WO1989011543A1

WO1989011543A1 - Detecting bacteria in urine of horses

Info

Publication number: WO1989011543A1
Application number: PCT/US1989/002116
Authority: WO
Inventors: Edward S. Hyman
Original assignee: Hyman Edward S
Priority date: 1988-05-25
Filing date: 1989-05-17
Publication date: 1989-11-30
Also published as: AU3834889A

Abstract

New and substantially improved methods for the detection of bacteria, bacterial fragments and/or bacterial antigens are described. Novel methods for treatment of arthritis in horses and in other animals, and a variety of diseases found to be associated with bacteriuria are also described. Additionally, the specification discloses that the new and improved methods for direct microscopic examination are advantageously used for examination of formed elements in samples of body fluids.

Description

DETECTING BACTERIA IN URINE OF HORSES

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of my earlier application serial No. 031,771 filed March 30, 1987, which, in turn is a continuation of my earlier application No. 603,088, filed on April 23, 1984, now U.S. Patent No. 4,673,637.

This invention relates to new and improved methods for the detection of bacteria in urine of horses and other animals; to the discovery of such heretofore unknown bacteria in the urine of horses suffering from arthritis and diseases that are apparently related; and to the treatment of these diseases as a result of the discovery of the presence of said bacteria. The methods are also applicable to the detection of bacteria in other body fluids, e.g. pleural fluid, peritoneal fluid, cerebrospinal fluid, synovial fluids, etc., and to the examination of formed elements (cytology) in urine and in such other body fluids.

This invention also relates to methods for the preservation of urine samples obtained from horses and other animals for the detection of bacteria at a later date.

BACKGROUND OF THE INVENTION 1. Conventional methods for Detecting bacteria in Urine Urine originates as an ultrafiltrate of plasma and is normally free of bacteria as it moves from the upper urinary tract to the bladder. Therefore, bladder urine obtained by suprapubic needle aspiration, and perhaps urine properly obtained by a catheter inserted via the urethral meatus, should contain no bacteria. As it is voided from the body however, the urine is frequently contaminated with microorganisms which colonize the distal urethra and/or the perianal area. The most common non-invasive method of obtaining urine samples which attempts to minimise, but rarely completely eliminates contamination, involves meticulous swabbing of the urethral meatus and periurethral areas with a bactericidal agent, followed by the collection of a mid-stream "clean-catch" specimen. The fresh specimen should be examined within 1 hour of collection (two hours if the unpreserved urine specimen is refrigerated). Although most contaminating microorganisms are eliminated by this "clean-catch" method, the collected samples may still contain some contaminants.

Although the art and science of renal or urinary tract disease is not so well developed for horses and other animals as it is for humans (reference 1), quantitation of the viable bacteria has been the recommended parameter used for determining the presence of clinically relevant bacteria in the urine of a horse as well as in that of the human. Thus the data for the human is relevant. As in the human, direct microscopic examination of urine sediment has been relegated to a presumptive test to detect and to approximate the number of bacteria in a urine specimen (reference 1).

As in human medicine, bacteria are demonstrated in urine by allowing the bacteria to grow in an appropriate culture medium until the colonies are visible to the naked eye. By counting the colonies and multiplying by the dilution of the urine, and by assuming that one colony consists of the progeny of a single bacterium (or a small cluster of bacteria such as a pair) in the original specimen, the number of bacteria (or more accurately, colony forming units of bacteria) in a given volume of urine may be estimated.

Using conventional urine culture techniques, the organisms isolated from the urine of horses are E. coli, Staphylococcus species, Corynebacterium species, and Pseudomonas aerugenosa. Except the Corynebacteria these are much the same as those isolated from the human urine: The latter are often seen in human urine on microscopy, are usually not reported in the human. The infection of the urinary tract of the horse is usually described as cystitis or a bladder infection (reference 1), and, as currently taught, kidney infections in the horse are due to invasion of those organisms from the bladder below. This too is very much the same as the teaching for the human. Also, much like the human, the evidences for that teaching are mostly based upon the bacteriological culture, and these evidences suffer the same frailty as the human in that not all bacteria that grow within, a horse will also grow in the artificial medium chosen. Moreover, the clinical description of urinary tract infections in horses (and other animals) is limited to the more florid infections, perhaps because of the limitation in communication. Thus the Staphylococcal infection of the urinary tract in the horse (and in other animals) is not merely subtle bacteriurla, but instead it is is much more florid. For the most part it is the result of an examination of urine of the animal whose doctor has made a clinical diagnosis of a bacterial infection in the urinary tract. Indeed, the organisms described in this application are not necessarily those which cause the florid infection with symptoms localizing to the urinary tract, but instead they are organisms which cause much more subtle disease. They rarely cause symptoms referable to the urinary tract (especially the bladder). They cause generalized illnesses such as arthritis and other related manifestations.

