Abstract:
Provided is a method for diagnosing a disease or pathological condition in an animal. An ion mobility spectrometry measurement (IMS) or a differential mobility spectrometry (DMS) is carried out on a body sample from the animal to determine an amount of ions formed by at least two biogenic amines contained in the sample. A ratio is calculated of the amounts of ions formed by the different biogenic amines in the sample, wherein the ratio is indicative of the presence or absence of the disease or pathological condition. Also provided is a device for collecting a body sample from an animal and a method for obtaining a body sample from an animal.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention relates to methods for diagnosing pathological conditions in animals. 
       BACKGROUND OF THE INVENTION 
       [0002]    Levels of biogenic amines in human body fluids have been used to diagnose pathological conditions. For example, elevated levels of certain biogenic amines in urine may indicate the presence or the likelihood of the presence of a cancer (there are many papers dealing with this—see, for instance, Suh, J W, Lee, S H, Chung, B C, Park, J, Urinary Polyamine Evaluation for Effective Diagnosis of Various Cancers, Journal of Chromatography B, 1997, Vol. 688, Iss 2, pp. 179 186). Several of the types of vaginal diseases may be expressed in elevated levels of biogenic amines in vaginal discharge and fluids (see, for instance, C. S. Chen, R. Amsel, D. A. Eschenbach and K. K. Holmes, Biochemical diagnosis of vaginitis: determination of diamines in vaginal fluid, J. Infectious Disease 145 (1982), pp. 337 345). 
         [0003]    Body fluids may include e.g. urine, blood, serum, saliva, vaginal discharge and fluids, etc. Further, samples in which the presence of biogenic amines may be determined may not be fluids, but, e.g., skin and tissues, sweat samples, etc. Even direct sniffing of skin or breath exhaled by a subject may provide information in this respect. This should be understood whenever body fluids are mentioned in this application. 
         [0004]    Biochemical reactions and degradation processes of dead cells are accompanied by the breakdown of peptides and DNA leading to the formation of amines. One of the processes of particular interest is the breakdown of amino acids and the production of diamines and polyamines. For example, decarboxylation of histidine, ornithine and lysine, produces histamine, putrescine and cadaverine, respectively. 
         [0005]    Ion Mobility Spectrometry (also, briefly, IMS) is an analytical method that has been applied to the determination of aliphatic and aromatic amines. See, for instance, Z. Karpas, Ion Mobility Spectrometry of Aliphatic and Aromatic Amines, Anal. Chem. 61 (1989), 684. An apparatus for carrying out this method—the Ion Mobility Spectrometer (IMS)—is used primarily for detection, identification and monitoring of trace amounts of gases and vapors. It is particularly suitable for detection of compounds that have high proton affinity and form stable positive ions, or for compounds that have a high electronegativity and readily form stable negative ions. IMS is discussed in J. I. Baumbach and G. A. Eiceman, Appl. Spectrosc. 1999, vol. 53, pp. 338A 355A and in the monograph “Ion Mobility Spectrometry: Second Edition”, by G. A. Eiceman and Z. Karpas, Taylor and Francis, Boca Raton, Fla., (2005). 
         [0006]    U.S. Pat. No. 7,056,745 to Lorber et al discloses a diagnostic method based on the amounts of biogenic amines that are contained in a human body fluid or other sample. A number of measured parameters related to the desired diagnostic information are derived from the amounts. For each diagnostic information desired, an input consisting of the identification of the diagnostic information is provided. The input is compared to the measured parameters and a diagnostic response is derived from the comparison. The measured parameters may be derived from the amounts of the biogenic amines according to a program stored in a memory. 
       SUMMARY OF THE INVENTION 
       [0007]    The present invention is based on the finding that detection of biogenic amines can be used for the diagnosis of pathological conditions, such as bacterial vaginosis, in animals. 
