Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/5695—Mycobacteria

Definitions

• the invention pertains to the field of diagnosing infection due to a bacterial organism.
• parat ⁇ berculosis may require 8 tol ⁇ weeks or more to perform due to the very slow growth rate of this organism.
• Another disadvantage to culture and identification methods of diagnosis is that the particular organism causing disease in a patient may fail to grow on standard culture media, leading to a negative culture result and a failure in diagnosis. Additionally, because such methods require the isolation of an infectious organism from a patient, these methods are inappropriate at times when the patient is not shedding the organism or if the organism is located in an inaccessible location within the body of the patient.
• molecular biological and immunological methods have been developed for the diagnosis of infectious diseases. These methods generally fall into three categories, detection of genome nucleic acids, detection of protein, and detection of antibodies directed against a pathogen.
• Diagnosis by identification of genome nucleic acids is typically performed using either or both amplification of DNA by polymerase chain reaction (PCR) followed by identification of PCR fragments produced or by use of probes that bind specifically to a portion of the genome of a suspected causative organism.
• PCR polymerase chain reaction
• These methods especially when used in combination, can be very sensitive and specific methods to establish a diagnosis of a causative organism.
• Another significant disadvantage associated with diagnosis by detection of genome nucleic acids is that an organism must be isolated in order to obtain the genome nucleic acids .
• diagnosis based on DNA sequence may fail to distinguish between closely related microbial pathogens, such as between different strains of Mycobacteri um, such as Mycobacteri um avi um subsp paratuberculosis and Mycobacterium avium subsp avium.
• Diagnosis by identification of proteins is typically performed by an enzyme-linked immunosorbent assay (ELISA) .
• ELISA enzyme-linked immunosorbent assay
• an antigen from a test sample typically a disrupted microorganism or a portion of a microorganism, is captured by a first antibody that is specific for the antigen of interest and which is bound to a solid support.
• a labeled second antibody that binds to antibodies in test serum is then exposed to the solid support complex to provide a means for identification of the presence of the antigen.
• ELISA tests suffer from several disadvantages including low sensitivity and the requirement to provide two different antibodies for the detection of an antigen. ELISA testing requires skilled laboratory technicians and can provide false results if samples are contaminated.
• An example of an infectious disease for which currently available diagnostic methods are inadequate is Johne's disease, a disease in cattle caused by Mycobacteri um avi um subsp. paratuberculosis (MAP) . Johne's disease results in decreased milk production and early culling of infected cows resulting in an annual loss of approximately $1.5 billion to the agricultural industry in the United States.
• MAP Mycobacteri um avi um subsp. paratuberculosis
• MAP is also the causative organism of Crohn' s Disease in humans.
• fecal culture is considered to be the most accurate means of diagnosing Johne's Disease.
• this diagnostic test has low sensitivity (less than 50%) and is capable of detecting infections only in animals that are actively shedding MAP in their feces.
• diagnosis of MAP by culture typically requires 8 to 16 weeks for growth of the organism.
• Other diagnostic tests for Johne's Disease include PCR, complement fixation, agar gel immunodiffusion, and ELISA.
• Figure 1 is a bar graph showing the high specificity of the method of the invention.
• Each bar represents the mean fluorescence intensity (mean +/- standard deviation of triplicate data) as determined by flow cytometry.
• Figure 1 shows that high levels of antibody binding were detected by fluorescent intensity on flow cytometry when serum from cows infected with MAP were mixed with MAP organisms. Flow cytometry following mixing of samples with closely related mycobacterial species resulted in minimal antibody binding and a negative test result for MAP infection.
• Figure 2 is a bar graph showing the high specificity of the method of the invention. Each bar represents the mean fluorescence intensity (mean +/- standard deviation of triplicate data) .
• Figure 3 is a bar graph showing the high sensitivity of the method of the invention.
• Each bar represents the mean fluorescence intensity (mean +/- standard error of triplicate data) as determined by flow cytometry.
• Figure 3 shows that serum from cows from farms determined to be MAP free (controls) binds only minimally to MAP organisms, serum from cows determined to be MAP positive show high levels of antibody binding, and that serum from cows from farms having MAP infection but which cows were determined by ELISA to be negative show levels of antibody binding higher than that of controls.
• Figure 4 is a bar graph showing the high sensitivity of the method of the invention utilizing the simplified procedure of Examples 2 and 7. Each bar represents the mean densimetric intensity (mean +/- standard deviation of triplicate data) as determined by flow cytometry.