It is further apparent that the teaching concerning the examination of urine in the horse has followed that in the human. As in human medicine, the direct visualization of bacteria in urine has largely been relegated to a presumptive test while awaiting the urine culture.

Any useful culture method requires that the bacteria will grow in the laboratory 1) in the medium selected, and 2) in the time allotted. If the medium used is inappropriate for the growth of the particular organisms present, they will not grow. If the time allotted is too short, colonies will not be visible. Positive cultures may be mistakenly reported as negative. And, if the oxygen tension or the oxidation potential of the culture medium is either too high or too low, fastidious anaerobic or aerobic organisms may be missed.

If the bacteria are dead when excreted from the body, then they will not grow. But, dead bacteria were once alive, and dead ones in the urine were probably alive in the body. Obviously dead bacteria are also unable to multiply in the bladder. There are many reasons why bacteria in urine might display less than optimal viability. For example, the ionic strength or osmolarity of the urine may be outside the requisite range. The wall of the bacterium or some vital biochemistry within the bacterium may be damaged so that it will require a special medium to grow. The oxidation potential potential of urine may be too high for growth of a particular bacterial species (e.g., the typical oxidation potential of human urine observed by me is about +.22 to +.25 volts as referenced to a saturated calomel electrode. This potential is sufficiently high to inhibit the growth of many bacteria). There may be metabolic products of the host or of antibiotics in urine which inhibit bacterial growth. Antibodies against bacteria have been identified in urine and they have been demonstrated to be deposited on bacteria in urine. Moreover, antibiotics administered to a patient may be excreted in urine in active form at a higher concentration than in other body fluids. The higher concentration of antibiotic, antibiotic metabolite, or host metabolite is more likely to inhibit bacterial growth than the less concentrated chemical. Urea and other metabolites present in all urine inhibits bacterial growth. Any one or a combination of these factors may inhibit or diminish bacterial growth in vitro.

2. Arthritis in Horses Arthritis in a horse is described only for "osteoarthritis" and for classic septic arthritis (reference 1). In septic arthritis bacteria actually invade the Joint. However, that osteoarthritis in the horse may be associated with tendonitis or bursitis as is the prominent inflammatory (rheumatoid) arthritis in the human. Moreover, the horse develops an inflammatory synovitis which resembles rheumatoid synovitis in the human. For this reason, it was found appropriate to examine the urine of a horse to look for the same organisms that I have associated with rheumatoid arthritis and osteoarthritis in the human. I have found small Gram positive cocci in the urine of the horse much like those I have found in the human. Moreover, as in the human, eradication of the bacteriurla of the horse has resulted in resolution of the swelling and tenderness of the joint, and the horse no longer favors the joint. Finally, it was found that the joint disease in the horse was associated with a loss of stamina, as in the human. Thus it is apparent that the arthritis, synovitis, tendonitis, etc. of the horse is quite similar to that in the human both in terms of the related subtle bacteriurla and in its response to the antibacterial agents given for the associated bacteriurla.

BRIEF SUMMARY OF THE INVENTION The present invention presents new and improved methods for detecting and identifying bacteria, bacterial fragments and/or bacterial antigens in samples of urine and other body fluids of horses and other animals. The present invention proposes improved methods 1) for the preparation of samples for rapid direct microscopic detection, identification, and quantitation of bacteria; 2) for the preservation of urine samples preparatory for delayed detection and identification of bacteria; 3) for the rapid, quantitative methods and assay kits employing monoclonal or polyclonal antibodies or other reagents specific for bacteria and/or bacterial antigens herein shown to be associated with arthritis and related diseases; 4) for the administration of antibiotics effective against such detected and identified bacteria in order to treat the above defined diseases; and 5) for the use of the system of detection to monitor the in-vivo effect of said antibacterial agents.

By virtue of the improved methods herein described, it has been demonstrated that certain diseases of hitherto unknown or uncertain etiology are associated with bacteriuria not detected by prior methods. These bacteria are generally Gram positive cocci, although the now- classic pyelonephritis due to Gram negative rods is also readily detected by the methodology reported herein. Application of antibiotic therapy appropriate for the organisms detected not only diminishes the detected and identified bacteriuria but it also offers therapeutic benefit for the associated disease. This therapeutic benefit and clinical improvement resulting from use of an agent whose primary effect is its antibacterial activity establishes a cause and effect relationship between the bacteria detected and the associated disease.

