AJTMH ASTMH MEMBERSHIP INFORMATION: astmh@astmh.org
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 78(1), 2008, pp. 117-121
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, C.-M.
Right arrow Articles by Igarashi, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, C.-M.
Right arrow Articles by Igarashi, I.
Related Collections
Right arrow Babesiosis
Right arrow Babesia

Development of a Rapid Immunochromatographic Test for Simultaneous Serodiagnosis of Bovine Babesioses Caused by Babesia bovis and Babesia bigemina

Chul-Min Kim, Lidia Beatriz Conza Blanco, Andy Alhassan, Hiroshi Iseki, Naoaki Yokoyama, Xuenan Xuan, AND Ikuo Igarashi*
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
With the objective of developing a simpler diagnostic alternative, a rapid immunochromatographic test (BoiICT) was constructed for the simultaneous detection of Babesia bovis- and Babesia bigemina-specific antibodies using B. bovis recombinant merozoite surface antigen-2c and B. bigemina recombinant rhoptry-associated protein-1. The BoiICT selectively detected specific antibodies to B. bovis and B. bigemina. All sera from cattle infected with other protozoan parasites (i.e., Cryptosporidium parvum, Neospora caninum, and Theileria orientalis) showed negative results in the BoiICT. The relative sensitivity and specificity for detecting antibody to B. bovis were 96.7% (29 of 30) and 91.3% (73 of 80), respectively. The relative sensitivity and specificity for detecting antibody to B. bigemina were 96.7% (29 of 30) and 92.5% (74 of 80), respectively. These findings indicate that the BoiICT is useful for fast field diagnostic assessment of bovine babesioses without any laboratory equipment.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine babesiosis, especially that caused by Babesia bovis or Babesia bigemina, is an important protozoan disease from both veterinary and economic viewpoints. It is estimated that bovine babesiosis may endanger half a billion cattle worldwide.1 The infections are generally characterized by fever, listlessness, anorexia, dehydration, and progressive hemolysis.2 Several serologic methods have been standardized for the diagnosis of babesiosis, such as the indirect fluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA), and the immunochromatographic test (ICT), and extensively used in epidemiologic studies.3–11 Compared with other serologic tests, the ICT is a simple and rapid method, which makes it suitable for clinical and field applications. It relies on migration of liquid across the surface of a nitrocellulose membrane and because of its convenience has been developed for a variety of applications over the past decade.12–14 Assays using this format take approximately 15 minutes to complete and require only a small volume (< 200 µL) of the tested serum on the test strip.8,15–18

Babesia bovis and B. bigemina are transmitted by the same tick vector, e.g., Rhipicephalus (Boophilus) microplus, R. annulatus, and R. geigyi.2,18 Previous studies have reported the simultaneous prevalence of both parasites in cattle.2,19 However, none of the serologic methods enabled simultaneous detection of specific antibodies to B. bovis and B. bigemina. Therefore, a simpler test capable of simultaneously detecting two types of antibodies in an ICT is needed. A rapid and accurate assay for differential diagnosis between B. bovis and B. bigemina would be extremely valuable in testing clinical specimens, not only for diagnostic purposes and controlling the diseases, but also for better understanding of the epidemiology of these organisms.2,6

We have developed two ICTs (BoICT and BiICT) for the individual detection of B. bovis- or B. bigemina-specific antibodies by placing either of the recombinant C-terminal portions of the rhoptry-associated proteins-1 (rRAP-1/CTs) of these parasites on a test strip.8 Because B. bovis and B. bigemina rRAP-1/CTs were available for the development of two kinds of diagnostic ICTs, we attempted to combine these two rRAP-1/CTs on a test strip and evaluate the activity of the simultaneous serodiagnosis of both infections using a dual ICT. However, the sensitivity and specificity of the dual ICT was < 80% compared with values for the separate tests, in which values were insufficient for diagnostic applications (Kim CM and others, unpublished data). Accordingly, to increase the sensitivity and specificity of the dual ICT, we replaced B. bovis rRAP-1/CT with recombinant merozoite surface antigen-2c (rMSA-2c), which was also known to be a potential antigen for serologic diagnosis of B. bovis,20 and then evaluated its use in the dual ICT.