         [0008]    In its first aspect, the present invention provides a method for diagnosing pathological disorders in animals. The pathological disorder may be, for example, a vaginal disorder such as bacterial vaginosis. In accordance with this aspect of the invention, a body sample is obtained from an animal and is analyzed to determine levels of one or more biogenic amines. A body fluid sample, such as saliva, vaginal discharge, etc., may be collected, for example, using a swab. Samples of other body fluids, such as blood, urine, semen, etc. can be collected in a vial or other vessel and analyzed. Samples that consist of tissues like feathers, hair, nails or soft body tissues can be removed from the animal and measured after proper pretreatment. 
         [0009]    The biogenic amine or amines to be determined may be any one or more of the amines, diamines and polyamines and their derivatives, such as acetylamines, trimethylamine (TMA), diamines like putrescine and cadaverine as well as polyamines like spermidine and spermine. 
         [0010]    One preferred embodiment of the invention comprises the following steps: 
         [0011]    (1) A sample is obtained from a animal subject, as explained above. The biogenic amines in the sample, particularly, but not only, in urine samples, may be pre-concentrated, for example, by contacting the sample with a solid support such as a fiber, with a suitable coating for selective absorption of the amines. Desorption of the pre-concentrated amines in vapor form may be carried out by heating (thermal desorption) or by applying chemicals that release the amines from the support. 
         [0012]    (2) Amines are emitted from the sample due to their natural volatility. Enhancement of vapor emanation may be carried out by the addition of an appropriate chemical reagent, such as an alkaline solution (e.g. KOH, NaOH, and/or ammonia), that transforms complex amine compounds, like salts and acidic forms, to more volatile forms. 
         [0013]    (3) Vapors emanating from the sample are ionized usually by attachment of one or several protons from a reagent gas that forms reactant ions. 
         [0014]    (4) The level of one or more ions in the vapor is determined by introducing the vapor into a device for the detection of amines in the sample. The device may be, for example, an ion mobility spectrometer (IMS) or differential mobility spectrometer (DMS) which separates the ions according to their mobility or mass by application of an electric field. Other devices for the detection gas phase ions may also be used in the invention. 
         [0015]    (6) The level of one or more amines in the sample is determined from the levels of ion signals derived from amines as determined in the previous step. 
         [0016]    (7) The presence of certain biogenic amines, and the ratio between them, is used for diagnosing a pathological condition of the animal from which the sample was collected. 
         [0017]    In one embodiment of the invention, the pathological condition to be diagnosed is bacterial vaginosis. In this embodiment, the level of trimethylamine (TMA) ions in vaginal fluid of a non-human animal is determined. In a preferred embodiment, the total amount of amine ions in the sample is measured, and if the number of TMA ions is above a predetermined threshold, such as about 40% of the total number of amine ions (or a signal intensity that is above a predetermined threshold, the presence of bacterial vaginosis in the animal is recognized, while if the number of TMA ions is below the threshold, the absence of bacterial vaginosis is recognized. 
         [0018]    In another embodiment, levels of putrescine and cadaverine are measured, and if the number of their ions is above a predetermined threshold of the total number of amine ions, various pathological conditions are suspected. 
         [0019]    The invention thus provides a method for diagnosing a disease or pathological condition in an animal comprising:
       obtaining a body sample from the animal;   carrying out an ion mobility spectrometry measurement (IMS) or a differential mobility spectrometry (DMS) on the sample thereby determining an amount of ions formed by at least two biogenic amines contained in the sample; and   calculating a ratio of the amounts of ions formed by the different biogenic amines in the sample, wherein the ratio is indicative of the disease or pathological condition.       
 