The present invention also presents novel methods for treatment of horses suffering arthritis and a variety of other diseases which are found by their resolution to be associated with bacteriuria according to the present invention.

Using the new and improved methods of detection of the present invention, bacteria, generally Gram positive cocci, have been found in the urine of horses suffering from arthritis, synovitis, and loss of stamina. Bacteriuria has also been demonstrated in horses suffering unexplained abdominal pain, and unexplained fatigue. Treatment with appropriate antibiotic agents resulted in not only a decrease in the number of bacteria in the urine but also improvement of clinical symptoms of the illness of unexplained etiology. It is believed that such horses suffer a bacterial infestation at some undisclosed body site.

The bacteria, dead or alive, whole or partial, have been detected in the urine of horses suffering from the above enumerated disorders by the novel methods of the present invention. It appears that the scope of illnesses which are associated with previously undetected bacteriuria is not limited by the boundaries of the conventional classification of .illnesses.

The present invention describes rapid and novel methods for determining the presence of live or dead bacteria or bacterial fragments in the urine. The methods described herein are useful as a general diagnostic technique. They can be utilized in the diagnosis of illnesses, and to monitor the effectiveness of antibacterial agents and their dosages in the treatment of the above-listed diseases or conditions. They are particularly useful for the detection of bacteria which are not reliably detected by conventional methods.

Once the bacteria have been found and identified, an amount of an antibiotic effective against the bacteria is administered. The amount necessary is determined by the response of the bacteriuria, an in-vivo test of the agent. Relatively large doses of antibiotic may be necessary. For example, the intravenous administration of 6 or more grams of lincomycin may effective, the dose being limited by the tolerance of the host for the chemical. The dosages may be adjusted for other routes of administration. It may be necessary to use two or more antibacterial agents at one time, administered separately or in combination, to reduce or eliminate the bacteriuria as monitored by the method herein disclosed, e.g., lincomycin may be used with gentamycin, tobramycin, piperacillin, one of several cephalosporins, tetracyclines, chloramphenicol, etc.

Typically, when cocci have been found in urine by the method of this invention, antibiotic therapy has effectively reduced or eliminated the cocci and has alleviated the symptoms, signs, and often the abnormal laborator y findings of the animal. One object of the invention is to detect bacteria in urine of horses and other animals that are not detected by conventional methods.

Another object of the invention is to detect bacteria rapidly so that the method may be useful as a clinical test.

Another object of the invention is to ensure that all bacteria and formed parts of bacteria in a urine sample are collected in the sediment . According to one embodiment of the present invention, this is ensured by adequate centrifugation.

Another object of the invention is to ensure retention of the urine sediment on the microscope slide throughout staining.

Another object of the invention is to alert the attending doctor to the possibility that antibiotic therapy, appropriate for the organisms found in the urine by the new and improved methods of this invention, might improve the patient's condition.

Another object of the invention is to provide therapeutic relief to horses and other animals in cases of arthritis and other diseases or conditions found by methods described herein to be associated with significant bacteriuria.

Other objects will be apparent to a person of ordinary skill in the ar t after studying the specification and claims .

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more fully understood by reference to the following detailed description of the invention, an example of clinical histories demonstrating treatment according to novel methods of the invention and the appended figures in which:

FIG. 1. is a schematic representation of the steps of an improved method for detecting bacteria in urine samples. FIG. 2. is .a photograph at 1000 diameters magnification of streptococci found in urine of a sick horse. The slide was prepared by the method described in this application. DETAILED DESCRIPTION OF THE INVENTION

IMPROVED METHODS FOR DIRECT

MICROSCOPIC EXAMINATION OF URINE SAMPLES

These are described in my U.S. Patent No. 4,673,637, and in the copending application , Serial No. 031,771, the disclosure of which is incorporated by reference.

Other positive embodiments of this procedure, not differing in essence from the invention herein disclosed, will be apparent to a person of ordinary skill in the art.

Use of antibodies: According to the present invention, bacteria, bacterial fragments and bacterial antigens demonstrated herein to be associated with equine arthritis and related diseases, etc., alternatively may be detected in urine samples using antibodies specific for soluble or insoluble antigens produced by such bacteria, these bacteria having been disclosed by this invention. As explained in detail above, such gram positive bacteria include but are not limited to: Streptococcus faecalis; S. faecium; S. mϊtis; S. mutans; S. mutans; S. viridans; S. intermedius; S. salivarius; Staphylococcus epidermidis; Staph. hemolyticus; Staph. hominis; Peptococcus, etc.