In the present study, recombinant antigens of B. bovis MSA-2c and B. bigemina RAP-1/CT were prepared as antigens for the dual ICT (BoiICT). We then evaluated the Boi-ICT for simultaneous serodiagnosis of two bovine babesioses.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites. The Texas T2B strain of B. bovis21 and the Argentine strain of B. bigemina22 were grown in purified bovine erythrocytes using a previously established continuous microaerophilous stationary-phase culture system.23 The parasites were used for DNA extraction as previously described.3

Preparation of recombinant antigens. In our previous study, B. bigemina rRAP-1/CT was prepared as a glutathione S-transferase (GST)-fusion protein and directly used for construction of BiICT because the fusion protein was not successfully cleaved into intact rRAP-1/CT and GST by thrombin protease.8 In the present study, a new recombinant C-terminal portion of the RAP-1 (rRAP-1/CT17) was designed (amino acids 326–480, GenBank accession no. M60878) and prepared by using oligonucleotide primers 5'-ccggaattcCTGGTCCCCGAAGAGCAC-3' and 5'-ataagaatgcggccgcTTACGCATCTGAATCATCTG-3' (the lowercase letters show the EcoRI and NotI restriction site linkers, respectively) as described previously.3 The GST protein was removed from the fusion protein using thrombin protease (Amersham Biosciences Corp., Piscataway, NJ), and an rRAP-1/CT17 without GST was successfully obtained.

Babesia bovis rMSA-2c was prepared with oligonucleotide primers 5'-cggaattcATGGTGTCTTTAACATAATAACC-3' and 5'-atagtttagcggccgcGAATGCAGAGAGAACGAAGTAGCAG-3' (the lowercase letters show the EcoRI and NotI restriction site linkers, respectively) on the basis of the nucleotide sequence of the MSA-2c gene (GenBank accession no. AY052542), and 798 basepairs of a DNA fragment containing the MSA-2c gene was amplified from the extracted DNA of the cultured parasite by polymerase chain reaction.3 The amplified DNA product was digested with EcoRI and NotI, purified with a QIAquick gel extraction kit (Qiagen, Inc., Hilden, Germany), and ligated into the EcoRI and NotI sites of a pGEX-4T E. coli expression plasmid vector (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). The resulting plasmid produced a recombinant MSA-2c fused with GST in the transformed E. coli BL21 strain, and non-fused rMSA-2c was successfully purified and cleaved as described previously.3

Production of rabbit antibodies to rMSA-2c and rRAP-1/ CT17. Three-month-old female Japanese white rabbits (CLEA Japan, Tokyo, Japan) were immunized with 1 mg of rMSA-2c or rRAP-1/CT17 protein mixed with an equal volume of TiterMax (CytRx Corporation, Norcross, GA) by multiple intradermal injections into their backs. Three booster immunizations were given at seven-day intervals. A week after the third booster immunization, sera were confirmed for the development of specific IgG titers (> 1/8,192) by ELISA8 and collected from immunized rabbits. Total IgG was purified from the sera with an Econo-Pac Protein A Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions.

Preparations of gold colloid-conjugated antigens and ICT strip. Gold colloid-conjugated antigens and an ICT strip were prepared as previously described16 with some modifications. Briefly, 200 µg/mL of purified rMSA-2c and rRAP-1/CT17 proteins were mixed gently with gold colloid (British BioCell International, Cardiff, United Kingdom), and the colloid-conjugated antigens were then sprayed on glass fiber (Schleicher and Schuell, Keene, NH). The assemblage of the ICT strip was then carried out as described previously with some modifications.15 Briefly, purified rabbit anti-MSA-2c IgG (2 mg/mL) and rMSA-2c (300 µg/mL) and rRAP-1/CT17 proteins (100 µg/mL) were jetted linearly on a nitrocellulose membrane as the procedural control, B. bovis, and B. bigemina test lines, respectively. The nitrocellulose membrane, as well as the conjugated pad, sample pad, and absorbent pad, was assembled on an adhesive card (Schleicher and Schuell) and then cut into 2-mm–wide strips using a BioJet 3050 quanti-dispenser (BioDot Inc., Irvine, CA). A single ICT for the individual detection of B. bovis- (BoICT) or B. bigemina-specific antibody (BiICT) was also prepared as previously described.8 The strips were stably stored with dehumidification in foil pouches at ambient temperature until use.