         [0023]    The body sample may be taken, for example, from male genitalia, female genitalia, udder, liver, heart, muscle, brain, tongue, throat, lungs, skin, and lymph node. The disease or pathological condition may be selected, for example, from bacterial vaginosis, inflammation, cancer, and bronchitis. The disease or pathological condition may be caused, for example, by bacteria, viruses, anaerobic microorganisms, fungi. 
         [0024]    The body sample may be, for example, a sample of vaginal fluid, wherein at least one of the amines comprises trimethylamine (TMA), and wherein the pathological conditions comprise vaginal disorders. The amines may comprise putrescine or cadaverine. In another embodiment, abnormally high amounts of putrescine or cadaverine indicate a pathological condition. 
         [0025]    In one embodiment of the invention, the method comprises calculating the ratio of the amounts of ions formed by trimethylamine and ions formed by all biogenic amines present in the sample, and diagnosing the presence of bacterial vaginosis if the ratio is above a predetermined threshold. The predetermined threshold may be, for example, 0.4-2. Alternatively, the method may comprise diagnosing the presence of bacterial vaginosis if the TMA signal is above a predetermined threshold. 
         [0026]    In another embodiment, the method comprises calculating the ratio of the amounts of ions formed by trimethylamine and ions formed by all biogenic amines present in the sample, and diagnosing the absence of bacterial vaginosis if the ratio is below a predetermined threshold. The predetermined threshold may be, for example, in the range of 0.4-2. [ 
         [0027]    In one embodiment of the invention, the method comprises diagnosing the absence of bacterial vaginosis if the TMA signal is below a predetermined threshold, which may be, for example, 2.0. 
         [0028]    In its second aspect, the invention provides a device for collecting a sample from an animal. The device of this invention includes a slender shaft having a proximal end and a distal end. At the distal end is a sample collector, which may be, for example, a wad of cotton. The device further includes a sheath around the shaft that is slidable between a first position in which the shaft covers the sample collector, and a second position in which the shaft doesn&#39;t cover the sample collector. For example, in order to obtain a sample from an inner part of the vagina of an animal, a device having a shaft of about 10 cm may be used. The inventors have found that this allows a sample to be collected from a deep location in the animal body with minimal contamination of the sample collector by contaminating factors commonly found on or near the surface of animal bodies. The sheath also tends to avoid transferring of bacteria from the area near the surface of the animal body deeper into the body cavity. 
         [0029]    In another of its aspects, the invention provides a method for collecting a body sample from an animal using the device of the invention. In accordance with this aspect of the invention, the distal end of the shaft of the device is inserted into an animal body cavity with the sheath covering the sample collector, so that the sample collector is protected from any contamination encountered during insertion of the device. Once the distal end of the shaft is positioned in the location where a sample is to be collected, the sheath is made to slide proximally over the shaft to expose the collector, thus allowing collection of a sample. After collection of a sample, the sheath is made to slide distally to recover the collector, and the device is removed from the animal body, again while protecting the collector from contamination. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0030]    In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: 
           [0031]      FIG. 1  shows the mobility spectrum obtained with TMA for calibration; 
           [0032]      FIG. 2  shows the ion mobility spectrum obtained from a sample of a cow having a high TMA level (peaks at 6.15 ms and 7.01 ms) indicative of vaginal infection; 
           [0033]      FIG. 3  shows the ion mobility spectrum obtained from a sample from the genitalia of an uninfected cow; 
           [0034]      FIG. 4  shows the ion mobility spectrum of a vaginal fluid swab with an extremely large amount of TMA indicating a severe infection; 
           [0035]      FIG. 5  shows the ion mobility spectrum from a vaginal discharge fluid of a sow indicative of a vaginal infection; 
           [0036]      FIG. 6  shows the ion mobility spectrum from a vaginal discharge fluid of an uninfected sow; 
           [0037]      FIG. 7  shows the mobility spectrum obtained from the udder of a cow having an udder inflammation; 
           [0038]      FIG. 8  shows the mobility spectrum obtained with a permeation tube with 2-Nonanone; 
           [0039]      FIG. 9  shows the mobility spectrum obtained from a sow not having a vaginal infection; 
           [0040]      FIG. 10  shows the mobility spectrum obtained from a sow having BV; 
           [0041]      FIG. 11  shows the mobility spectrum obtained from a sow with BV 
           [0042]      FIGS. 12   a  and  12   b  show a collecting device for collecting a body sample from an animal; | and 