Both conventional polyclonal antibodies (antisera) and monoclonal antibodies can be used. Monoclonal antibodies offer the advantage that large amounts of monoclonal antibody specific for a single bacterial antigen can easily and inexpensively be produced.

Kits employing such antibodies for use in enzyme-linked immunosorbent assays, fluorescence quenching, fluorescence polarization assays or other methods for the detection of Gram positive bacteria in urine are also encompassed within the scope of the present invention. Thus, it can be seen that the present invention has several basic advantages over the prior art methods, including:

1. The detection of bacteria (dead or alive, whole or partial) not earlier observed in the urine of horses and other animals suffering from the above recited illnesses.

2. The rapid positive identification of both bacteria and bacterial fragments detected and stained by the methods of this invention within a treatment time frame measured in minutes or hours from the time of taking an initial urine sample.

3. The treatment of the various illnesses by the administration, of therapeutically effective dosages of antibiotics specific to the illness.

4. The monitoring of treatment by the detection and identification of bacteria in the urine of the horses and other animals being treated.

The methods of the present invention to obtain these four basic advantages are characterized by several specific improvements over the prior bacteria detection and identification techniques.

One improvement of the present invention is based on the discovery that urine contains lipids which act as detergents and interfere with adherence of bacteria and bacterial fragments to a slide. Thin layer chromatography of these lipids in a solvent system appropriate for lipids commonly found in human tissues such as phosphatidylcholine (lecithin), phosphatidyl-ethanolamine, phosphatidylserine, and sphingomyelin, reveals that these lipids are in the same range of polarity as human phospholipids. These lipids however, do not contain appreciable phosphorus, and thus they are for the most part not phospholipids. If these lipids are allowed to remain with dried urine sediment on a slide, they will cause the sediment to be partially or completely released from the slide when an aqueous solution is applied. The bacteria in the sediment would be lost before they could be seen. This is one reason why past methods of direct microscopy have failed to detect significant members of bacteria in the urine of horses and other animals with diseases such as rheumatoid arthritis, etc.. and one reason why the teaching with regard to these diseases has been that significant bacteria are not present in the urine. Standard methods of preparing and staining urine specimens for microscopic examination do not provide, for the precautionary removal of these lipids. Another improvement of this invention involves the further fixation of bacteria and other urinary sediment onto a glass slide. Whereas delipidation of the dried sediment improves adherence of bacteria to a glass slide, much stronger adherence is obtained by the use of certain chemicals which do not interfere with the staining of the adherent sediment. This includes fixation of proteins of the sediment with glutaraldehyde and the fixation of glycoproteins of the sediment with alcian blue.

Another improvement involves the removal of crystals and of excesses of water soluble releasing agents such as glucose and soluble proteins by washing the urine sediment.

The positive identification of the various types and states of bacteria (i.e., live bacteria, dead bacterial and bacterial fragments) is also an improvement of this invention. Only live bacteria (and usually non-fastidious) can be detected and identified by the present conventional cultural methods. Culture methods for fastidious bacteria and usually too cumbersome and too expensive for routine use. Dead or fragmented bacteria simply will not grow in any of the culture media.

Yet the presence of the previously undetected dead or fragmented bacteria is Mghly relevant to diagnosis and treatment by the present invention. If dead or fragmented bacteria are present in the urine, these bacteria were once alive and came from a source within the patient. Cessation of the excretion of both live and dead bacteria in urine of a patient, whose urine had contained these bacteria or bacterial parts, is evidence that the source of live bacteria has been eradicated or sealed off (even if not permanently), and suggests that and the patient has been adequately treated for that time. (N.B. Recurrence of bacteriuria after treatment suggests that the bacteria are "protected" in some locus within the body of the host, or it suggests reinfection)

The non-cultural methods of the present invention result in a rapid, yet positive detection and identification of bacteria and bacterial fragments within a time span to accomplish treatment during the same day or even during the same office visit, as opposed to the 3. to 6 days required for culture growth and analysis. The advantages to the practitioner and to the patient are obvious. The treatment aspect of the present invention is undertaken only after detection and identification of the bacteria (alive or dead, whole or fragmented) has been completed. Thus, a proper antibiotic to be administered and the proper dosage is predetermined from the prior analysis.

The monitoring of the treatment by the same detection and identification method ensures the eradication of bacteria (live or dead, whole or fragmented) from urine excreted prior to cessation of treatment. It also may indicate the desirability of a change in the treatment, either in the specific antibiotic, the use of a supplemental antibiotic or a variance in the dosage level.

Using appropriate antibacterial agents, treatment of horses and other animals suffering from the above-mentioned diseases or conditions, has resulted in improvement or resolution of the clinical manifestations of symptoms as the bacteriuria lessened or eliminated.