Enzyme-linked immunosorbent assay. The ELISA using either rMSA-2c (BoELISA) or rRAP-1/CT17 (BiELISA) protein diluted with carbonate buffer (pH 9.6) at a final concentration of 0.5 or 0.3 µg/mL, respectively, was performed as described previously.3,5 The optical densities were measured at a wavelength of 415 nm (OD415) using an MTP-120 ELISA reader (Corona Electric, Ibaraki, Japan).

Sera. Bovine sera used for the evaluation of ICTs were as follows: sequential bovine sera from experimentally infected cattle with B. bovis (n = 13, 0–93 days post-infection) or B. bigemina (n = 13, 0–274 days post-infection) provided by the National Institute of Animal Health (Tsukuba, Ibaraki, Japan); non-infected control sera (n = 10) obtained from healthy cattle that had been bred at Obihiro University of Agriculture and Veterinary Medicine in Japan, where no B. bovis and B. bigemina infections have been reported; Neospora caninum– (n = 5) and Cryptosporidium parvum–positive bovine sera (n = 5) diagnosed by ELISA24,25; Theileria orientalis–positive bovine sera (n = 5) diagnosed using a microscopic test and polymerase chain reaction using blood. For ICTs, 50 µL of the diluted serum with phosphate-buffered saline (1:1) was applied on the sample pad, followed by evaluation within 10 minutes at room temperature as described previously.8 The relative sensitivities and specificities of ICTs and ELISAs were determined with 30 B. bovis- or B. bigemina-positive field bovine sera or 50 B. bovis- and B. bigemina-negative field bovine sera that had been previously evaluated by IFAT and different ELISAs using recombinant B. bovis and B. bigemina antigens (RAP-1/CTs).8

Statistical analysis. The strength of agreement between the BoiICT and single ICT was estimated by calculating the kappa statistic. Kappa statistic values > 0.75, 0.40–0.75, and < 0.40 represent excellent agreement, good to fair agreement, and poor agreement, respectively.26


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluations of ELISAs using B. bovis rMSA-2c and B. bigemina rRAP-1/CT17. To evaluate the usefulness of B. bovis rMSA-2c and B. bigemina rRAP-1/CT17 proteins as serodiagnostic antigens, these proteins were subjected to the diagnostic ELISAs (BoELISA and BiELISA). The BoELISA for B. bovis infection detected antibodies in two negative sera and 29 B. bovis- and 2 B. bigemina-positive sera at an OD415 > 0.22 (Figure 1AGo), whereas 3 negative sera and 2 B. bovis- and 29 B. bigemina-positive sera showed positive responses in the BiELISA for B. bigemina infection at an OD415 >0.18 (Figure 1BGo). The BoELISA and BiELISA showed identical relative sensitivity (96.7%, 29 of 30), and the relative specificities were 95% (76 of 80) and 93.8% (75 of 80) for the BoELISA and BiELISA, respectively, compared with data evaluated by previously described methods.18 We then examined the usefulness of the ELISAs by using sequential sera obtained from cattle experimentally infected with B. bovis or B. bigemina. The BoELISA and BiELISA detected antibodies to B. bovis and B. bigemina from 14 to 93 days and from 13 to 274 days post-infection, respectively.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		    FIGURE 1. Scatter chart of optical density (OD) values in an enzyme-linked immunosorbent assay (ELISA) using A, Babesia bovis recombinant merozoite surface antigen-2c (rMSA-2c) and B, B. bigemina recombinant rhoptry-associated protein-1 (rRAP-1/CT17). Samples from 30 B. bovis- or 30 B. bigemina-positive field bovine sera, or 50 B. bovis- and B. bigemina-negative sera that had been previously evaluated by indirect fluorescent antibody test and different ELISAs using recombinant RAP-1/CTs were studied using two ELISAs (BoELISA or BiELISA, respectively). Dotted lines represent the cut-off lines of the ELISAs. A sample was considered positive at an OD > 0.22 at 415 nm in the BoELISA (A) and > 0.18 in the BiELiSA (B).