           [0043]      FIGS. 13   a  to  13   d  show a method for collecting a body sample from an animal. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0044]      FIGS. 12   a  and  12   b  show a device  2  for collecting a body sample from an animal in accordance with one embodiment of the invention. The device  2  has a slender shaft  4  having a proximal end  6  and a distal end  8 . At the distal end  8  is a sample collector, which may be, for example, a wad of absorbent material, such as cotton  10 . The device further includes a sheath  12  surrounding the shaft  4  that is slidable between a first position shown in  FIG. 12   a  in which the shaft covers the sample collector  10 , and a second position shown in  FIG. 12   b  in which the shaft doesn&#39;t cover the sample collector. 
         [0045]    As explained below, this allows a sample to be collected from a deep location in the animal body with minimal contamination of the sample collector by contaminating factors commonly found on or near the surface of animal bodies. For example, in order to obtain a sample from an inner part of the vagina of an animal, a device having a shaft of about 10 cm may be used. During insertion of the distal end of the shaft into the vagina, the sheath covers the sample collector, so that the sample collector is protected from any contamination encountered during insertion of the device. Once the distal end of the shaft is positioned in the location where a sample is to be collected, the sheath is made to slide proximally over the shaft to expose the collector, thus allowing collection of a sample. After collection of a sample, the sheath is made to slide distally to re-cover the collector, and the device is removed from the animal body, again while protecting the collector from contamination. 
         [0046]      FIG. 13  shows a method for collecting a body sample from an animal using the device  2 . In  FIG. 13   a  the distal end  8  of the shaft  4  of the device  2  is inserted into a body cavity of an animal  20 , such as a vagina  22 , with the sheath covering the sample collector, so that the sample collector is protected from any contamination encountered during insertion of the device. Once the distal end of the shaft is positioned in the location where a sample is to be collected, the sheath is made to slide proximally over the shaft to expose the collector, thus allowing collection of a sample ( FIG. 13   b ). A sample is then collected, for example, by wiping the vaginal surface with the cotton wad  10 . After collection of a sample, the sheath is made to slide distally to re-cover the collector ( FIG. 13   c ), and the device is removed from the animal body, again while protecting the collector from contamination. 
       EXAMPLES 
     Methods 
       [0047]    Vaginal swabs were obtained from organs of slaughtered cows, and from live sows in a breeding unit, using a cotton tipped swab by a certified experienced veterinary surgeon and an ion mobility spectrum was obtained of vapors emitted by each sample using an ion mobility spectrometer (IMS) device after addition of an alkaline solution to the sample on the cotton swab. 
         [0048]    In one set of tests, vaginal swabs were collected from 21 sows at the Lahav Research Institute of Kibbutz Lahav, Israel, and analyzed on Mar. 4, 2009 for bacterial vaginosis (BV). Each sample was placed in a 25 ml polystyrene vial and five drops of KOH were added. The spectra were obtained using a reagent (2-Nonanone) and calibration was carried out with 10 μl of trimethylamine (TMA) that were added to a 25 ml polystyrene vial for calibration as shown in  FIG. 1 . Permeation tube with 2-nonanone was connected to the drift air during all the trials. Samples were also collected from the throat of hens with the aid of a swab. 
         [0049]    Results 
         [0050]    Cows 
         [0051]    Over 60 samples were collected from three groups of cows. All of the samples collected from the organs of cows (genitalia, lymph nodes, udder, and liver) were obtained in the abattoir and were inspected on site by an experienced veterinarian. One group consisted of 21 genitalia of which five (23.8%) had elevated levels of trimethylamine (TMA), indicating that they had a pathological condition in the form of a vaginal infection. A representative spectrum from genitalia having elevated levels of TMA is shown in  FIG. 2 .  FIG. 3  shows a representative spectrum from genitalia having a normal spectrum and is thus negative for BV. Thirteen samples of vaginal discharge fluid were collected from the second group of genitalia and one (7.7%) had an elevated TMA level, while the rest were negative. The third group consisted of 24 cow organs all of which were without elevated TMA levels. 
         [0052]    An even more extreme spectrum is shown in  FIG. 4  where the TMA concentration was so high, indicative of a severe infection, that all other ions in the sample transferred their charge to the TMA. 
         [0053]    In a similar fashion, vaginal discharge fluid samples were collected from 21 sows in the breeding unit and analyzed. Two samples (9.5%) were found to have elevated levels of TMA indicating vaginal infections, as shown in the representative spectra of  FIGS. 5  (positive for BV) and  6  (negative for BV). 
         [0054]    Twenty one organ samples were collected from cows on Mar. 8, 2009. The methodology described above for the pigs (2-nonanone as a reagent, calibration with TMA) was used in this test as well. The results are summarized in Table 2. 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Summary of diagnostic results for 21 cows. 
               