According to the present invention, bacteria, dead or alive, detected in urine do not necessarily represent active bacterial infections of the urinary tract. Rather it is currently thought that the majority of presently detected bacteria originate from sites of bacterial infections or infestations of other parts of the body and that the bacteria or bacterial parts are excreted by the kidney.

"Damaged" or "exploded" cocci are seen in the company of either large or small Gram positive cocci. Experimentally these forms can be simulated by performing the same staining method on a smear of broken cell walls of staphylococci or streptococci grown in a culture. "Exploded" cocci are thought to be the cell walls of cocci whose contents have been lost when the cell wall (a shell) was opened by host defenses or by previously administered drugs.

As in the human, the method of the present invention has demonstrated a much higher incidence of bacteriuria in horses as compared to that seen with conventional methods for detecting bacteriuria, It can be readily shown by staining and microscopy that many of the bacterial forms observed under the microscope using methods of the present invention were not alive at the time the specimen was obtained. For example, some do not contain any nucleic acid, either DNA or RNA, a biochemical component essential to life. Should all of the bacteria in a given specimen be devoid of nucleic acid, then none will grow and the culture of urine is sterile. Indeed urine specimens containing huge numbers of bacteria as demonstrated by the novel methods of the present invention may not yield a positive culture in the bacteriology laboratory. When the laboratory reports "no growth" the doctor usually abandons the possibility of significant bacteriuria. Nonetheless, though they do not grow in routine culture, these dead, damaged or fastidious bacteria are or were once alive, and they have caused or exacerbated the illness.

A NOVEL METHOD FOR TREATMENT OF EQUINE ARTHRITIS As explained above, in equine arthritis, large numbers of small cocci have been detected in urine samples. Typical photomicrographs often reveal many cocci per oil immersion field. Moreover, without any other change or any other introduction of medication, horses have had significant improvement of the illness as a result of antibacterial therapy which greatly reduces or eliminates the cocci. Some have even had a full remission of the illness with eradication of the bacteriuria.

Among the useful therapeutic agents, the antibiotics lincomycin and clindamycin seem to be the most useful, whether given alone alone or with an am inoglycoside antibiotic such as streptomycin, tobramycin, kanamycin, neomycin, or amikacin. This has been used with full knowledge that this group of antibacterials have been nearly dismissed from equine medicine because of their adverse effects (colitis and consequences) in horses in particular. Other useful therapeutic agents are cephalexin, cephradine, cefazolin, cephalothin, chloramphenicol, novobiocin, fusidic acid, the quinolones, metronidazole and some of the penicillins. More antibacterial agents may also be found to be useful, and the methodology described herein is valuable to determine whether any given agent or new agent is effective in vivo. When the drugs are given parenterally, the dosage can be controlled and the response has been predictable. Better and more predictable results are had by intravenous administration of about 6.0 to 12.0 gm of lincomycin. The drug dosage is continued at least until the urine sediment, examined by the methods as disclosed above, no longer shows the presence of cocci.

Novel Methods for Treating Other Conditions According to the present invention, large numbers of cocci or "exploded cocci" have been detected in urine samples from horses suffering from skin abscesses, "dulling of the coat", an illness resembling the human "Chronic Fatigue Syndrome", other manifestations of sickness, etc. Antibiotic therapy appropriate for such microorganisms, usually given in the same or in an analogous protocol offers therapeutic benefit in those disorders associated with bacteriuria. Moreover, Gram positive bacteria have been found in the urine from horses suffering from renal disorders, including nephritis with casts and red blood cells in the urine.

The antibiotics of the lincosamine group (lincomycin and clindamycin), alone or in combination with an aminoglycoside such as streptomycin, tobramycin, kanamycin, neomycin, or amikacin seem to be the most useful. Other beneficial therapeutic agents include the antibiotics cephalexin, cephradine, cefazolin, chloramphenicol, novobiocin, metronidazole, the quinolones, fusidic acid and penicillin. When the antibiotics are given parenterally, the dosage can be better controlled. Other routes of administration, however, are also useful.

EXAMPLE

A 3 year old gelding race-horse, "Paseo", was suffering from arthritis in his hip, but also his appetite had waned and he was tired, lazy, and unable to sustain a run. Additionally he had the beginnings of "founder" in that he had warmth of the digits of his foreleg occasionally with a palpable pulse in that area. He had been examined by veterinarians who had found no abnormality in his urine. By the method disclosed in this invention, his urine contained diplococci in significant numbers. Along with proper precautions for bacterial overgrowth, he was given three courses of six grams of lincomycin intravenously ten days apart, after which his urine cleared, he was much more alert, all of his joints subsided, his appetite and muscle mass improved dramatically, his coat improved, the minimal evidence of founder went away, he was far more energetic, and he began training to race.