 
Evaluation of the BoiICT for simultaneous serodiagnosis for B. bovis and B. bigemina infections. Figure 2AGo shows three types of detectable lines for procedural control and B. bovis and B. bigemina tests produced on the BoiICT after the application of various kinds of serum samples. The BoiICT can detect simultaneously, but selectively, two kinds of specific antibodies to target parasites on a single test strip. We then examined the usefulness of BoiICT on the simultaneously diagnostic activity in sequential bovine sera from cattle infected with B. bovis or B. bigemina. The BoiICT detected the antibodies to B. bovis in the sera collected from 14 to 93 days post-infection (Figure 2BGo) and those to B. bigemina from 13 to 274 days post-infection (Figure 2CGo). In addition, no cross-reaction was observed between the B. bovis-and B. bigemina-infected positive sera, and all sera collected from cattle infected with other protozoan parasites (Cryptosporidium parvum, Neospora caninum, and Theileria orientalis) showed negative reactions in the BoiICT.


Figure 2
View larger version (48K):
[in this window]
[in a new window]

  		    FIGURE 2. Rapid immunochromatographic test (BoiICT) strips for simultaneous detection of specific antibodies to Babesia bovis and B. bigemina. A, Examples of BoiICT strips before (lane 1) and after (lanes 2–5) application. Lane 1, strip before application of serum; lane 2, strip after application of negative serum against B. bovis and B. bigemina; lane 3, strip after application of serum positive only against B. bovis; lane 4, strip after application of serum positive only against B. bigemina; lane 5, strip application of serum positive against B. bovis and B. bigemina. B and C, Specific antibody responses in sequential bovine sera obtained from cattle experimentally infected with B. bovis (B) and B. bigemina (C). The numbers on the strips represent days post-infection. BoiICT detected antibodies to B. bovis and B. bigemina in sera collected from 14 to 93 and from 13 to 274 days post-infection, respectively.

 
To determine the relative sensitivity and specificity of the BoiICT compared data evaluated by previously described methods,18 30 B. bovis- or 30 B. bigemina-positive sera or 50 B. bovis- and B. bigemina-negative sera were examined using the BoiICT, and the results were compared with those obtained with BoICT and BiICT, two tests that were developed for the individual diagnosis of either B. bovis or B. bigemina infection. The relative sensitivities of both BoICT and Boi-ICT for detecting antibody to B. bovis were 96.7% (29 of 30), and the relative specificities were 93.8% (75 of 80) and 91.3% (73 of 80), respectively (Table 1Go). The relative sensitivities of BiICT and BoiICT for detecting antibody to B. bigemina were 96.7% (29 of 30), and the relative specificities were 93.8% (75 of 80) and 92.5% (74 of 80), respectively (Table 2Go). When these results were compared statistically, the agreement between BoiICT and single ICTs was 98.2% (108 of 110) with a kappa statistic of 0.96 (P < 0.001).


View this table:
[in this window]
[in a new window]

 
TABLE 1
Comparison of BoICT and BoiICT for the detection of specific antibodies against Babesia bovis in bovine sera*
 

View this table:
[in this window]
[in a new window]

 
TABLE 2
Comparison of BiICT and BoiICT for detection of specific antibodies against Babesia bigemina in bovine sera*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In recent years, there have been several reports on the serodiagnosis of bovine babesiosis.3,6,9,19 However, these methods, such as the ELISA and IFAT, involve complex procedures that require laboratory materials, equipment, and trained personnel. In the present study, we developed a new rapid assay (BoiICT) using rMSA-2c and rRAP-1/CT17 antigens for serodiagnosis. The obvious advantage of ICT is that this assay provides accurate results in 15 minutes or less. Another important aspect of the BoiICT developed in the present study, compared with other available tests, is its multiplex characteristic. With this approach, it was possible to simultaneously and selectively detect two kinds of specific antibodies to B. bovis or B. bigemina using one reaction strip. Simultaneous detection is extremely useful because these species have often shown an overlapping geographic distribution.8,19