• Figure 5 is a bar graph showing results of the method of the invention for diagnosing MAP infection using a dot blot procedure. Each bar represents the mean fluorescence intensity. Figure 5 also contains an insert showing images of stained MAP organisms on dot blot.
• A sera from cows from MAP negative farm.
• B sera from cows found to be MAP positive by IDEXX ELISA.
• Figure 6 is a graph showing the correlation between serum and milk antibody binding as analyzed by the method of the invention. Each point represents the average intensity of reaction obtained from serum and milk samples from one cow.
• Figure 7 is a bar graph showing the specificity of milk antibody binding to three strains of MAP as compared with negative reactions that were obtained with Mycobacterium scrofulaceum (MS) and three isolates of M.
• MS Mycobacterium scrofulaceum
• FIG. 8 is a graph showing the detection of MAP in pooled serum by the method of the invention.
• the cut-off value (dotted line) was determined as the mean fluorescence obtained from six serum samples from a MAP-free farm +3 standard deviations for IgG binding.
• Figure 9 is a bar graph showing the detection of MAP infections in the American bison. All bison except VA 105 (arrow) were known to be MAP-positive as determined by fecal culture, ELISA, and histological examination.
• the invention is a method for diagnosing an infection in an animal caused by a microorganism.
• the diagnostic method of the invention is based on antibody binding to one or more specific antibody binding sites that exist or existed on the surface of a particular microorganism.
• a test sample preferably a serum sample
• the test sample is obtained from an animal suspected of being infected with a microorganism.
• the test sample thus obtained is exposed to a population of the microorganism. It is then determined if the test sample contains an antibody that binds to the microorganism, preferably to the surface of the microorganism.
• the test is positive for infection with the microorganism if antibodies in the test sample bind to the microorganism.
• the method of the invention is distinct from presently utilized methods for microbial diagnosis and provides several advantages that are unobtainable from such methods. Unlike culture methods, the method of the invention does not require isolation of an organism from an infected animal or the need to culture an organism in vitro. Therefore, the method of the invention provides results in a much shorter time period than is achievable with culture methods and, in contrast to culture methods, can provide a positive diagnosis even during times when the microorganism is not able to be isolated from a host animal . Unlike recent innovations in microbial diagnosis such as those based on nucleic acid or protein identification, the method of the invention is not based on the determination of the presence of any specific macromolecule peculiar to a particular organism.
• the method of the invention does not present an antibody to determine if it binds to an extract of a microorganism or portion of a microorganism that is present in a host animal. Rather, the method of the invention is based upon determining that one or more antibodies present in a test sample isolated from the body of a host animal binds to a particular microorganism that is brought into contact with the test sample.
• the method of the invention provides several advantages previously unobtainable by present diagnostic methods.
• the method of the invention may be performed rapidly. In a field version of the method of the invention, a positive or negative test result may be obtained rapidly, typically within about two hours.
• the method of the invention is extremely sensitive, more sensitive than presently available methods.
• the method of the invention can be used to provide a positive diagnosis even during periods when a microbial pathogen is not detectable in, or isolatable from, a host animal. Additionally, the method of the invention has a specificity that is higher than is obtained with other presently available methods of diagnosis.
• the method of the invention is useful for the diagnosis of microbial infections in animals.
• Such animals include mammals, such as humans and non-human primates, carnivores such as dogs, cats, bears, and weasels, ungulate ruminants and non-ruminants such as horses, cattle, goats, sheep, pigs, non-ungulate ruminants such as camels and llamas, pinnipedia such as seals and sea lions, lagomorpha such as rabbits and hares, rodentia such as squirrels, rats, and mice, cetacea such as whales, dolphins, and porpoises, and proboscidea such as elephants.
• Such animals also include non-mammalian vertebrates such as birds, reptiles, amphibians, and fish.
• test sample that is obtained from an animal in accordance with the method of the invention may be any fluid or tissue in which an antibody that specifically binds to a suspected causative organism would be present if the animal were infected with that organism.
• the test sample is blood or a portion thereof, such as plasma or preferably serum.
• sample sources may be utilized in accordance with the invention. The selection of such source of test sample will vary depending, primarily, on the symptoms and signs of an infected animal and the suspected cause of such symptoms or signs.
• the test sample may be obtained from fluids such as saliva, milk, pus, tears and other ocular discharges, nasal discharges, sputum, cerebrospinal fluid, peritoneal or pleural fluid, urine, feces, and vaginal, uterine, or urethral secretions and discharges. Fluids may also include those that are produced as part of a pathologic process such as exudates or transudates, such as from the skin, the pleural or peritoneal cavity, the oral cavity, or from the digestive, respiratory, or genital system.
