Patent Application
20190271697
This invention relates to a high throughput modified indirect enzyme-linked immunosorbent assay (MI-ELISA) developed to diagnose diverse strains of Babesia bovis in all bovine infection stages in order to effectively control intra- and inter-herd transmissions of B. bovis and to prevent the spread of B. bovis from endemic to non-endemic herds.
Bovine babesiosis, caused by protozoan parasites of the genus Babesia (order Piroplasmida, phylum Apicomplexa), is an economically important tick-borne disease, particularly in tropical and subtropical areas of the world (Bock et al. 2004. Parasitol. 129 Suppl: S247-S269; Bovine Babesiosis. 2012. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 7th Edition, World Organization for Animal Health (OIE), Paris). Among the various Babesia species, Babesia bovis and Babesia bigemina are widely distributed and of major importance in Africa, Asia, Australia, and Central and South America (OIE 2012, supra). Babesia species causing severe disease in naïve cattle are a potential threat to Babesia-free or non-endemic areas of the world, including the United States, given the large numbers of cattle that are moved across borders each year. The higher prevalence of B. bovis and B. bigemina in tropical and subtropical areas is closely associated with the availability of the Rhipicephalus microplus tick, the main vector of B. bovis and B. bigemina transmission to cattle and related species (Bowman and Nutall. Babesiosis of Cattle. 2008. In: Ticks: Biology, Disease and Control, Cambridge University Press, Cambridge, UK, pages 281-307). Babesia bovis infection is characterized by high fever, ataxia, anorexia, circulatory shock, and sometimes central nervous system signs due to sequestration of parasitized erythrocytes in cerebral capillaries. Babesia bovis is generally more pathogenic than other bovine Babesia species (OIE 2012, supra).
A few reports demonstrated the persistence of B. bovis in cattle infected with a moderately attenuated strain or vaccinated with a modified live vaccine (MLV) strain. Calves infected with the Mo7 strain, which is moderately attenuated by cloning and in vitro passages, were persistently infected for at least 10 months, based on PCR and other evidence (Suarez et al. 2012. Mol. Biochem. Parasitol. 185:52-57). Friesian cattle vaccinated with an attenuated strain showed evidence of persistent parasitemia for up to 47 months as determined by sub-inoculation into splenectomized calves (Pipano et al. 2002. Vet. J. 164:64-68). These two experiments, though limited, nevertheless provide strong evidence of long-term persistence of B. bovis infection in cattle. In view of this, an ideal serological assay should target a B. bovis antigen that is stably expressed for an extended period and should thus be able to detect specific antibodies throughout all stages of infection. However, to our knowledge, there is no report on long-term (>100 days post-infection or vaccination) serological monitoring of B. bovis-specific antibody responses in persistently infected cattle. One study monitored B. bovis-specific antibody responses through 98 days after challenge with a highly virulent strain, T2Bo, using the cELISA (competitive Enzyme-Linked Immunosorbent Assay) based on rhoptry-associated protein-1 (RAP-1) epitope (Goff et al. 2003. Clin. Diagn. Lab. Immunol. 10:38-43). Another study monitored antibody responses based on an immunofluorescence assay (IFA) through 60 days post-vaccination (Guglielmone et al. 1997. Vet. Parasitol. 70:33-39). In these two studies, both the cELISA and IFA were reliable for short-term monitoring of B. bovis-specific antibody responses; however, their reliability for monitoring longer post-infection periods has not yet been determined. Further defining long-term persistence of B. bovis infection and longevity of B. bovis-specific antibody responses in cattle after infection with various attenuated vaccine strains and pathogenic strains is crucial for developing control measures including higher performance diagnostics and safer vaccines against this tick-borne disease.
Several serological diagnostic assays to detect B. bovis-specific antibodies have been used as in-house assays with limited validation and quality control. The IFA test has been widely used, but low throughput and subjectivity in result interpretation are major disadvantages to its use for serological diagnosis of B. bovis infection. The complement fixation (CF) test has been used in some countries to qualify animals for importation as well as for general diagnosis (OIE 2012, supra). However, the CF test is labor-intensive, time-consuming to perform, and suffers from poor reproducibility among laboratories, perhaps attributable to inadequate standardization of both test reagents and procedure as reported in other intraerythrocytic species (Aubry and Geale. 2011. Transbound. Emerg. Dis. 58:1-30). Further, the CF test as formatted using guinea pig complement does not detect all bovine IgG antibody isotypes (Calder et al. 1996. J. Clin. Microbiol. 34:2748-2755) possibly contributing to poor diagnostic sensitivity. Due to these disadvantages, IFA and CF tests have largely been replaced by ELISAs as the sero-diagnostic test of choice for B. bovis (Bono et al. 2008. Vet. Parasitol. 157:203-210; Boonchit et al. 2004. J. Clin. Microbiol. 42:1601-1604; Goff et al. 2003, supra; Goff et al. 2006. Clin. Vaccine Immunol. 13:1212-1216; Molloy et al. 1998a. Prev. Vet. Med. 33:59-67; Molloy et al. 1998b. Parasitol. Res. 84:651-656; (OIE 2012, supra); Terkawi et al. 2011a. Clin. Vaccine Immunol. 18:337-342). Early ELISA formats for B. bovis included indirect ELISAs using whole merozoite antigen (Molloy et al. 1998a, supra) or recombinant subunit proteins (Bono et al., supra; Boonchit et al., supra). More recently, a competitive blocking ELISA (cELISA) based on an epitope of RAP-1 has been developed and evaluated with limited sera (Goff et al. 2003, supra; Goff et al. 2006, supra). Presently, there is no ELISA or other high throughput assay that has been systematically validated against diverse sera from cattle in different infection stages from various geographical areas (OIE 2012, supra). Cross-reactivity with closely-related Babesia species or other species results in relatively lower specificity for ELISAs using whole parasite antigens (Aubry and Geale, supra; Duzgun et al. 1988. Vet. Parasitol. 29:1-7). If sufficiently-conserved subunit proteins containing multiple B cell epitopes are not utilized, ELISAs using subunit proteins of B. bovis tend to have the opposite problem of relatively lower diagnostic sensitivity due to antigenic variation among B. bovis strains. To overcome the challenges in sero-diagnosis of B. bovis and to better control B. bovis infection globally, a novel, high-throughput assay having excellent diagnostic sensitivity (>95%) and excellent B. bovis species specificity (>95%) is needed. Such a serodiagnostic assay would detect antibody throughout all stages of the infection with the vast majority (ideally all) of global B. bovis isolates and would not detect antibody induced by other closely related Babesia species.