Merozoite surface proteins of apicomplexan hemoparasites, such as Babesia and Plasmodium spp., often provide potential targets for the immune-mediated control.27–29 Babesia bovis MSA-2c is also exposed on the surfaces of both merozoites and sporozoites.30 Previous studies showed that B. bovis MSA-2c has a high degree of genetic and antigenic conservation among geographically distant strains.20 Thus, this antigen appears to be an adequate diagnostic candidate for the serodiagnosis of bovine babesiosis. The adaptation of rMSA-2c, instead of rRAP-1/CT, could lead to the successful development of an alternative test. Because a highly specific recombinant antigen in ELISA has showed promise for its use in the diagnostic ICT,15,16 the diagnostic potential of B. bovis rMSA-2c and B. bigemina rRAP-1/CT17 was first evaluated in ELISA prior to construction of the ICT. The developed ELISAs using the rMSA-2c and rRAP-1/CT17 proteins (BoELISA and BiELISA, respectively) showed high relative sensitivity (96.7%) and specificities (95% and 93.8%, respectively). Therefore, we applied these two proteins in developing a simultaneous ICT, BoiICT, for B. bovis and B. bigemina infections.

From the results of the BoiICT, the relative sensitivity and specificity for B. bovis infection were 96.7% and 91.3%, respectively, and those for B. bigemina were 96.7% and 92.5%, respectively. The results suggest that the BoiICT is sensitive and specific for serodiagnosis of both infections, as indicated in previously reported ICTs for various protozoan infections with sensitivities ranging from 72% to 100% and specificities ranging from 61% to 100%.15–17,31–33 In addition, no significant differences were observed in the sensitivities and specificities between the BoICT and the BoELISA and those between the BiICT and the BiELISA, although discordant cases were observed in two sera between the BoICT and the Boi-ICT and one serum between the BiICT and the BoiICT. These discordant sera showed high absorbance (OD415 > 1.2) above the cutoff value against only one species of either B. bovis or B. bigemina in the ELISA. Therefore, non-specific reactions might influence the antigen or antibody cross-reactivity, and the BoiICT might result in a misdiagnosis.

For clinical use, the ICT strip must have high specificity to distinguish other infections of related pathogenic protozoa. The BoiICT could accurately differentiate the B. bovis or B. bigemina infections from other infections. Therefore, the present configuration of the BoiICT is useful for simultaneously detecting B. bovis- and B. bigemina-specific antibodies under a variety of infective circumstances. Although the specificity of the BoiICT was slightly lower than that of single ICTs, the kappa value (0.96) demonstrated excellent agreement between them. Moreover, the BoiICT detected antibodies to B. bovis and B. bigemina from 14 and 13 days to the last days post-infection, respectively, without cross-reactivity. These results indicate that this rapid test could simultaneously detect the early stage to latent infection of B. bovis and B. bigemina.

In conclusion, the BoiICT is a simple and rapid method that provides accurate and simultaneous detection of B. bovis and B. bigemina infections, thereby saving time and eliminating the need for special training. This rapid test can therefore be practically implemented in epidemiologic surveys for bovine babesiosis, although evaluation on a larger scale with various cattle sera is still necessary.

Received July 14, 2007. Accepted for publication October 4, 2007.

Acknowledgments: We thank Yoshio Nakamura (National Institute of Animal Heath) for providing sequential bovine sera of cattle experimentally infected with B. bovis and B. bigemina.

Financial support: This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and grants from the Program for the Promotion of Basic Research Activities for Innovative Biosciences and the 21st Century COE Program (A-1), Ministry of Education, Culture, Sports, Science, and Technology, Japan.