Normalization of the Dilution of Urine

It is not possible to get a voluntary urine specimen from a horse or from other animals. One way that a person in attendance will get a urine specimen from a horse when wanted is to give the horse a hypodermic dose of a potent "loop diuretic", such as furosemide. Soon thereafter the horse's kidneys respond by passing large quantities of urine which rapidly fills the urinary bladder, and the horse soon urinates. However, this urine specimen is dilute, i.e., it contains an excess of water. Thus the "bladder-full" of this urine represents a much shorter time interval than the normal bladder-full. Urine sediment is most meaningful when the examiner can relate the quantity of sediment shed by the kidney into a specimen of urine to the time it took the kidneys to create that urine. A normal voiding may represent several hours of urine which in turn means that the sediment in that specimen represents the cells, casts, and bacteria sediment shed by the kidney during that time interval. Should the urine follow a diuretic, then the same volume will represent a much shorter time interval and will contain less sediment. The important parameter is sediment per time, not sediment per volume of urine. The common diuretic used is furosemide, which largely increases the water excreted per time, leaving the salts per time of urine formation not changed very much. Salt added to water makes the water heavier, and the more salt the heavier. The "specific gravity" of the salt solution is a nearly linear function of the salt concentration for any given salt. In other words, if an equal quantity of pure water is added to a salt solution whose specific gravity is 1.024, then the diluted salt solution will have a specific gravity of about 1.012. This principle may be used to normalize the salt content of urine, and thereby partially correct for the extra water in a urine specimen after a loop diuretic, which in turn corrects for the shorter time of the urine collection. If a natural voiding were to have a specific gravity of 1.024, and if the diuretic caused enough water to be added to that urine to reduce its specific gravity to 1.008, then the concentration of salt in that urine is about l/3rd that in the undiluted specimen, and the sediment is probably diluted to 1/3 per volume. Then 3 times the volume of the diluted urine should contain the same amount of sediment as in 1 volume of the undiluted urine. By measuring the specific gravity, the examiner may normalize the specimen of urine even after a diuretic has been given to induce a horse to void.

In actual practice, this is very easy. The measurement of specific gravity can be read in a simple optical device that reads the refractive index on a scale calibrated in specific gravity. This takes only a minute. Either the necessary multiple of volume of urine may be centrifuged for sediment, or more practical, a single centrifuge tube may used. The sediment of the first volume is retained after the supernatant is decanted and the tube is refilled enough times so that the accumulated sediment represents the total volume desired. This principle has been used with consistent results. In specimens preserved for mailing to a central laboratory, a preservative has been used which does not modify the measured specific gravity (as measured by reading the specific gravity of the fresh urine at the barn and comparing that number to the same reading on a similar instrument at the central laboratory several days later).

REFERENCES:

1) Equine Medicine and Surgery (two volumes), Third Edition, R.A. Mansmann and E.S. McAllister eds., American Veterinary Publications, Drawer KK, Santa Barbara, California, 1982. a) The Urinary System, Chapter 18 by R.P. Hackett, J.T. Vaughan, and B.C. Tennant, pp 907-922. b) Arthritis on pp 1053-1055. c) Lincosamines In Chapter 6, "Clinical Pharmacology", page 141, contains the only reference to the lincosamines (i.e., lincomycin and clindamycin). In its entirety, it reads,

"Macrolides and Lincosamides.

An attractive feature of these two groups of antibiotics is their activity against penicillinase producing Staphylococci. However, their usefulness in horses is limited because of their tendency to induce severe diarrhea in this species. This is particularly true for lincomycin. Erythromycin commonly induces diarrhea in horses regardless of the route of administration after 3-4 days of use." Fusidic Acid- There is no mention of this agent.

2) Current Therapy in Equine Medicine, N.E. Robinson ed., W.B. Saunders Company, Philadelphia, 1987, a) Cystitis and pyelonephritis, pp 708-712. b) Osteoarthritis, pg. 289. c) Lincomycin, Clindamycin, and Fusidic Acid. There is no mention.

There is no mention of the lincosamines as a group of antibiotics, or of either lincomycin or clindamycin, in either the index of the book or in the Table of Common Drugs (pp. 730-734). There is no mention of the lincosamines, or of either lincomycin or clindamycin, in the "Table 1. Antimicrobial agents suitable for use in urinary tract infections of the horse." on page 710. There is no mention of the use of FUSIDIC ACID in either section.