             
          
           
               
                 Sample No. 
                 IMS Results 
               
               
                   
               
             
          
           
               
                 1 
                 negative 
               
               
                 2 
                 negative 
               
               
                 3 
                 Positive(preg.) 
               
               
                 4 
                 negative 
               
               
                 5 
                 negative 
               
               
                 6 
                 negative 
               
               
                 7 
                 negative 
               
               
                 8 
                 positive 
               
               
                 9 
                 negative 
               
               
                 10 
                 positive 
               
               
                 11 
                 positive 
               
               
                 12 
                 positive 
               
               
                 13 
                 negative 
               
               
                 14 
                 negative 
               
               
                 15 
                 negative 
               
               
                 16 
                 negative 
               
               
                 17 
                 negative 
               
               
                 18 
                 negative 
               
               
                 19 
                 negative 
               
               
                 20 
                 negative 
               
               
                 21 
                 negative 
               
               
                   
               
             
          
         
       
     
         [0055]    Clinical observations in situ by an experienced veterinarian 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                   
               
               
                 Sample No. 
                 Observations 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 5 
                 blood 
               
               
                 9 
                 pus 
               
               
                 12 
                 pus 
               
               
                 20 
                 gave birth recently, 
               
               
                   
                 mucus with blood 
               
               
                   
               
             
          
         
       
     
         [0056]    A swab sample collected from the udder of a cow with visible udder inflammation was tested. The mobility spectrum of the udder inflammation is shown in  FIG. 7 . Elevated levels of TMA were clearly seen in the mobility spectrum, while samples collected from a cow without such an inflammation did not have TMA. 
         [0057]    A sample collected from lymph nodes of an inflamed organ of a cow also had elevated levels of biogenic amines. 
       Sows 
       [0058]    A representative ion mobility spectrum of a sample from a sow is displayed in  FIG. 8 . The results are summarized in Table 1. Two of the 21 samples clearly contained TMA indicating the presence of a BV infection, and this was confirmed by the veterinarian. Three of the samples were with blood, one had pus and one was extremely malodorous, but this did not affect the diagnosis by IMS. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Summary of diagnostic results for 21 sows. 
               
             
          
           
               
                   
                   
                   
                 No. of times 
                 IMS 
               
               
                 Sample No. 
                 Pig No. 
                 Observations 
                 giving birth 
                 Result 
               
               
                   
               
             
          
           
               
                 1 
                 1160 
                 Blood 
                 3 
                 negative 
               
               
                 2 
                 1762 
                   
                 1 
                 negative 
               
               
                 3 
                 1550 
                   
                 2 
                 negative 
               
               
                 4 
                 9896 
                   
                 4 
                 negative 
               
               
                 5 
                 1557 
                   
                 2 
                 negative 
               
               
                 6 
                 1238 
                   
                 2 
                 negative 
               
               
                 7 
                 1095 
                 Contaminated 
                 3 
                 negative 
               
               
                   
                   
                 with feces* 
               
               
                 8 
                 1527 
                   
                 2 
                 negative 
               
               
                 9 
                 1812 
                   
                 1 
                 negative 
               
               
                 10 
                 8960 
                   
                 7 
                 negative 
               
               
                 11 
                 1344 
                 Blood 
                 2 
                 negative 
               
               
                 12 
                 1756 
                   
                 1 
                 negative 
               
               
                 13 
                 1686 
                   
                 1 
                 negative 
               
               
                 14 
                 9539 
                 blood 
                 5 
                 positive 
               
               
                 15 
                 9174 
                   
                 5 
                 negative 
               
               
                 16 
                 9544 
                   
                 5 
                 negative 
               
               
                 17 
                 1526 
                   
                 2 
                 negative 
               
               
                 18 
                 1515 
                   
                 2 
                 negative 
               
               
                 19 
                 9552 
                 Turbid and 
                 5 
                 positive 
               
               
                   
                   
                 malodorous* 
               
               
                 20 
                 9817 
                   
                 4 
                 negative 
               
               
                 21 
                 8417 
                 pus 
                 9 
                 negative 
               
               
                   
               
               
                 *Contamination did not interfere with the test. 
               
             
          
         
       
     
         [0059]    All of the uninfected samples tested produced the same spectrum, a representative spectrum of which is shown in  FIG. 9 . The positive samples were tested twice, in order to confirm the results. A representative spectrum obtained from the second measurement of a vaginal sample of a pig infected with BV in shown in  FIG. 10 . Another example from an infected pig is shown in  FIG. 11 . 
         [0060]    Diagnosis of Bronchitis in Chicken 
         [0061]    Swab samples were collected from the throats of 10 chickens and were tested by IMS. One of the samples contained elevated levels of TMA and the chicken was diagnosed by the veterinarian as having a bronchitis infection. 
         [0062]    While embodiments of the invention have been described by way of illustration, it will be apparent that the invention may be carried into practice with many modifications, variations and adaptations, without departing from the scope of the claims.