai, China). The SapA-N DNA fragments from T-SapA-N were digested with EcoRI and SalI (all restriction endonucleases were from Fermentas) and inserted into the pET32a (+) vector (Novagen). The resulting recombinant plasmid (pET-SapA-N) was transformed into DH5a cells and purified from the positive clones. Similarly, the same subclone procedure of SapA-C DNA fragments from T-SapA-C were performed, and the resulting positive recombinant plasmid was named pET-SapA-C. 2.4. Expression, purification, SDS–polyacrylamide gel electrophoresis, and Western blotting analysis of the recombinant protein The pET-SapA-N and pET-SapA-C recombinant plasmids were transformed into E. coli strain BL21 (DE3; from stock from the authors’ laboratory). The transformed E. coli cells were plated on lysogeny broth (LB) solid medium containing ampicillin (50 g/ mL) and incubated overnight at 37 °C. One clone was selected and inoculated in 5 ml of LB containing ampicillin. It was then cultivated overnight at 37 °C on a shaker. A 1-ml sample of the overnight culture was then transferred into 100 ml of LB for further growth and cultivated at 37 °C on a shaker. When the OD600 (optical density at 600 nm) value reached 0.5, isopropyl–d-thiogalactoside (Sigma) was added to a final concentration of 0.8 mmol. The mixture was grown for another 5 h, and 1 ml of the culture was set aside for SDS–polyacrylamide gel electrophoresis (PAGE) analysis. The remaining cells were harvested by centrifugation (Beckman centrifuge; J2–21 M) at 5000g for 15 min at 4 °C. The cell

Table 1 The primer sequences of the SapA-N and SapA-C genes.*

SapA-N(U-EcoRI) SapA-N(L-SalI) SapA-C(U-EcoRI) SapA-C(L-SalI) *

PCR = polymerase chain reaction.

Oligonucleotides primer sequence

PCR product size (bp)

50 -GAATTCGCAAGTGAGGGTGATGGT-30 50 -AAGTCGACAACCTTAGCAGCAGCTC-30 50 -GAATTCCTCTACAGCAGCAAAAGAC-30 50 -GTCGACAATTACGCTTCCATCAT-30

1398 1422

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A 50

100

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200

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350

400

450

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600

650

700

750

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900

4.5

0

Hydrophilicity Plot - Kyte-Doolittle

-4.5 1.7

0

Antigenic Index - Jameson-Wolf

-1.7 6

1 0

Surface Probability Plot - Emini

B

SapA(2820bp) SapA-C(1422bp) SapA-N(1398bp)

Fig. 1. Prediction of antigenicity of SapA (A) and expression of SapA-N and SapA-C fragments. (B) In comparison to SapA-C, SapA-N formed the main antigenic region in SapA.

pellets were stored at –70 °C. The recombinant proteins rSapA-N and rSapA-C were purified using Bug Buster Ni–NTA His Bind Purification Kit (Novagen). The eluted product was collected in 7 tubes, and the fractions were analyzed by SDS–PAGE. Immunoblot analysis was carried out according to established procedures. After electrophoresis, the protein bands on the gel were transferred onto a nitrocellulose (NC) membrane (pore size, 0.45 um) using a transblotting apparatus (Bio-Rad) at 5 V for 3 h. The reactive sites of the NC membrane were blocked by incubation in 5% skimmed milk (Boster Bio-Technology Co.) in phosphate buffered saline (PBS). After washing with PBS, strips of the membrane containing the separated proteins were cut and incubated overnight at room temperature with sera that were positive for C. fetus at a dilution of 1:100 in PBS. The bound protein was detected by subsequent incubation for 2 h with horseradish peroxidase (HRP)-conjugated rabbit anti-bovine immunoglobulins (Sigma) at a dilution of 1:5000. After repeated washes, the labeled bands were visualized by the reaction of HRP with the diaminobenzidine substrate (DAB; Watson Biotechnologies, Inc.).