• the test sample may also be a solid tissue sample if appropriate for diagnosis of a particular disease. The test sample may be obtained by whatever method is appropriate to obtain such a sample.
• the test sample may be obtained by methods such as syringe withdrawal of fluid, including vascular puncture, such as by venipuncture, or by withdrawal of fluid from other sources as described above, or by biopsy.
• the organism that is diagnosed by the method of the invention is any microorganism that is capable of eliciting an antibody response in an animal infected by such microorganism.
• infective microorganisms that may be diagnosed by the method of the invention include bacteria, fungi, viruses, protozoa, rickettsia, and chlamydia.
• the invention is described in detail herein with reference to mycobacterial infections, and particularly with reference to Mycobacteri um avi um, and most particularly with reference to Mycobacterium avi um subsp.
• test sample may be exposed to a population of microorganism in any way that permits antibodies that are contained in the test sample to interact with the microorganisms.
• the test sample and the microorganism are combined in a vessel such as a test tube or a well and are mixed together, such as by stirring or tapping the exterior of the test tube or well.
• test sample and the microorganism may also be reacted together on a surface such as on a slide, filter, or membrane, such as a nitrocellulose membrane.
• the test sample is exposed to a population of intact whole microorganisms. In this way, antibody binding sites on the entire surface of the microorganism are available for binding to antibodies in the test sample. It is preferred, if intact microorganisms are used, that the microorganisms be killed so as to avoid the risk of infection to humans and to other animals.
• a preferred method for killing the microorganisms is by exposure of the microorganisms to a chemical fixative.
• One preferred chemical fixative is formaldehyde which, when used to kill MAP organisms, maintains the ability of surface antibody binding sites of MAP to bind with antibodies in serum from animals infected with the organism.
• a preferred concentration of formaldehyde is about 1% to 10% v/v, with a concentration of about 2% most preferred.
• Other chemical fixatives that may be used to kill microorganisms for use in the method of the invention include non-coagulant fixatives such as acetone, glyceraldehydes, glutaraldehyde, and paraformaldehyde, and less preferred coagulant fixatives such as ethanol and mercuric chloride.
• Ethanol in concentrations tested by the inventors (70% v/v) , destroyed the ability of surface antibody binding sites of MAP to bind with antibodies in serum from MAP infected animals. It is conceived that coagulant fixatives such as ethanol may be useful for killing microorganisms to be used in the method of the invention, especially if used to diagnose infections by microorganisms other than MAP, or that certain concentrations of such fixatives may be suitable for killing microorganisms and may not render the killed microorganisms unsuitable for the method of the invention. Because of this uncertainty concerning ethanol, coagulant fixatives such as ethanol are less preferred.
• the method of the invention may alternatively be performed by exposing the test sample to a population of disrupted or partial microorganisms, such as microorganisms that have been fractionated, or to one or more isolated antibody binding sites of the surface of a microorganism.
• a population of microorganisms includes exposing the test sample to intact microorganisms, to disrupted or partial organisms, or to one or more isolated antibody binding sites of a microorganism.
• antibody- microorganism binding is determined by flow cytometry. Such flow cytometry determination may be performed by analysis of a sample obtained by mixing a suspension containing a serum sample and a population of microorganisms with a labeled anti-antibody, typically a fluorescein-labeled anti-antibody.
• antibody-microorganism binding is determined by blot analysis, such as dot blot or Western blot analysis.
• Such dot blot determination may be performed by mixing a suspension containing a serum sample and a population of microorganisms with an anti-antibody which is labeled, such as with biotin or colloidal gold, spotting this mixture on a membrane, such as a nitrocellulose or polyvinylidene fluoride (PVDF) membrane, and determining the presence of labeled microorganism fixed on the membrane.
• a membrane such as a nitrocellulose or polyvinylidene fluoride (PVDF) membrane
• diagnosis of infection with such methods is accurate, sensitive, and specific. Determination of infection with methods such as dot blot analysis permits diagnosis to be made by visual inspection and such methods are therefore capable of being performed by individuals who are not technically trained in sophisticated laboratory techniques .
• the invention is further illustrated in the following non-limiting examples.
• Example 1 - Technique for Serological Diagnostic Test Using Flow Cytometry Two microliters of serum samples obtained from bovine subjects and 49 microliters of phosphate-buffered
• Tween buffer A
• buffer A a population of whole organisms of Mycobacteri um species (5 microliters packed volume) to form a suspension of organisms. The suspension was incubated at room temperature for 1 hr, washed three times with 100 microliters buffer A by centrifugation at 5000 rpm for 10 in, mixed with fluorescent-labeled rabbit anti-bovine IgG antibody (MP Biomedicals (formerly ICN Biomedicals) , Irvine, CA, USA) diluted 1:50 in buffer A, incubated at room temperature for 1 hr, and washed twice with buffer A by centrifugation at 5000 rpm for 10 min.
• MP Biomedicals previously ICN Biomedicals
• Example 2 Alternative Technique for Serological Diagnostic Test by Flow Cytometry
• the method of the invention was performed utilizing a simplified alternative technique for serological diagnosis by flow cytometry.
• This alternative technique requires a shorter time than the technique described in Example 1 and does not require centrifugation .
• One microliter of bovine serum, 5 microliters of fluorescent-labeled rabbit anti-bovine IgG antibody (1.5 mg/ l) , and 44 microliters of buffer A were added to whole organisms of MAP (5 microliters packed volume) , and incubated at room temperature for 1 hr.
• One tenth of the treated organisms were suspended in 1 ml buffer A and fluorescence on 10,000 organisms was analyzed using a flow cytometer.
• Example 3 Serological Diagnostic Test Using a Dot Blot Technique
• MAP MAP
• 9 microliters of buffer A were added to whole organisms of MAP (5 microliters packed volume), and incubated at room temperature for 1 hr.
• the organisms were washed three times with 100 microliters of buffer A by centrifugation at 5000 rpm for 10 min, mixed with undiluted colloidal gold-labeled rabbit anti-bovine IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), incubated at room temperature for 1 hr, and washed twice with 100 microliters of buffer A by centrifugation at 5000 rpm for 10 minutes.
• the treated organisms were resuspended in 100 microliters of buffer A and spotted on a PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by using a dot blot apparatus (Bio-Rad) . Images of the stained organisms on the dot blot were captured, and their densitometric intensities measured, using a gel documentation system (ChemiDoc XRS, Bio-Rad) .
• Example 4 Specificity of the Method of Invention Pooled and individual serum samples from cows known to be infected with MAP were • analyzed as described in Example 1. Serum samples from cows known to be infected with MAP were mixed with organism populations that were one of 5 strains of Mycobacteri um avi um subsp. avi um (MAA) , one strain of Mycobacteri um scrofulaceum, or 4 strains of MAP, respectively prior to detection of binding by flow cytometry. Results are shown in Figure 1. As shown in Figure 1, the method of the invention correctly identified infection with MAP in all samples and showed a lack of false positive diagnoses as the method of the invention did not show binding when bacterial populations closely related to MAP were used as the test organism.
• MAA Mycobacteri um avi um subsp. avi um
• Results are shown in Figure 1. As shown in Figure 1, the method of the invention correctly identified infection with MAP in all samples and showed a lack of false positive diagnoses as the method of the invention did not show binding when bacterial populations closely
• Example 5 Specificity of the Method of the Invention Serum samples from 8 cows known to be infected with MAP were pooled. The pooled serum sample was tested as described in Example 1 by combining individual 2 microliter samples of the pooled serum sample with one of
• Example 6 Sensitivity of the Method of the Invention
• the method of the invention utilizing whole MAP organisms, as described in Example 1, was tested in comparison with an ELISA-based test HerdChek ⁇ Mycobacteri um paratuberculosis Test Kits (Johne's disease) (IDEXX Laboratories, Inc., Westbrook, ME, USA) .
• the method of the invention was performed according to the procedure of Example 1 utilizing (1) sera from cows in farms that were determined to be free of MA?
• FIG 3 the average antibody binding, as determined by fluorescent intensity, is shown for each of three types of samples, cows from MAP free farms, cows from MAP positive farms but which have been determined by IDEXX ELISA test to be MAP negative, and cows determined to be positive for MAP by IDEXX ELISA test.
• the data of Table 1 and Figure 3 establish that the method of the invention is more sensitive than that of the most commonly used non-culture method presently in use for diagnosing Johne's disease in cattle.
• the IDEXX ELISA test had a false negative rate of 40% as established when later confirmed with the method of the invention, in which the false negative rate was 0%.
• Example 7 Alternative Technique for Diagnosis of MAP by Flow Cytometry
• Sera from cows determined by IDEXX ⁇ LISA to be MAP negative were pooled and sera from cows determined by ID ⁇ XX ⁇ LISA to be MAP positive by IDEXX ELISA test were pooled to provide two samples.
• the samples were tested for the presence of MAP infection by the simplified, alternative technique described in Example 2.
• MAP organisms, pooled serum, and fluorescent-labeled secondary antibody were mixed, incubated for 1 hr, and antibody binding to MAP was determined by flow cytometry. The results were compared with a control sample which contained MAP organisms and secondary antibody but no serum. Results are shown in the bar graph of Figure 4.
• the control sample (None) showed a low level of fluorescent activity as determined by flow cytometry. Additionally, the MAP positive serum showed a very high level of fluorescent activity indicating a high level of serum antibody binding with the MAP organisms. Unexpectedly, the fluorescent activity of the MAP free pooled sera, that found to be negative by ELISA test, showed a level of fluorescent activity about twice that of control. This result indicates that the ELISA test incorrectly identified MAP infected cows as being free of infection. The diagnostic test according to the invention, however, correctly identified that antibodies due to MAP infection were present in the pooled serum from this group.
• Example 8 Diagnosis of MAP by Dot Blot Technique
• Sera from cows found to be MAP negative by ⁇ LISA test were pooled and sera from cows found to be MAP positive by ⁇ LISA test were pooled to provide two samples. The samples were treated as described above in ⁇ xample 3 and diagnosed by dot blot technique. The results are shown in Figure 5.
• the dot blot technique of the invention showed a high degree of antibody binding in sera from cows previously found to be MAP positive by ELISA test. Additionally, antibody binding to MAP was detected by this method even in sera from cows previously found to be negative by ELISA testing, establishing the high sensitivity of the method of the invention as performed by dot blot technique.
• the dot blot technique is a simple method that can be performed easily in the laboratory by trained personnel. Additionally, because results of dot blot technique are evaluated visually, this technique can be performed in field conditions even by individuals not trained in laboratory techniques. Thus, the method of the invention is useful for both laboratory and field diagnosis of MAP infected individuals, and such diagnosis may be performed by either technical or non-technical personnel.
• Example 9 Diagnosis of MAP in Milk Samples Serum and milk samples were obtained from 48 cows and analyzed by the method of the invention using a flow cytometer. The serum and milk samples were diluted 1:50 and 1:2, respectively, with a phosphate buffered saline solution. The samples were tested for the presence of antibodies against MAP using the technique described in Example 1. The results are shown in Figure 6. As shown in Figure 6, horizontal and vertical lines represent the cut-off positive values for serum and milk antibody binding, respectively. The data show a strong correlation in sensitivity levels between serum and milk samples. Of the 18 cows that tested MAP- positive (100%) by the method of the invention using serum samples, 16 (88.9%) of the corresponding milk samples tested positive.
• Example 10 Specificity of the Method of the Invention in Milk Samples Milk from a cow diagnosed as positive for MAP by IDEXX ELISA was tested as described in Example 9 except that the milk samples were divided into samples and each sample was then mixed with organisms from one of three strains of Mycobacteri um avi um subsp. avi um, one strain of M. scrofulaceaum (MS) , or three strains of MAP, as described above in Example 4. The results are shown in Figure 7. The results clearly show that the method of the invention specifically detected MAP infections in milk samples and did not produce false positives by reacting with species of Mycobacterium that are closely related to MAP. This study establishes the high subspecies specificity of the method of the invention for diagnosing MAP (Johne's disease) in milk as well as serum.
• MAP Johne's disease
• ⁇ xample 11 - ⁇ ffects of diluting MAP-positive serum samples Each well of a 96-well plate was inoculated with 100 microliters of a MAP suspension as described in ⁇ xample 1 and then pelleted by centrifugation at 3500 x g for 10 min. After aspiration, the MAP pellets were resuspended and incubated with serum from a MAP-positive cow that had been diluted by adding serum from a MAP- negative cow to obtain dilutions of 1:2, 1:4, 1:8, 1:16 and 1:32. The samples were then analyzed by flow cytometry. Results are shown in Figure 8.
• Example 12 Diagnosis of MAP infections in the American bison Serum samples were obtained from 24 bison and analyzed by the method of the invention using a flow cytometer. Serum samples were diluted 1:50 with a phosphate buffered saline solution and tested for the presence of antibodies against MAP using the technique described in Example 1. Results are shown in Figure 9.

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