We have developed and validated a spherical body protein-4 (SBP4) MI-ELISA to diagnose bovine species infected with antigenically diverse isolates of Babesia bovis in order to effectively control intra- and inter-herd transmissions of B. bovis and to prevent the spread of B. bovis from endemic to non-endemic herds.
In accordance with this discovery, it is an objective of the invention to provide a method of detecting acute and long-term carrier infection of diverse Babesia bovis isolates with improved sensitivity and specificity over the existing methods of detection of B. bovis in animals, resulting in a more rapid and accurate high throughput diagnosis.
It is thus an objective of this invention to provide a novel fusion protein comprising a modified Babesia bovis-specific protein (SBP4) wherein the expressed protein comprises a signal peptide-deleted SBP4 and glutathione S-transferase (GST) (GST-SBP4 antigen) fusion protein.
It is further an objective of the invention to provide an enhanced method of recombinant GST-SBP4 antigen coating and presentation using glutathione-BSA (G-BSA). This method improves the presentation quality of epitopes in the coated antigen to detect Babesia bovis-specific antibodies in test samples.
Another objective of the invention is to provide a recombinant GST-SBP4 labelled with horse-radish peroxidase (HRP) as the detection conjugate in the MI-ELISA. This novel conjugate is crucial for improved sensitivity and specificity of the MI-ELISA over the other ELISA methods in detecting B. bovis antibodies. This conjugate system allows the MI-ELISA to detect Babesia bovis antibodies in test samples from non-bovine species. This feature is not available in other indirect ELISAs based on specific-specific conjugate system.
Other objectives and advantages of this invention will become readily apparent from the ensuing description.
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Most cattle survive acute B. bovis infection, and some studies have demonstrated that recovered cattle become clinically inapparent carriers that could serve as reservoirs for intra- and inter-herd transmission (Bock et al., supra; Calder et al., supra; Goff et al. 2003, supra; Suarez et al. 2012, supra). However, long-term kinetics and durations of B. bovis parasitemia and antibody responses remain to be defined. Defining these aspects in cattle infected with diverse B. bovis strains in variable doses is crucial for establishing effective control measures against transmission of this important trans-boundary disease, particularly from endemic to non-endemic and Babesia-free countries.
A typical characteristic of B. bovis, but not of B. bigemina, is the ability to escape the immune system of the host using rapid antigenic variation, cytoadhesion and capillary sequestration, and binding of host proteins to the surface of infected red blood cells (Allred, D. R. 2003. Trends. Parasitol. 19:51-55). It is believed that these mechanisms contribute to the establishment of persistent infections, which usually result in the development of long term and fluctuating immune responses. Therefore, if persistent infections are characterized frequently by very low levels of circulating antibodies and often undetectable parasitemia, then detection of persistently infected animals or unknown infected carriers may require exquisitely sensitive methods of detection.
A recent comparative study with five subunit proteins of B. bovis in the conventional indirect ELISA format reported that spherical body protein-4 (SBP4) was relatively better than MSA-2c (merozoite surface antigen-2c), RAP-1/CT (rhoptry-associated protein-1/carboxy terminal), TRAP-T thrombospondin-related anonymous protein-truncated) and SBP1 (spherical body protein-1) in both diagnostic sensitivity and specificity (Terkawi et al. 2011a, supra). Further study defined that SBP4 was produced and secreted in a stable and dominant manner through all stages of the B. bovis life cycle (Terkawi et al. 2011 b, Mol. Biochem. Parasitol. 178:40-45). However, only 85% diagnostic concordance (84.5% sensitivity and 86.2% specificity) was observed between an SBP4-based conventional indirect ELISA and IFA with 469 sera collected from five countries suggesting the need for further improvement (Terkawi et al. 2011a, supra). Our main objectives are to 1) develop and evaluate a novel SBP4 MI-ELISA format using recombinant SBP4 as the coating antigen and detection conjugate to produce higher diagnostic performance than previously reported antibody assays, 2) compare the performance of this novel SBP4 MI-ELISA to the reference IFA and to the previously developed RAP-1 cELISA using cattle sera from non-endemic, endemic and epidemic areas, and 3) demonstrate that the novel SBP4 MI-ELISA would detect antibody in sera from cattle that had long term infections documented by at least occasional nested PCR positive tests and then compare these results with those from IFA and RAP-1 cELISA.