2.5. ELISA: screening of recombinant proteins The concentrations of rSapA-N and rSapA-C were determined by ultraviolet spectrophotometry and were found to be 2.964 mg/ml and 2.726 mg/ml, respectively. rSapA-N was first diluted to the same dilution as rSapA-C (2.726 mg/ml) and the two proteins were further diluted 1:800 for use in coating ELISA plates (50 ll/well). The plates were incubated overnight at room temperature followed by incubation with 1% gelatin, primary antibody (dilution, 1:200), and secondary antibody (HRP-conjugated rabbit anti-bovine immunoglobulins; dilution, 1:10,000; all antibodies were diluted with PBST containing 0.05% Tween 20) at 37 °C for 1 h in a moist chamber. After color development and cessation of the reaction, the optical density (OD) value of each well was measured using ELx800 microplate reader (BioTek) at a wavelength of 450 nm. The OD values yielded by rSapA-N and rSapA-C were com-

pared and the difference was analyzed using the Statistical Analysis System (SAS). The recombinant protein with better immunological activity was screened and used as the antigen for ELISA. A C. fetus-negative serum sample and a blank were used as the controls. 2.6. ELISA cut-off value A total of 80 negative bovine serum samples were tested to determine the cut-off value. The mean OD450 value of this negative population plus standard deviations (SD) was established as the positive cut-off value (Cp), while the mean OD450 value plus 2 SD was the negative cut-off value (Cn). Based on the OD450 values of the samples (S), positive controls (P), and negative controls (N), the following formula was used to calculate the titer (S/P) of the serum sample: S/P = (S–N)/(P–N). If S/P P Cp, the C. fetus antibody reaction was considered to be positive, and if S/P < Cn, the reaction was negative. If Cp > S/P P Cn, infection was categorized as suspect and the assay was repeated. 2.7. Testing the cross-reactivity Established indirect ELISAs were used to detect sera (all sera were identified by themselves ELISA) that were positive for the following 5 common bovine diseases: C. fetus infection, infectious bovine rhinotracheitis (IBR), bovine brucellosis, bovine paratuberculosis, and bovine tuberculosis. Negative sera were used as the controls. 2.8. Testing the specificity and sensitivity After preparing 200-fold dilutions of 140 normal bovine serum samples and clinically or subclinically C. fetus-infected serum samples, the OD values were determined by the indirect ELISA. Based on the results, the specificity and sensitivity of the method was determined.

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2.9. Testing of clinical samples The antigen rSapA-N was used to test 102 bovine serum samples by the indirect ELISA. The samples included 70 bovine brucellosis-positive sera and 32 IBR-positive sera. The absorbance of all samples was measured at 450 nm, and the results were analyzed.

Table 2 The result of immunogenicity of the 2 proteins on the enzyme-linked immunosorbent assay plates.

Positive serum 1 Positive serum 2 Positive serum 3 Negative Control

3. Results

SapA-N and SapA-C were PCR amplified from the C. fetus genome, and the PCR products were confirmed by agarose gel electrophoresis. The recombinant plasmids T-SapA-N and T-SapA-C thus obtained were subjected to nucleotide sequencing and identified by double digestion with SalI and EcoRI. DNA sequence alignment showed that the insertion of the fragments was as expected. The fragments were then inserted into pET32a (+) resulting in the recombinant plasmids pET-SapA-N and pET-SapA-C. 3.2. Expression, purification, and immunoblot analyses of the fusion protein The recombinant plasmids pET-SapA-N and pET-SapA-C were transformed into E. coli strain BL21. The fusion proteins were expressed in E. coli as soluble His-tagged recombinant proteins and appeared as bands of approximately 66.2 kDa on a 12% polyacrylamide gel (Fig. 2A). The expressed fusion proteins (SapA-N and SapA-C) were purified on Ni–NTA His Bind resin (Fig. 2B), and their concentrations were 2.964 mg/ml and 2.726 mg/ml, respectively. Visualization with DAB revealed prominent bands in the strips incubated with the sera from C. fetus-infected animals (Fig. 2C). The ELISA results indicated that the immunological activity of rSapA-N was higher than that of rSapA-C. Statistical analysis using the SAS software showed that the difference between the OD450 values of rSapA-N and rSapA-C was significant (P < 0.05). Therefore, in the present study, rSapA-N was used as the test antigen in the ELISA (Table 2). 3.3. Cut-off value of the ELISA The average of the OD450 values of 80 serum samples that were negative for C. fetus was 0.299 with an SD of 0.05. The average OD + 3  SD was 0.45, while the average OD + 2  SD was 0.4. Based on the OD450 values of the samples (S), positive control sera (P), and negative control sera (N), the titers of the serum samples (S/P) were calculated by the formula S/P = (SN)/(PN). The test 2

3

4

5

6

7

rSapA-C

rSapA-N + rSapA-C

0.741b 0.874b 0.496b 0.131a 0.055

0.816c 1.036c 0.568c 0.124a 0.052

Superscripts ‘‘a, b, c” indicate that the differences among OD450 (optical density at 450 nm) values of rSapA-N, rSapA-N, and rSapA-N + rSapA-C were significant. a Indicates that the OD450 values of rSapA-N, rSapA-N, and rSapA-N + rSapA-C showed no differences.

3.1. PCR amplification of the target genes and identification of the recombinant plasmids pET-SapA-N and pET-SapA-C

1

rSapA-N 0.892a 1.143a 0.673a 0.137a 0.053

samples should have S/P values P0.45. In other words, when SOD450 P 0.45  (PN) + N, the reaction was regarded to be positive. On the other hand, when S/P < 0.4 (i.e., SOD450 < 0.4  (PN) + N), the reaction was regarded to be negative. 3.4. Sensitivity and specificity testing Sixty-two out of 70 known positive serum samples were positive (S/P P 0.45) and the remaining 8 were negative, yielding a sensitivity of 88.6% (Table 3). Four out of 70 known negative sera were positive, yielding a specificity of 94.3% (66/70) (Table 3). Receiver operating characteristics (ROC) analysis of the ELISA plates for rSapA-N (OD450) using the SAS software showed that the sensitivity and specificity were better correlative (Fig. 3). Sera that were positive for the 5 common bovine infectious diseases were tested using indirect ELISA. As shown in Table 4, all the serum titers were less than 0.4. This included samples that were positive for IBR, bovine brucellosis, M. paratuberculosis, and M. bovis. Only the C. fetus-positive serum titer was higher than 0.45, indicating a positive response (Table 4). 3.5. Clinical sample testing The results from the testing of clinical serum samples are shown in Fig. 4. Four C. fetus-positive sera (+) were found among the 70 bovine brucellosis-positive serum samples, and 2 C. fetuspositive serum samples (++) were found among the 32 IBR-positive serum samples. 4. Discussion Indirect ELISA can be a very useful tool in the diagnosis of infectious diseases in animals. The technique seems to be particularly useful in regions where little epidemiological information is available on the disease and where large numbers of sera samples have

1 2 3 4 5 6 7

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A

B

C

Fig. 2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the expression of the recombinant protein (A) and purified protein (B), along with Western blot analysis (C). Lanes A7, B1, C1: protein molecular weight markers; lanes A1, A2: insoluble part of the bacterial lysate SapA-N; lane A3: soluble part of the bacterial lysate SapAC; lane A4: soluble part of the bacterial lysate SapA-N; lanes A5, A6: the vector of pET32a; lanes B2–5: purified fusion protein of SapA-N; lanes B6, B7: purified fusion protein of SapA-C; lanes C2, C3: nitrocellulose (NC) membrane incubated with sera from cattle infected with Campylobacter fetus.

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Table 3 The result of the specificity and sensitivity testing of the enzyme-linked immunosorbent assay (ELISA) plates for rSapA-N (OD450).* ELISA positive

ELISA negative

C. fetus positive (A) 62

C. fetus negative (B) 4

C. fetus positive (C) 8

C. fetus negative (D) 66

*

OD450 = optical density at 450 nm; sensitivity = [A/A + C]  100 = 88.6%; specificity = [D/B + D]  100 = 94.3%; positive ratio = [A/A + B]  100 = 93.9%; negative ratio = [D/ D + C]  100 = 89.2%; n = 140.

1 0.9

sensitivity

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.1

0.2

0.3

0.4

0.5 0.6 1-specificity

0.7

0.8

0.9

1

Fig. 3. Receiver operating characteristics (ROC) analysis of the the enzyme-linked immunosorbent assay (ELISA) plates for rSapA-N (OD450). The blue line indicates the test curve, and while the pink line shows a noninformative test curve.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 4 The results of the specificity testing of the enzyme-linked immunosorbent assay plates for rSapA-N (OD450).*

Negative Campylobacter fetus Brucella Mycobacterium bovis Mycobacterium paratuberculosis Infectious bovine rhinotracheitis *

rSapA-N (OD450)

Result

0.137 1.038 0.308 0.352 0.324 0.249

– + – – – –

OD450 = optical density at 450 nm; – = negative; + = positive.

Fig. 4. Results from the indirect enzyme-linked immunosorbent assay that used rSapA-N as the diagnostic antigen to test 102 bovine serum samples: 4 C. fetuspositive sera among the 70 bovine brucellosis-positive serum samples and 2 C. fetus-positive serum samples among the 32 infectious bovine rhinotracheitis (IBR)positive serum samples. OD450 = optical density at 450 nm.

to be tested to obtain such information (Francisco et al., 1995). Conventional diagnostic tests for C. fetus are too time-consuming to be used in large-scale surveys. In contrast, ELISAs are rapid, sim-

ple, sensitive, and specific and can be used to detect antibodies to C. fetus. This technique can be easily applied to large-scale surveys. Considering the advantages of ELISA, many researchers have tried to establish the ELISA method for detecting C. fetus infection. For example, Brooks et al. (2004) used an ELISA with 4 monoclonal antibodies to test the lipopolysaccharide core antigen of C. fetus. The lipopolysaccharide was digested with proteinase K and washed with liquid carbolic acid. The results showed that the specificity of these monoclonal antibodies for C. fetus was very high. Although this method had good specificity, the procedure was complicated. However, the approach presented in this study is more convenient for obtaining the antigens and preparing the antibodies. Moreover, good specificity and sensitivity were achieved by this method. The ELISA and Western blot results showed that both fusion proteins had immunological activity, but the activity of rSapA-C was lower than that of rSapA-N (P < 0.05). The 2 proteins were also mixed and coated simultaneously; however, the results from ELISA confirmed earlier observations. The results showed that the antigenic epitopes of the surface antigen are centralized in the SapAN region. In other words, the N-terminal of SapA has higher immunological activity, which was in accordance with the results derived from commercial software (DNAstar). rSapA-N was therefore used as the diagnostic antigen to test the specificity, sensitivity, and cross-reactivity. The results from the receiver operating characteristics (ROC) analysis showed that when the critical value was 0.45, there was a balance between the sensitivity and specificity of the tests. Both the specificity (94.3%) and sensitivity (88.6%) were high and were consistent with the critical values obtained with our formula. We also calculated the area under the ROC curve (AUC) and obtained a value of 0.909. The results also showed that this test had high accuracy. In the cross-reaction test, we chose five kinds of disease-positive cattle serum. There were two diseases with C. fetus could lead to abortion, and two other diseases whose symptoms were different from those of C. fetus. The ELISA results confirmed that there was no cross-reactivity between rSapA-N and other sera that tested positive for other bacterial diseases. This suggests that rSapA-N can be used as a specific diagnostic antigen in ELISA to test for the presence of bovine C. fetus. We collected serum samples and pathological materials of disease as well as some semen samples. All pathological materials of disease were identified by bacterial culture and biochemical tests. We used indirect ELISA to detect positive and negative sera, and the results were confirmed by bacterial culture and biochemical tests. There was good agreement between the results of ELISA and those of bacterial culture and biochemical tests. The indirect ELISA detection method used in this study had high sensitivity and can be used for large-scale testing of samples. Many pathogens can lead to abortions among pregnant livestock such as cows and sheep. Therefore, indirect ELISA was used to test sera that were positive for pathogens such as those causing bovine brucellosis and IBR. The results of the tests indicated that among all the samples of bovine brucellosis and IBR, positive reactions were observed in only 4 and 2, respectively, which suggests that mixed infections of bacteria and viruses are widespread in cases of abortion due to infections. rSapA-N expressed in the cur-

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rent study is a novel diagnostic antigen for ELISA and can be used for the development of rapid and sensitive immunodiagnostic techniques. Acknowledgments This work was granted by National ‘‘973” program (No. 2006CB504401) and the National Key Technology R & D Program during the 11th Five-Year Plan Period (No. 2007BAD40B01). References Blaser, M.J., Smith, P.F., Repine, J.E., Joiner, K.A., 1988. Pathogenesis of Campylobacter fetus infection. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance. J. Clin. Invest. 81, 1434–1444. Brooks, B.W., Devenish, J., Lutze-Wallace, C.L., 2004. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. Vet. Microbiol. 103 (1-2,5), 77–84. Devenish, J., Brooks, B., Perry, K., Milnes, D., Burke, T., McCabe, D., Duff, S., LutzeWallace, C.L., 2005. Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus. Clin. Diagn. Lab. Immunol. 12, 1261–1268. Dubreuil, J.D., Logan, S.M., Cubbage, S., Eidhin, D.N., McCubbin, W.D., Kay, C.M., Beveridge, T.J., Ferris, F.G., Trust, T.J., 1988. Structural and biochemical analyses of a surface array protein of Campylobacter fetus. J. Bacteriol. 170 (9), 4165– 4173. Dworkin, J., Tummuru, M.K., Blaser, M.J., 1995. A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs. J. Bacteriol. 177, 1734–1741. Francisco, A., Uzal, Carrasco, A.E., Echaide, S., Nielsen, K., Robles, C.A., 1995. Evaluation of an indirect ELISA for the diagnosis of bovine brucellosis. J. Vet. Diagn. Invest. 7, 473–475.

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