Identification and Expression of a 50-Kilodalton Surface Antigen of Babesia gibsoni and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in the B. gibsoni genome. The native P50 protein of B. gibsoni with a molecular mass of 50 kDa was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface of B. gibsoni merozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected with B. gibsoni was developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies to B. gibsoni in dogs.     

Identification of Secreted Antigen 3 from Babesia gibsoni

A cDNA expression library of Babesia gibsoni was screened with the serum collected from a dog experimentally infected with B. gibsoni. A novel antigen sharing homology with secreted antigen 1 of B. gibsoni was isolated. The genomic analysis indicated that the BgSA3 gene exists as multicopies in the genome of B. gibsoni. The putative peptide encoded by the BgSA3 gene showed some characteristics of secreted proteins. The serum raised in mice immunized with the recombinant BgSA3 expressed in Escherichia coli could recognize a native parasite protein with a molecular mass of 70 kDa. Moreover, a sandwich enzyme-linked immunosorbent assay with anti-BgSA3 antibodies could detect the circulating BgSA3 in the blood plasma of dogs experimentally infected with B. gibsoni. The identification of BgSA3 provided a useful target for the development of a diagnostic test for detecting specific antibodies and circulating antigens.Babesia canis and Babesia gibsoni are recognized as the two species that cause canine babesiosis, a clinically significant hemolytic disease of dogs. Individual Babesia species are typically classified by the size and morphological appearance of the intraerythrocytic forms, often referred to as piroplasms. B. gibsoni is considered to be a small babesial parasite with round or oval intraerythrocytic piroplasms. The infection caused by B. gibsoni is characterized by serious clinical problems, including remittent fever, progressive anemia, hemoglobinuria, and sometimes death (4, 6). This disease is epidemic in many areas of Asia, North America, and Northern and Eastern Africa but rarely so in Europe (4, 12, 18).For the diagnosis of this disease, several serological methods have been developed, such as indirect fluorescent antibody tests (IFAT) (24), enzyme-linked immunosorbent assays (ELISAs) (13, 22), and immunochromatographic tests (14). However, the detection of antibodies is unreliable for determining the infection status of dogs because the titer of the antibodies against the parasites can remain very high for a long time, even when the parasites have been completely eliminated. However, the secreted proteins, which include a broad variety of antigens released by the parasites and circulating in the bloodstream of the hosts during the asexual stage, can be used as diagnostic targets in antigen detection tests in order to avoid such problems. In an attempt to identify these circulating antigens, a method to screen a cDNA library was designed in a previous study (13). Several antigen candidates were isolated, and one of them, named secreted antigen 1 of B. gibsoni (BgSA1), was identified and evaluated as a useful antigen for further serologically diagnostic tests (13). Circulating BgSA1 could be detected in the plasma of a dog infected with B. gibsoni. In addition, the ELISA using the recombinant BgSA1 expressed in Escherichia coli showed advantages in sensitivity and specificity when it was used in field samples.In this study, we describe the identification of another member of secreted antigens, which share homology with BgSA1, here designated as secreted antigen 3 of B. gibsoni (BgSA3). The gene encoding BgSA3 was isolated from a cDNA library by immunoscreening with serum from a dog experimentally infected with B. gibsoni. As expected, the native BgSA3 also circulated in the bloodstream of the dogs infected with B. gibsoni. Our data indicate that BgSA3 could be useful as an antigenic marker of active B. gibsoni infection.     

High-Level Expression of Truncated Surface Antigen P50 of Babesia gibsoni in Insect Cells by Baculovirus and Evaluation of Its Immunogenicity and Antigenicity

Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10⁸ cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.     

Development of Rapid Immunochromatographic Test with Recombinant NcSAG1 for Detection of Antibodies to Neospora caninum in Cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

Detection ofEchinococcus coproantigens by enzyme-linked immunosorbent assay in dogs,dingoes and foxes

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection ofEchinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with eitherE. granulosus orE. multilocularis. The ELISA was based on protein-A-purified polyclonal antibodies [anti-E. granulosus excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n=37) or infected with non-Echinococcus cestodes (n=76) or with various nematodes (n=42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homologous worm burden. All 13 samples from foxes harboring >1,000E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing >200E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring <1,000E. multilocularis and 9 of 10 samples from dogs or dingoes bearing <200E. granulosus tested negative. Experimental prepatent infections of dogs withE. granulosus revealed positive ELISA reactions within the prepatent period (10–20 days post-infection) for six animals bearing >1,000E. granulosus each; a low worm burden (<1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest. All five dogs that had been experimentally infected withE. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.Dedicated to Prof. K.T.F. Friedhoff on the occasion of his 60th birthday     

Evaluation of Babesia bigemina 200?kDa recombinant antigen in enzyme-linked immunosorbent assay

A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.     

Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.     

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Seroprevalence of Neospora caninum infection in dogs in Chaharmahal-va-Bakhtiari Province, Iran

The aim of this study was to determine seroprevalence of Neospora caninum infection in dogs in Chaharmahal-va-Bakhtiari province in Iran. One hundred serum samples were collected from dogs of different sex, age, and breed. Collected serum samples were examined using an indirect fluorescent antibody test. Thirty-two of the examined samples showed detectable IgG antibodies in titers of 1:50. Dogs older than one year old and stray dogs had a higher seroprevalence than other dogs. No sex or breed predispositions to N. caninum infection were detected during this serological assay (P ≤ 0.05).     

CONGENITAL CMV INFECTION; DIAGNOSIS IN SYMPTOMATIC INFANTS

Background: Samples from babies exhibiting clinical symptoms suggestive of congenital infection are referred regularly to NICD, New Delhi, from Government Hospitals located in Delhi and a home for abandoned children (Palna), for the diagnosis of etiological agents like toxoplasma, rubella, CMV and herpes. Blood samples of mothers of most of the affected babies are also received. Objective: Evaluation of rapid and accurate technique for the diagnosis of congenital CMV infection. Materials and Methods: One hundred and twenty five blood samples suggestive of symptomatic congenital CMV infection were selected from samples received at NICD during the period June 2005-March 2007. A request to collect and send the urine samples of the selected babies was sent to the respective hospitals. Serum samples of the babies were tested for CMV-IgM antibodies using µ-capture ELISA. Mothers’ serum samples were subjected to CMV-IgM and IgG class antibodies assay by commercial ELISA kits. DNA isolation and amplification was performed in urine samples and some of the serum samples using a commercial PCR kit for detection of HCMV. Blood and urine samples from 20 normal babies were included in the study. Results: Twenty Seven serum samples (21.6%) of infants, of the 125 tested, were positive for CMV-IgM antibodies. Twenty five samples (20%) showed amplification of CMV –DNA. All 25 samples positive for PCR were positive for CMV IgM antibodies. Sera of 73 mothers, out of 75 tested (97.3%), were positive for CMV IgG antibodies. However, none of them was positive for CMV IgM antibodies. Mothers of all 27 positive babies were positive for CMV-IgG antibodies. Serum and urine samples from 20 normal babies were negative for ELISA and PCR. Conclusion: µ-capture ELISA technique was found to be more sensitive than PCR (92.6%) for detection of congenital CMV infection. ELISA is also rapid, less cumbersome and cost effective for diagnosis of CMV infection.     

Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool

The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis.     

Antibody reactivity to Omp31 from Brucella melitensis in human and animal infections by smooth and rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection.

Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.     

Development and laboratory evaluation of a lateral flow device (LFD) for the serodiagnosis of Theileria annulata infection

Several DNA-based and serological tests have been established for the detection of Theileria annulata infection, including polymerase chain reaction, reverse line blot and loop-mediated isothermal amplification, indirect enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. In this study, we have applied knowledge from the development and application of a recombinant protein-based indirect ELISA and competitive ELISA to establish a rapid test for point-of-care diagnosis of T. annulata infection in the field to be used by the veterinarian. For the development of a lateral flow test, the recombinantly expressed T. annulata surface protein (TaSP) was applied as the test antigen and anti-TaSP antiserum as the control line. TaSP antigen conjugated to colloidal gold particles was used as the detection system for visualization at the test line for the binding of anti-TaSP antibody present in the serum of infected animals. The developed test specifically detected antibodies in the serum of animals experimentally infected with T. annulata and showed no cross-reactivity with serum from animals infected with other tested bovine pathogens (Trypanosoma brucei, Anaplasma marginale, Babesia bigemina, Babesia bovis, and Theileria parva). Testing of field samples was compared to results obtained by other serological tests, resulting in a sensitivity and specificity of 96.3% and 87.5% compared to indirect fluorescence antibody test, 98.7% and 81.8% compared to indirect ELISA, and 100% and 47.6% compared to competitive ELISA. In conclusion, a rapid test for the detection of T. annulata infection (T. annulata lateral flow device, Ta-LFD) has been developed, which is easy to perform, delivers results to be read by the naked eye within 10 min, and is suitable for the detection of infection in field samples.     