3) Raisbeck, M.F., Holt, G;R., and Osweiler, G.D., Lincomycin-associated colitis in horses., Journal of The American Veterinary Medical Association, vol. 4, pp. 362-3, 1981.

(Horses which by accident ate hay infested with the fungus - Streptomyces lincolnensis - which makes lincomycin, became ill with colitis from the lincomycin produced. A significant number of them died of that colitis.)

4) G.P. Holmes, J.E. Kaplan, N.M. Gantz et al.. Chronic Fatigue Syndrome: A Working Case Definition, Annals of Internal Medicine, 108, 387-389, 1988.

Claims

IN THE CLAIMS:

1) A method of treating arthritis in a horse or other animal suffering from arthritis, comprising:

(a) administering a therapeutically effective amount of an antibiotic effective against bacteria identified by collecting a sample of urine from a from a horse or other animal suffering from arthritis and detecting any abnormal bacteria or bacterial fragments in that urine sample; and

(b) monitoring the urine for the presence of bacteria or bacterial fragments to determine efficacy of the treatment.

2. An improved method for the direct microscopic detection of bacteria or bacterial fragments in a urine sample from a horse or other animal including staining the bacteria or bacterial fragments with an aqueous soluble dye and microscopically observing the bacteria, in which the improvement comprises removal of any lipid components of the sample prior to staining.

3. An improved method for direct microscopic detection of bacteria or bacterial fragments in a urine sample from a horse or other animal including the steps of staining the bacteria or bacterial fragments with an aqueous soluble dye and microscopically observing the bacteria, in which the improvement comprises centrifuging the sample at about 3,500-11,000 times gravity to sediment bacterial or bacterial fragments in the sample prior to staining.

4. The method according to claim 3, in which the lipid soluble components are removed by use of a lipid solvent system.

5. The method according to claim 3, in which the lipid solvent system is a mixture of methanol and halogenated hydrocarbon, or methanol alone.

6. The method according to claim 5, wherein the lipid solvent system is a mixture of methanol and 1,1,1-trichloroethane.

7. A method of detecting bacteria or bacterial fragments in a urine sample from a horse or other animal , comprising: a) centrifuging the sample at a relative centrifugal force of about 3,500-11,000 times gravity to sediment bacteria and bacterial fragments; b) separating the sediment from the supernatant; c) spreading the sediment on a surface; d) washing the sediment on the surface with a lipid solvent composition to remove any lipid components of the sediment preparatory to staining; e) fixing and staining the washed sediment; and, f) microscopically observing the washed and stained sediment.

8) The method of claim 7, further comprising contacting the sediment with a tagged anti-equine IgG antibody to demonstrate the presence of equine IgG on the bacteria of the sediment.

9) The method of claim 8 wherein the antibody is tagged with a fluorescent dye.

10) The method in claim 7, wherein the sediment is contacted with and acridine dye to demonstrate nucleic acids by fluorescence.

11) The method in claim 7, further comprising contacting the sediment with a proteolytic enzyme to aid in identifying any bacteria in the sediment.

12) The method of claim 11, wherein the proteolytic enzyme is selected from a group consisting of bacterial and fungal proteases, crystalline trypsin, and chymotrypsin.

13) The method of claim 11, wherein the enzyme is selected from a group consisting of amylases, DNases, RNases, lipases, lecithinaεes, sialases, neuraminidases, hyaluronidases, sphingomyelinases. . 14) The method in claim 13, wherein the antibiotic is selected from the group consisting of clindamycin, lincomycin, cefazolin, cefamandole, cephradine, cephalexin, cephalothin, moxalactum, metronidazole, the quinolones, the penicillins, fusidic acid, and novobiocin, and is administered according to the table below:

DRUG ROUTE OF ADMINISTRATION DOSAGE MG/DAY clindamycin oral 600 - 2700 clindamycin intravenous 1200 - 8400 lincomycin oral 1500 - 2500 lincomycin intravenous 3000 - 18000 cefazolin intravenous 1000 - 8000 cefamandole intramuscular 1000 - 3000 cefamandole intravenous 1000 - 3000 cephradine oral 1000 - 2000 cephalexin oral 1000 - 2000 cephalothin intravenous 2000 - 8000 moxalactum intravenous 2000 - 12000 novobiocin intravenous 1000 - 2000 gentamycin intravenous 240 - 800 metronidazole oral 2000 - 5000 metronidazole intravenous 500 - 4000 ampicillin intravenous 1000 - 10000 ampicillin oral 2000 - 10000 ciprofloxacin oral 500 - 4000 fusidic acid oral 1000 - 2000 fusidic acid intravenous 1000 - 2000