A well-defined, reliable target antigen and robust assay format are critical to the development of a diagnostic assay with high specificity and sensitivity against sera from long-term unapparent carriers. Several sero-diagnostic assays including the CF test (Mahoney, D. F. 1962. Aust. Vet. J. 38:48-52), the indirect hemagglutination (Goodger, B. F. 1971. Aust. Vet. J. 47:251-256), the rapid card agglutination test (Todoric and Kuttler. 1974. Am. J. Vet. Res. 35:1347-1350), the immunofluorescence assay (IFA, Goff et al. 1982. Vet. Parasitol. 11:109-120) and the cELISA (Goff et al. 2003, supra) have been developed, and some have been used extensively in diagnostic labs. Although most of these assays provide diagnostic performance appropriate for herd monitoring that requires moderate diagnostic accuracy, they are not sufficient for disease and disease-free certification of individual animals. Currently the CF test, IFA and ELISA are OIE-alternative tests for international trade of bovines. However, whether these OIE-alternative assays are acceptable for uses in disease or disease-free certification requiring high diagnostic specificity and sensitivity has not been validated.
A cELISA using an epitope of RAP-1 has been developed and tested with limited sera (Goff et al. 2003, supra; Goff et al. 2006, supra). By their very design, single epitope-based cELISAs are susceptible to low diagnostic sensitivity when used for infectious agents with high antigenic variation such as B. bovis. Conventional indirect ELISAs using well-characterized subunit proteins or well-purified whole parasites may have an advantage in diagnostic sensitivity due to leveraging far more B cell epitopes than cELISAs. Nevertheless, antigens for both cELISAs and indirect ELISAs must be carefully selected for species-limited conservation to avoid cross reactivity against other Babesia or related species that could have a deleterious effect on specificity. A recently published comparative analysis of several B. bovis proteins in the conventional indirect ELISA format suggested enormous variations in diagnostic performance depending on subunit proteins. This study also indicated relatively better diagnostic performance of the indirect ELISA based on SBP4 (85% agreement with IFA results) than other B. bovis proteins when tested against sera from five different countries (Terkawi et al. 2011a, supra). A moderate (85%) diagnostic agreement of the SBP4-based conventional indirect ELISA against 469 sera validated in an IFA test in this comparative study indicates the need for further improvement and optimization in format and other variables followed by extensive validation using more diverse sera.
To this end, in the present study, the SBP4-based modified indirect ELISA, SBP4-MI-ELISA, was developed using the recombinant fusion protein rGST-SBP4. The rGST-SBP4 fusion protein (SEQ ID NO: 1) is comprised of glutathione S-transferase (GST) and recombinant spherical body protein-4 modified by having the signal sequence deleted. The rGST-SBP4 fusion protein is used as the coating antigen in the SBP4-MI-ELISA and also as the detection antigen (rGST-SBP4-HRP) when conjugated with horseradish peroxidase. The cutoff for positive or negative antibody detection used in this assay was 3.0 S/N ratio which gave an excellent combination of 100% specificity and 97.6% sensitivity when tested with diverse sera collected from endemic, epidemic and non-endemic areas. Surprisingly, the SBP4-based MI-ELISA had a significantly better diagnostic performance than results reported for the indirect ELISA based on SBP4 (Terkawi et al. 2011a, supra) and the cELISA based on RAP-1 (Goff et al. 2003, supra; Goff et al. 2006, supra) in several aspects. First, the SBP4-based MI-ELISA detected B. bovis antibody earlier after infection than IFA (˜1.5 day delayed positive detection) and the RAP-1-based cELISA (˜9.5 day delayed positive detection). Second, the MI-ELISA was more reliable for long term monitoring of antibody responses in the carrier stage of B. bovis infection than the cELISA, which was unreliable after 5-10 months post-infection when parasitemia was still detectable by PCR. Third, the MI-ELISA had 37% and 9.2% higher diagnostic sensitivity than the cELISA against sera collected from B. bovis-endemic regions in Mexico and Australian herds, respectively. Fourth, diagnostic specificity (100%) of the MI-ELISA was comparable to the cELISA when tested against sera from northwestern U.S. herds kept in tick-free barns. In addition, the MI-ELISA had superior diagnostic specificity to the cELISA against sera from the southern U.S. (100% versus 90.4%) and Mexico (100% versus 93.8%). Fifth, the MI-ELISA had no positive cross-reactivity with sera from cattle infected with B. bigemina or Anaplasma marginale. Persistence of antibody responses specific for B. bovis SBP4 even at DPIs with no detectable parasitemia by nested PCR clearly indicates the relative advantage of certain antibody-based diagnostics over parasitemia detection assays for this disease. This of course depends on the availability of an accurate, high-throughput serology assay with more objective interpretation than IFA, such as the SBP4 MI-ELISA described here. This assay is a more reliable tool for identifying animals in the carrier stage of B. bovis infection than PCR-based parasitemia detection or antibody detection by the RAP-1 cELISA.
It is understood that the present invention also encompasses rGST-SBP4 variants. A preferred rGST-SBP4 variant is one having at least 90% amino acid sequence similarity to the rGST-SBP4 amino acid sequence (SEQ ID NO: 1), a more preferred rGST-SBP4 variant is one having at least 95% amino acid sequence similarity to SEQ ID NO: 1 and a most preferred rGST-SBP4 variant is one having at least 99% amino acid sequence similarity to SEQ ID NO: 1 as defined by the algorithm, CLUSTRAL or PILEUP.
A “variant” of rGST-SBP4 may have an amino acid sequence that is different by one or more amino acid “substitutions”. The variant may have “conservative substitutions”, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software. The term “biological activity” refers to rGST-SBP4 having structural, regulatory or biochemical functions of the naturally occurring protein. Likewise, “immunological activity” defines the capability of the natural, recombinant or synthetic rGST-SBP4, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The phrase “conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue. “Chemical derivative” refers to the chemical modification of a nucleic acid sequence encoding rGST-SBP4 or the encoded rGST-SBP4 wherein the subject nucleic acid or polypeptide has one or more residues chemically derivatized by reaction of a functional side group. Examples of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group; however, replacements are not limited to these groups. A nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of natural rGST-SBP4. Also included are those peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids, e.g., 5-hydroxylysine or ornithine may be substituted for lysine.
The term “peptide” as used herein refers to a molecular chain of amino acids with a biological activity (e.g., capable of binding antibody specific for B. bovis), and does not refer to a specific length of the product. Thus, inter alia, proteins, oligopeptides, polypeptides and fusion proteins as well as fusion peptides are included. Further, GST-SBP4+ and rGST-SBP4 are interchangeable as reagents for detecting
B. bovis-specific antibodies, for generating B. bovis-specific antibodies, and for vaccine development. Thus, inter alia, reference to GST-SBP4+ encompasses rGST-SBP4, and reference to rGST-SBP4 encompasses GST-SBP4+.
The term “antibody,” as used herein, includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The phrases “specifically binds to” or “specifically immunoreactive with”, when referring to an antibody or other binding moiety refers to a binding reaction which is determinative of the presence of the target analyte in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target analyte and do not bind in a significant amount to other components present in a test sample. Specific binding to a target analyte under such conditions may require a binding moiety that is selected for its specificity for a particular target analyte. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immuno-reactivity. Typically a specific or selective reaction will be at least twice background signal to noise and more typically more-than 10 to 100 times background.
The DNA sequences of the invention can be used to prepare recombinant DNA molecules by cloning in any suitable vector. A variety of vector-host combinations may be employed in practicing the present invention. Host cells may be either prokaryotic or eukaryotic, and, when the host cells are bacterial cells, they may be either gram-negative or grain-positive bacteria. Without being limited thereto, examples of hosts suitable for use herein are prokaryotic and eukaryotic hosts such as E. coli K12 or XL1 Blue cells and related bacteria, Saccharomyces cerevisiae, Sf9 or Sf21 insect cells (Spodoptera frugiperda), Chinese hamster ovary cells, and plant cells in culture. However, other hosts may also be utilized.
Vectors used in practicing the present invention are selected to be operable as cloning vectors or expression vectors in the selected host cell. Numerous vectors are known to those of skill in the art, and selection of an appropriate vector and host cell is a matter of choice. This invention encompasses a hybrid vector, that comprises a vector capable of replication, transcription and expression of DNA segments operably coupled thereto; and a DNA segment encoding a polypeptide of this invention comprising the peptide disclosed herein operatively coupled thereto, wherein when the vector is placed in an appropriate host it can express the polypeptide encoded by the DNA segment. Examples of such vectors are pGex (Pharmacia), baculovirus, pET-9d (Novagen), pRSET T7 (Invitrogen), pTriplEx2 plasmid vector and pMal-c2 plasmid vector (Invitrogen). The vector may be a eukaryotic or a prokaryotic vector depending on the host selected for transfection and in which the gene product is going to be expressed.
Still part of this invention is another hybrid vector, that comprises a vector capable of replication, transcription and expression of DNA segments operably coupled thereto; and a DNA segment comprising a DNA fragment encoding at least one of the polypeptides of the invention and a second unrelated DNA segment, both sequences being operably coupled to one another and to the vector. The preparation of the hybrid vector described above is known in the art and need not be further described herein (Smith, D., and K. Johnson, “Single Step Purification of Polypeptides Expressed in E. coli as Fusions with Glutathione S-transferase”, Gene, 67:31(1988); Studier, F. W., et al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes”, Meth. Enzymol., 185:60-89 (1990)).
The antigenic peptides of the invention are produced by growing host cells transformed by the expression vectors described above under conditions whereby the antigen is produced. The antigens are then isolated from the host cells.
Also an important part of this invention is a method of diagnosing B. bovis infection that comprises contacting a body substance with rGST-SBP4 of this invention; and detecting any selective binding of the rGST-SBP4 to any anti-B. bovis antibodies in a body substance using the MI-ELISA of the invention. The present antibody-polypeptide binding complex may be detected by a variety of methods such as is described above. Examples of body fluids are blood, serum, saliva, urine, and the like, including aqueous humor, vitreous humor, blood plasma, cerebrospinal fluid, perilymph, endolymph, lymph, mucus, pericardial fluid, pleural fluid, synovial fluid, milk, colostrum, or oral fluids. Methods for the preparation of the body fluid are standard in the art and need not be further detailed herein (see, for example, Manual of Clinical Microbiology, Chapter 8, “Collection, Handling and Processing of Specimens”, 4th edition, Eds, Lennette, E. H., et al., American Society for Microbiology (1986)).
Still part of this invention is a kit for the diagnosis of B. bovis infection, that comprises the rGST-SBP4 peptide of this invention; and instructions for use of the kit. This kit may be utilized for the detection of endogenous antibodies produced by a subject that is afflicted with babesiosis. Even at the early stages where the parasite is commencing invasion of a subject's cells, some amount of B. bovis-specific antibody may be detected in serum. In addition to the above, the kits may also comprise a control, anti-antibodies, protein A/G, and the like, suitable for conducting the different assays referred to above.
In another aspect of the invention, immunoassays using the disclosed rGST-SBP4 peptide are provided. In the context of this aspect of the invention, the rGST-SBP4 peptide that has specificity for the antibodies in the biological sample is bound to a substrate. The term “bound” refers to both covalent and non-covalent attachment of a peptide to a substrate. Thus, polypeptides can be covalently bound to the substrate via a linker physically attached to a substrate or non-covalently bound to a substrate (e.g., adsorbed to a substrate surface, for example, a polystyrene surface). In various embodiments, the substrate can be selected from tubes, cylinders, beads, discs, silicon chips, microplates, nitrocellulose membrane, nylon membrane, porous membranes, non-porous membranes, plastic, polymer, silicon, polymeric pins, a plurality of microtiter wells, or any combinations thereof. The composition of the substrate can also be varied. For example, substrates (alternatively referred to as a support) can comprise glass, cellulose-based materials, thermoplastic polymers, such as polyethylene, polypropylene, or polyester, sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane film composed of nitrocellulose, nylon, polysulfone, or the like. Thus, the substrate may be any surface or support upon which a peptide, such as the rGST-SBP4 peptide, can be immobilized, including one or more of a solid support (e.g., glass such as a glass slide or a coated plate, silica, plastic or derivatized plastic, paramagnetic or non-magnetic metal), a semi-solid support (e.g., a polymeric material, a gel, agarose, or other matrix), and/or a porous support (e.g., a filter, a nylon or nitrocellulose membrane or other membrane). In some embodiments, synthetic polymers can be used as a substrate, including, e.g., polystyrene, polypropylene, polyglycidylmethacrylate, aminated or carboxylated polystyrenes, polyacrylamides, polyamides, polyvinylchlorides, and the like. In preferred embodiments, the substrate comprises a microtiter immunoassay plate or other surface suitable for use in an ELISA.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
A cDNA encoding the recombinant fusion protein rGST-SBP4 comprised of glutathione S-transferase (GST) and recombinant spherical body protein-4 (SBP4) of B. bovis (T2Bo strain) modified by having the signal sequence deleted (GenBank accession number: KX524469) was cloned into the pGEX-2T vector (New England BioLabs, Ipswich, Mass., USA). This recombinant (r) GST-SBP4-containing vector was transformed to BL21 cells (New England BioLabs, Ipswich, Mass., USA) and expressed as follows. Briefly, 200 mL of an overnight culture of pGEX-2T/SBP4-transformed BL21 cells were inoculated into 1.8 liters of LB medium (Becton, Dickinson and Company, Sparks, Md., USA) containing 0.01% ampicillin and grown at 37° C. for 3 hr. Following addition of 0.048 g of isopropyl-β-D-thiogalacto-pyranoside (IPTG; US Biologicals, Salem, Mass., USA), the bacteria were incubated at 37° C. for an additional 5 hr. The bacteria were harvested by centrifugation at 4.5×103 g for 25 min and then resuspended in phosphate buffered saline. The resuspended pellet was sonicated on ice and Triton X-100 (Sigma-Aldrich, St. Louis, Mo., USA) was added to a final concentration of 1%. The sonicated suspension was centrifuged at 5.7×104 g for 30 min. Finally, the clarified supernatant containing the rGST-SBP4 fusion protein was collected and stored at −80° C. until used for antigen coating or further purification to make the horseradish peroxidase (HRP)-conjugated recombinant SBP4.
The recombinant GST-SBP4 fusion protein was purified from the supernatant described above using glutathione-agarose beads (Sigma G4510) according to the manufacturer's instruction. Briefly, glutathione-agarose beads equilibrated with PBS containing 1% Triton-X 100 were resuspended in the clarified lysate to agitate for 60 minutes at room temperature. The beads capturing recombinant GST-SBP4 were pelleted by centrifugation and washed with PBS three times. After the last wash of the beads, the recombinant GST-SBP4 was eluted from the beads using the elution buffer containing 30 mM reduced Glutathione in 50 mM Tris-HCl (pH 9.0). The eluted rGST-SBP4 was conjugated with horse-radish peroxidase according to the method previously described (Nakane and Kawaoi. 1974. J. Histochem. Cytochem. 22:1084-1091). The conjugate concentrate was stabilized by adding a final concentration of 10% heat-inactivated goat serum and stored at 2-7° C.
The IFA was performed as previously described (Goff et al. 2006, supra; Goff et al. 1982, supra) using 50 μl of a 1/50 dilution of serum in serum dilution buffer (1×PBS) and substrate slides prepared using red blood cells parasitized by two B. bovis strains Mo7 and T2Bo. A positive result was defined as fluorescence equal (1+) to or greater than (2 to 3+) that of a weak positive control sample defined by IFA, western blot and RAP-1 cELISA after collection from a bovine experimentally-infected with Mo7 stain. A negative result was defined as comparable to the background fluorescence of a negative control serum collected from a B. bovis-negative herd in the northwestern U.S.
Western blot analysis to detect B. bovis-specific antibodies directed against RAP-1 in bovine sera was performed using recombinant RAP-1 (rRAP-1) according to the previously described method (Nakane and Kawaoi, supra) with some modifications. Briefly, rRAP-1 was boiled for three minutes in sample buffer (DGel Sciences, Montreal, Canada) and separated by electrophoresis using sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad, Hercules, Calif., USA). Transfer to nitrocellulose was performed by standard techniques (Chung et al. 2014. J. Vet. Diagn. Invest. 26:61-71) and membranes were blocked in tris-tween-20 buffer containing 5% skim milk. Bovine serum antibody bound to the rRAP-1 band was detected with HRP-conjugated goat anti-bovine IgG (KPL, Gaithersburg, Md., USA) (Terkawi et al. 2011a, b, supra).
Negative sera were collected from 302 uninfected cattle in northwestern US herds that were maintained in barns free of B. bovis-transmitting ticks and that had no history of clinical babesiosis. Ninety-four sera were collected from Texas herds with unknown B. bovis status, 93 of these sera were negative in the indirect immunofluorescence assay (IFA). Thirty-two sera, negative by IFA, were selected as additional negative samples from B. bovis endemic areas. These 427 negative sera were used to evaluate the diagnostic specificity of the RAP-1-based cELISA and the newly developed rSBP4-based MI-ELISA.
B. bovis-positive sera (n=826) were obtained from cattle with positive results by IFA, and were used to evaluate the diagnostic sensitivity of the MI-ELISA and the RAP-1 cELISA. Two hundred twenty-eight of these 826 sera were from herds in Australia vaccinated with an attenuated B. bovis Dixie strain (n=180) or challenged with virulent W strain (n=48) in Australia, and the remaining 598 sera were collected from several areas in Mexico. Four hundred and two additional IFA-positive sera were selected from sequential serum collections from six experimentally-infected cattle and used to further evaluate relative sensitivity of the MI-ELISA and the RAP-1 cELISA. Excluding the 32 negative samples at early post-infection days and 402 positive samples from experimentally-infected animals, a total of 1,254 field serum samples were used to evaluate likely diagnostic sensitivity and specificity resulting in a statistical power of 95% confidence within a 2% margin of error.
Two main features of the rGST-SBP4-based MI-ELISA for highly specific and sensitive diagnosis of B. bovis infection in cattle include 1) glutathione-BSA catcher-based purification/coating of rGST-SBP4 as antibody capture antigen and use of HRP-conjugated rGST-SBP4 as detection system (
For the SBP4 MI-ELISA testing procedure, 50 μl/well of test serum were added to wells of the plate and incubated at room temperature for 30 minutes. Wells were then washed three times with 250 μl of wash buffer (PBS+0.05% Tween 20) per well. Fifty μl of HRP-conjugated rGST-SBP4 diluted in conjugate diluting buffer (PBS containing 1% bovine serum albumin) were added to each well of the plate, and the plates were incubated at room temperature for 30 min. After wells were again washed three times with 250 μl of wash buffer per well, 50 μl of tetramethylbenzidine substrate (SurModics, Eden Prairie, Minn., USA) were added to each well, and the plates were incubated at room temperature for 15 min. The reactions were then stopped with 50 μl of 1.5% sodium fluoride solution (SurModics, Eden Prairie, Minn., USA) per well. The optical densities (OD) of the assay wells were read at 450 nm (OD450) with a microplate spectrophotometer and results were calculated as the ratio of sample OD to negative control OD (S/N). Specifically, the OD450 of the sample in question was divided by the OD450 of a negative control analyzed in the same assay run.
The SBP4 MI-ELISA was optimized by evaluation of several format variables including the antigen coating protocol, serum incubation time, wash buffer, and concentration of HRP/GST-SBP4 conjugate used to detect antibody binding. Optimization utilized a small set of 6 samples that included two known negative sera and four two-fold dilutions of a B. bovis-positive serum in negative serum ranging above and below the end-point detection (Table 1). The optimized format included the use of 50 μl undiluted serum to maximize assay simplicity.
Using this optimized SBP4 MI-ELISA format, S/N ratios were determined for 1,254 sera that were already categorized as either B. bovis-IFA positive or -IFA negative. Scatter plot and ROC curve analysis were carried out using the S/N ratio data and IFA categorization (
To compare the specificity of the SBP4 MI-ELISA and the RAP-1 cELISA, 302 sera collected from northwestern U.S. herds maintained in barns free of B. bovis tick vectors and negative by IFA using two different B. bovis strains as IFA substrate were tested. All 302 sera were negative using both ELISA assays, generating no false positives and a resultant diagnostic specificity of 100% for both assays (Table 2). Furthermore, the SBP4 MI-ELISA had 100% diagnostic specificity when evaluated with 32 IFA-negative sera collected from B. bovis-endemic regions of Mexico (Table 2). However, the RAP-1 cELISA had a lower diagnostic specificity of 93.8% as two of the same 32 IFA-negative sera were positive (Table 2).
Ninety-three of 94 additional sera collected from Texas herds as potential negative samples were negative by IFA, with the remaining sample being weakly IFA positive. Nine of the 93 IFA negative sera were positive by RAP-1 cELISA (90.3% specificity) while none were positive by SBP4 MI-ELISA (100% specificity)(Table 2). All nine IFA-negative sera with discrepant results between the RAP-1 cELISA and the SBP4 MI-ELISA were negative in RAP-1 western blot analysis (
To evaluate the sensitivity of the SBP4 MI-ELISA and the RAP-1 cELISA against well-characterized positive sera, 402 IFA-positive sera collected at various DPIs from six cattle experimentally infected with one of three different B. bovis strains were analyzed. Of the 402 sera, 335 sera were strong IFA positives while the remaining 67 sera were weak IFA positives. The RAP-1 cELISA had a sensitivity of 84.6%, with 340 of the 402 IFA positive sera being RAP-1 cELISA positive (Table 3). The SBP4 MI-ELISA had a sensitivity of 100% as all 402 IFA positive sera were positive. For these experimentally infected calves, the SBP4 MI-ELISA had significantly (p<0.001) better sensitivity than the RAP-1 cELISA.
One hundred seventy-seven of 228 sera collected from cattle herds in Australia that were vaccinated with an attenuated B. bovis strain or challenged with the pathogenic W strain were strong positive by IFA and the remaining 51 sera were weak positive. Analyses with all of these 228 sera resulted in 95.2% and 86.0% diagnostic sensitivity for the SBP4 MI-ELISA and the RAP-1 cELISA, respectively (Table 3). Thus, the SBP4 MI-ELISA had significantly (p≤0.004) better diagnostic sensitivity than the RAP-1 cELISA against these sera.
Three hundred eighty-eight of 598 IFA positive sera collected from endemic regions in Mexico were strongly IFA-positive and the rest were weak IFA-positive. The diagnostic sensitivity of the SBP4 MI-ELISA was 98.7% while diagnostic sensitivity of the RAP-1 cELISA was 60.0% for all 598 IFA-positive sera (Table 3). The SBP4 MI-ELISA has significantly (p<0.001) better diagnostic sensitivity than the RAP-1 cELISA for these sera.
The analytical specificity of the SBP4 MI-ELISA was evaluated for sera positive to B. bigemina (n=25) and Anaplasma marginale (n=25), which are other tick-borne intraerythrocytic parasites commonly co-infecting with B. bovis in cattle from endemic areas. All of these sera were negative in the SBP4 MI-ELISA, demonstrating suitable analytical specificity against closely related and/or frequently co-infecting intra-erythrocyte parasites (Tables 4 and 5).
bigemina antibody-positive sera.
B. bovis
B. bigemina
B. bovis (+)
B. bovis (−)
B. bigemina (+)
B. bigemina (−)
Anaplasma marginale antibody-positive sera.
B. bovis SBP4
A. marginale MSP5
B. bovis (+)
B. bovis (−)
Anaplasma
Anaplasma (−)
The results of the present study demonstrate that the SBP4 MI-ELISA has very high diagnostic specificity and sensitivity when evaluated with diverse sera collected from bovine herds in non-endemic, endemic, and epidemic areas. This SBP4 MI-ELISA can consistently detect B. bovis-specific antibodies in calves with acute as well as long-term carrier infections making it a pivotal tool for controlling B. bovis infection in cattle in endemic areas and preventing their movement to non-endemic and free areas.
Data analysis: The diagnostic specificities of the SBP4 MI-ELISA and the RAP-1 cELISA were calculated as the percentage of IFA-negative sera for B. bovis that were also negative by the assay in question. Diagnostic sensitivity was the percentage of the IFA-defined B. bovis-positive sera having a positive result in the assay being evaluated. Receiver operating characteristic (ROC) curve and scatter plot analysis were performed using spreadsheet software (Excel software, Microsoft, Seattle, Wash., USA) and R software from the R Foundation for Statistical Computing (Hornik 2016. “The R FAQ”, Retrieved from the internet: CRAN.R-project.org/doc/FAQ) to evaluate the cutoff for positive and negative detection by the newly developed SBP4 MI-ELISA through comparison with the IFA-positive and -negative serum reference panels described above (Greiner, M. 1995. J. Immunol. Methods 185:145-146; Greiner et al. 1995. J. Immunol. Methods 185:123-132; Griner et al. 1981. Ann. Intern. Med. 94:557-592).
Statistical analysis: Binomial test (Newcombe, R. G. 1998. Stat. Med. 17:873-890) was used to determine if there were significant (p<0.05) differences in sensitivity and specificity between the SBP4 MI-ELISA and RAP-1 cELISA or IFA test when testing various sets of serum samples. All statistical analyses were performed using R software described above.
In a previous study (Suarez et al. 2012, supra) showing strong evidence of the long-term persistence of B. bovis in cattle, two calves were infected with the B. bovis Mo7 attenuated strain (Rodriguez et al. 1983. Infect. Immun. 42:15-18) and two calves were infected with the B. bovis Tf-137-4 strain (Hines et al. 1989. Mol. Biochem. Parasitol. 37:1-9; Levy and Ristic. 1980. Science 207:1218-1220). These four calves received a low dose (5×103) of infected erythrocytes intravenously and were monitored for signs of acute babesiosis, including parasitemia, fever, and low packed cell volume (PCV) on a daily basis for the first twenty days post-inoculation (DPI) and on a biweekly basis thereafter until 11 months post-inoculation.
Two calves were infected with a high dose (5×105) of erythrocytes infected with either the B. bovis Mo7 attenuated strain or the B. bovis T2Bo pathogenic strain (Suarez et al. 2006. Int. J. Parasitol. 36:965-973) and then were monitored as above on a daily basis for the first fifty DPI followed by a biweekly basis thereafter until 10 months post-inoculation. All six experimentally-infected animals had recurrent parasitemia lasting more than 10 months post-infection, even those infected with a low dose of attenuated B. bovis strains, indicating persistent infection capacity of all three B. bovis strains regardless of pathogenicity and challenge dose. Five animals challenged with attenuated strains had no clinical signs or thrombocytopenia after the first acute parasitemia. However, the calf challenged with the pathogenic B. bovis (T2Bo strain) had consistent parasitemia, thrombocytopenia and retarded weight gain during the 12 month monitoring period (data not shown). The consistent parasitemia in the calf challenged with pathogenic strain, T2Bo, and the significantly lower frequency of parasitemia in calves challenged with the attenuated strains, Mo7 and Tf-137-4, suggest lack of reliability of parasitemia detection-based diagnosis, e.g., PCR, due to the narrow window for detection of parasitemia in unapparent carriers.
Here, in this study, long-term duration and kinetics of parasitemia and B. bovis-specific antibody response are systematically defined in calves infected with high and low doses of three different B. bovis strains including attenuated and pathogenic ones. The protocol of infection used in these animal studies and all animal handling was approved by the Institutional Animal Care and Use Committee (IACUC) in Washington State University (#03735-008 approved on 12/9/2009).
DNA Extraction and PCR:
DNA was extracted with a purification reagent from FTA cards spotted with whole blood of test according to the manufacturer's instructions (FTA card and DNA purification reagent, GE Healthcare, Piscataway, N.J., USA). Briefly, blood samples were dotted on FTA cards and treated with a purification reagent to lyse erythrocytes and remove cellular proteins. DNA of each sample was eluted in PCR buffer and nested PCR to detect B. bovis DNA was performed according to previously reported methods (Suarez et al. 2012, supra).
Interestingly, all six calves maintained robust B. bovis antibody responses during all 12 months of the monitoring period when analyzed by the low throughput and relatively subjective sero-diagnostic tool, IFA. Persistence of antibody responses specific for B. bovis even at DPIs with no detectable parasitemia clearly indicates the relative advantage of an antibody-based diagnostic over an antigen detection assay, particularly when an accurate, high-throughput serology assay is available and results are interpreted more objectively than IFA results.
One calf was experimentally infected with a high dose (5×105 infected erythrocytes) of B. bovis Mo7 and another calf was infected with 5×105 erythrocytes infected with the T2Bo strain. Both had detectable parasitemia longer than 12 months when tested daily for the first 33 days post-infection and biweekly until 12 months post-inoculation by nested PCR (
The timing of initial antibody detection in experimentally-infected calves was slightly variable depending on the assay and the B. bovis strain used for infection. The calf infected with a high dose of moderately attenuated Mo7 strain had detectable antibody beginning at 10, 11 and 14 days DPI by the SBP4 MI-ELISA (
B. bovis-specific antibody responses after initial positive detections were maintained for longer than 12 months post-infection according to all three assays (
Two calves experimentally infected with a low dose (5×103 infected erythrocytes) of B. bovis Mo7 strain (C1287 and C1291) and two calves infected with the same number of erythrocytes infected with the attenuated Tf-137-4 strain (C1290 and C1292) had variable parasitemia for longer than 11 months when tested by the nested PCR, demonstrating long-term persistence of B. bovis after infection (
Following initial detection in these four calves, the long-term pattern of parasitemia was variable, as was B. bovis-specific antibody responses detected by all three serologic assays. After the first positive SBP4 MI-ELISA result at 13 DPI, the Mo7-infected calves had 84.7% (50 of 59 time points) and 69.5% (41 of 59) PCR-positive time points (
All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15210792 | Jul 2016 | US |
| Child | 15886915 | US |