Evaluation of a serological method for the detection of <Emphasis Type="Italic">Taenia saginata</Emphasis> cysticercosis using serum and meat juice samples

Two peptides, HP6-2 and Ts45S-10, were used as antigens for the detection of antibodies against Taenia saginata cysticercosis in serum and meat juice samples using enzyme-linked immunosorbent assay (ELISA). Positive control samples were obtained from animals experimentally infected (serum) and from animals naturally infected (meat juice). The two peptides and a pooled preparation of both peptides were evaluated, and their cut-off points with both sample categories were calculated. ELISA results from these different peptides were compared. Sensitivity and specificity of HP6-2 using serum were calculated as being 100 and 98%, respectively, showing to be higher than the values for the other antigens used. The average optical density (OD) value for negative samples was 0.646, whereas it was 1.702 for the positive control samples. This peptide was used to examine serum samples from animals with cysts and random field serum samples. For meat juice samples the pooled peptides showed the highest sensitivity and specificity, as they were 100 and 95%, respectively. The average OD values for the negative and the positive reference meat juice samples were 0.379 and 1.291, respectively. The optimal dilution of the meat juice samples for the ELISA was very low, as it was 1:20 using the pooled peptides, compared with 1:800 serum dilution using HP6-2. To the authors’ knowledge, this is the first report of a successful testing for T. saginata cysticercosis using meat juice.     

Development of rapid immunochromatographic test with recombinant NcSAG1 for detection of antibodies to Neospora caninum in cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

Seroprevalence of canine babesiosis in Hungary suggesting breed predisposition

Six hundred fifty-one blood samples were collected from urban and rural dogs in various parts of Hungary to measure antibody levels to Babesia canis with indirect fluorescent antibody test. Thirty-seven (5.7%) of the sera showed positivity with titers between 1:80 and 1:10,240. Seroconverted dogs were found in 13 locations of the country. It is concluded that canine babesiosis is becoming more prevalent in Eastern Hungary. Seropositivity increased then declined with age, reaching a maximum in case of 3.1- to 5-year-old dogs. Prevalence of antibodies to B. canis was significantly higher among german shepherds and komondors. This suggests a genetic predisposition of german shepherd dogs to chronic babesiosis (carrier status) with long-term maintenance of their seropositivity. On the other hand, heavy-coated komondors are phenotypically more suitable for repeated exposure to ticks, potentially infected with B. canis. This is the first report on the seroprevalence of canine babesiosis in Hungary.     

Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis

The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies in the area. In this study, we describe the possible use of the clone-9 protein for this purpose, which was identified as a potential immunogenic piroplasm protein by random sequencing of cDNA library clones followed by bioinformatic analyses. The clone-9 gene was partially recombinantly expressed and used for the development of an indirect ELISA for the detection of circulating antibodies in sera of T. uilenbergi-infected sheep. No cross-reactivity was observed in serum from animals infected with Theileria lestoquardi. The cut-off was calculated at 48.6% positivity using 25 serum samples from uninfected animals. A total of 101 field samples collected from an endemic area in China were used to evaluate the clone-9 ELISA for its use in the field.     

Humoral immune responses during experimental infection with <Emphasis Type="Italic">Fascioloides magna</Emphasis> and <Emphasis Type="Italic">Fasciola hepatica</Emphasis> in goats and comparison of their excretory/secretory products

This study investigated the humoral immune responses of goats experimentally infected with Fascioloides magna and Fasciola hepatica to F. magna excretory/secretory products (FmESP) or F. hepatica excretory/secretory products (FhESP), respectively. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum antibody responses and for possible discrimination of F. magna and F. hepatica infections in goats. Comparison of ESPs of both flukes and evaluation of ESP antigenicity was also studied applying immunoblotting techniques. In all infected goats, antibody level was significantly increased (against negative control) since 2 weeks post infection (WPI). However, the dynamics of antibodies varied between F. magna and F. hepatica groups during the course of the infection. The cross-reaction of antibodies developed against F. magna and F. hepatica with ESP proteins was recorded by ELISA. The species-specific proteins 40, 120 kDa from FmESP and 80, 160 kDa from FhESP (with no antibody cross-reaction) were detected by two dimensional electrophoresis and immunoblot as the potential immunodiagnostic markers. Our results suggest that F. magna and F. hepatica infection could be distinguished by common immunological techniques based on species-specific antigen–antibodies interaction.     

Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay

Sera collected from dogs experimentally infected with Borrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR(1) to IR(6)) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C(1) to C(6)), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR(2) and IR(6), were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR(6) appears earlier and is stronger than that to IR(2). Thus, the IR(6) sequence alone appeared to be sufficient for serodiagnosis. When C(6) alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C(6) ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C(2) and C(6) together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C(6) alone, confirming that C(6) suffices as a diagnostic probe. Moreover, the C(6) ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C(6) ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.