15) A method of treating a horse or other animal suffering from bursitis, tendonitis, arthritis (and related ills), a disorder resembling human "Chronic Fatigue Syndrome", urinary tract infections, urinary tract stones, brittle diabetes mellitus, lymphangiitis, and proteinuria, comprising administering to the animal a therapeutically effective amount of an antibiotic effective against bacteria identified in a sample of urine according to the method of claim 7.-- 16) A method of diagnosing and treating arthritis and related disorders in a horse or other animal comprising:

(a) obtaining a urine sample from the animal;

(b) preparing the urine sample for microscopic examination by centrifuging the sample at about 3500 - 11000 times gravity, separating the sediment from the supernatant, spreading the sediment on a surface, washing the sediment with a lipid solvent to remove any lipid components of the sediment, fixing, and staining the washed sediment;

(c) microscopically detecting and identifying any bacteria or bacterial fragments in the stained sediment;

(d) administering to the horse or other animal a therapeutically effective amount of an antibiotic effective against the bacteria identified in step (c); and

(e) monitoring the treatment by periodically repeating steps (a), (b), and (c) while continuing the treatment of step (d) until the performance of step (c) shows the substantial absence of the identified bacteria. --

17) A method as identified in Claim 16, wherein the antibiotic amount is at least 600 mg./day. --

18) A method as defined in Claim 16, wherein the antibiotic is selected from the lincosamine group.

19) A method of treating a horse or other animal having the symptoms of arthritis or a related disorder including obtaining a urine sample from the horse, preparing the urine sample for microscopic examination by centrifuging the sample, separating the sediment from the supernatant, and spreading the sediment on a surface, the improvement comprising:

(a) washing the sediment with a lipid solvent to remove any lipid components from the sediment;

(b) fixing and staining the washed sediment;

(c) microscopically detecting and identifying any bacteria or bacterial fragments in the stained sediment; and

(d) administering to the horse a therapeutically effective amount of an antibiotic effective against the detected and identified bacteria. --

20) A method as defined in Claim 19, wherein the antibiotic amount is administered orally at a dosage of at least about 600 mg/day. --

21) A method as defined in Claim 19, wherein the antibiotic amount is administered parenterally at a dosage of at least about 1200 mg/daγ. --

22) A method as defined in Claim 21, wherein the antibiotic is selected from the group consisting essentially of clindamycin and lincomycin. --

23) A method of treating a horse or other animal having the symptoms of arthritis by the steps of:

(a) determining the. presence of bacteria or bacterial fragments in a urine sample obtained from the horse according to the method of claim 3; and

(b) administering to the horse at least 600 mg/day of an antibiotic which is effective against any bacteria detected by step (a).

24) A method of treating arthritis in a horse or other animal, comprising:

(a) collecting a sample of urine from the horse or other animal;

(b) detecting any abnormal bacteria or bacterial fragments in the sample of urine according to the method of Claim 3;

(c) administering a therapeutically effective amount of an antibiotic effective against the bacteria; and

(d) monitoring the urine for the presence of bacteria or bacterial fragments, according to the method of claim 3, to determine efficacy of the treatment

25) The method of Claim 24, in which one or two antibiotics are selected from the group consisting of lincomycin, clindamycin, other lincosamines, cephalexin, cephradine, cefazolin, cephalothin, chloramphenicol, novobiocin, the quinolones, fusidic acid, metronidazole, and penicillins. 26) The method according to Claim 25, further comprising administering another antibiotic selected from the group consisting of streptomycin, tobramycin, kanamycin, neomycin, and amikacin in combination with the first antibiotic.

27) A method for treating arthritis and related diseases in a horse or in other animals, comprising administering to a horse a therapeutically effective amount of an antibiotic effective against Gram positive cocci.

28) The method according to Claim 25, in which the amount of antibiotic comprises about 600 to 18,000 mg per day.

29) The method according to Claim 27, in which the amount of antibiotic comprises about 600 to 18,000 mg per day.

30) A method for preservation of a sample of urine, comprising adding to the sample an effective amount of liquefied phenol.

31) The method according to Claim 30, in which the amount of liquefied phenol comprises about 1% by volume.

32) A kit for detecting Gram positive bacteria associated with arthritis and related diseases in a horse or other animal, comprising the following components:

(a) an antibody specific for the Gram positive bacteria or a component of said bacteria; and

(b) a substrate capable of reacting with an antibody-antigen complex such that its reaction can be monitored; in which the antibody and substrate are present in relative amounts sufficient for the determination of the Gram positive bacteria.