* Address correspondence to Ikuo Igarashi, Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. E-mail: igarcpmi{at}obihiro.ac.jp Back

Authors’ address: Chul-Min Kim, Lidia Beatriz Conza Blanco, Andy Alhassan, Hiroshi Iseki, Naoaki Yokoyama, Xuenan Xuan, and Ikuo Igarashi, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Reprint requests: Ikuo Igarshi, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan, Telephone: 81-155-49-5641, Fax: 81-155-49-5643, E-mail: igarcpmi{at}obihiro.ac.jp.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. McCosker PJ, 1981. The global importance of babesiosis. Ristic M, Kreier JP, eds. Babesiosis. New York: Academic Press, 1–24.
  2. Bock R, Jackson L, de Vos A, Jorgensen W, 2004. Babesiosis of cattle. Parasitology 129 (Suppl): S247–S269.
  3. Boonchit S, Alhassan A, Chan B, Xuan X, Yokoyama N, Ooshiro M, Goff WL, Waghela SD, Wagner G, Igarashi I, 2006. Expression of C-terminal truncated and full-length Babesia bigemina rhoptry-associated protein 1 and their potential use in enzyme-linked immunosorbent assay. Vet Parasitol 137: 28–35.[Web of Science][Medline]
  4. Boonchit S, Xuan X, Yokoyama N, Goff WL, Wagner G, Igarashi I, 2002. Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle. J Clin Microbiol 40: 3771–3775.[Abstract/Free Full Text]
  5. Boonchit S, Xuan X, Yokoyama N, Goff WL, Waghela SD, Wagner D, Igarashi I, 2004. Improved enzyme-linked immunosorbent assay using C-terminal truncated recombinant antigens of Babesia bovis rhoptry-associated protein-1 for detection of specific antibodies. J Clin Microbiol 42: 1601–1604.[Abstract/Free Full Text]
  6. Bose R, Jorgensen WK, Dalgliesh RJ, Friedhoff KT, de Vos AJ, 1995. Current state and future trends in the diagnosis of babesiosis. Vet Parasitol 57: 61–74.[Web of Science][Medline]
  7. Ferrer D, Castella J, Gutierez JF, Lavin S, Marco I, 1998. Seroprevalence of Babesia ovis in mouflon sheep in Spain. J Wildl Dis 34: 637–639.[Abstract]
  8. Kim CM, Alhassan A, Verdida RA, Yokoyama N, Xuan X, Fujisaki K, Kawazu S, Igarashi I, 2007. Development of two immunochromatographic tests for the serodiagnosis of bovine babesiosis. Vet Parasitol 148: 137–143.[Web of Science][Medline]
  9. Molloy JB, Bowles PM, Bock RE, Turton JA, Katsande TC, Katende JM, Mabikacheche LG, Waldron SJ, Blight JW, Dalgliesh RJ, 1998. Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe. Prev Vet Med 33: 59–67.[Web of Science][Medline]
  10. Reiter I, Weiland G, 1989. Recently developed methods for the detection of babesial infections. Trans R Soc Trop Med Hyg 83: 21–23.[Medline]
  11. Wright IG, 1990. Immunodiagnosis of and immunoprophylaxis against the hemoparasites Babesia sp. and Anaplasma sp. in domestic animals. Rev Sci Tech Off Int Epizoot 9: 345–356.
  12. de Saeger S, van Peteghem C, 1996. Dipstick enzyme immunoassay to detect Fusarium T-2 toxin in wheat. Appl Environ Microbiol 62: 1880–1884.[Abstract]
  13. Shiff CJ, Premji Z, Minjas JN, 1993. The rapid manual Para-Sight-F test. A new diagnostic tool for Plasmodium falciparum infection. Trans R Soc Trop Med Hyg 87: 646–648.[Web of Science][Medline]
  14. Sumathy S, Thyagarajan SP, Latif R, Madanagopalan N, Raguram K, Rajasambandam P, Gowans E, 1992. A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections. J Virol Methods 38: 145–152.[Web of Science][Medline]
  15. Huang X, Xuan X, Verdida RA, Zhang S, Yokoyama N, Xu L, Igarashi I, 2006. Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses. Clin Vaccine Immunol 13: 553–555.[Abstract/Free Full Text]
  16. Huang X, Xuan X, Hirata H, Yokoyama N, Xu L, Suzuki N, Igarashi I, 2004. Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats. J Clin Microbiol 42: 351–353.[Abstract/Free Full Text]
  17. Mills CD, Burgess DCH, Taylor HJ, Kain KC, 1999. Evaluation of a rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria. Bull World Health Organ 77: 553–558.[Web of Science][Medline]
  18. Wright IG, Goodger BV, 1988. Pathogenesis of babesiosis. Ristic M, ed. Babesiosis of Domestic Animals and Man. Boca Raton, FL: CRC Press, 99–118.
  19. Barros SL, Madruga CR, Araujo FR, Menk CF, de Almeida MA, Melo EP, Kessler RH, 2005. Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays. Mem Inst Oswaldo Cruz 100: 513–517.[Medline]
  20. Wilkowsky SE, Farber M, Echaide I, Torioni de Echaide S, Zamorano PI, Dominguez M, Suarez CE, Florin-Christensen M, 2003. Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle. Mol Biochem Parasitol 127: 133–141.[Web of Science][Medline]
  21. Suarez CE, Florin-Christensen M, Hines SA, Palmer GH, Brown WC, McElwain TF, 2000. Characterization of allelic variation in the Babesia bovis merozoite surface antigen 1 (MSA-1) locus and identification of a cross-reactive inhibition-sensitive MSA-1 epitope. Infect Immun 68: 6865–6870.[Abstract/Free Full Text]
  22. Hotzel I, Suarez CE, McElwain TF, Palmer GH, 1997. Genetic variation in the dimorphic regions of RAP-1 genes and rap-1 loci of Babesia bigemina. Mol Biochem Parasitol 2: 479–489.
  23. Levi MG, Ristic M, 1980. Babesia bovis: continuous cultivation in a microaerophilus stationary phase culture. Science 207: 1218–1220.[Abstract/Free Full Text]
  24. Chahan B, Gaturaga I, Huang X, Liao M, Fukumoto S, Hirata H, Nishikawa Y, Suzuki H, Sugimoto C, Nagasawa H, Fujisaki K, Igarashi I, Mikami T, Xuan X, 2003. Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. Vet Parasitol 118: 177–185.[Web of Science][Medline]
  25. Takashima Y, Xuan X, Kimata I, Iseki M, Kodama Y, Nagane N, Nagasawa H, Matsumoto Y, Mikami T, Otsuka H, 2003. Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits. J Parasitol 89: 276–282.[Medline]
  26. Pottumarthy S, Morris AJ, Harrison AC, Wells VC, 1999. Evaluation of the tuberculin gamma interferon assay; potential to replace the Mantoux skin test. J Clin Microbiol 37: 3229–3232.[Abstract/Free Full Text]
  27. Chitnis CE, Blackman MJ, 2000. Host cell invasion by malaria parasites. Parasitol Today 16: 411–415.[Web of Science][Medline]
  28. Cowman AF, Baldi DL, Duraisingh M, Healer J, Mills KE, O’Donnell RA, Thompson J, Triglia T, Wickham ME, Crabb BS, 2002. Functional analysis of Plasmodium falciparum merozoite antigens: implications for erythrocyte invasion and vaccine development. Philos Trans R Soc Lond B Biol Sci 357: 25–33.[Abstract/Free Full Text]
  29. Egan AF, Burghaus P, Druilhe P, Holder AA, Riley EM, 1999. Human antibodies to the 19kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro. Parasite Immunol 21: 133–139.[Web of Science][Medline]
  30. Cowman AF, Bernard O, Stewart N, Kemp D, 1984. Genes of the protozoan parasite Babesia bovis that rearrange to produce RNA species with different sequences. Cell 37: 653–660.[Web of Science][Medline]
  31. Chan R, Chen J, York MK, Setijono N, Kaplan RL, Graham F, Tanowitz HB, 2000. Evaluation of a combination rapid immunoassay for detection of Giardia and Cryptosporidium antigens. J Clin Microbiol 38: 393–394.[Abstract/Free Full Text]
  32. Mohebali M, Taran M, Zarei Z, 2004. Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick rk39 test and direct agglutination. Vet Parasitol 121: 239–245.[Web of Science][Medline]
  33. Reithinger R, Quinnell RJ, Alexander B, Davies CR, 2002. Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick test, enzyme-linked immunosorbent assay, and PCR. J Clin Microbiol 40: 2352–2356.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, C.-M.
Right arrow Articles by Igarashi, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, C.-M.
Right arrow Articles by Igarashi, I.
Related Collections
Right arrow Babesiosis
Right arrow Babesia

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS