veterinary clinical microbiology: part i

Clinical Microbiology Newsletter Vol. 12, No. 20 October 15, 1990

Veterinary Clinical Microbiology: Part I

Patrick McDonough, Ph.D. New York State School of Veterinary

Medicine Ithaca, NY

Andrew B. Onderdonk, Ph.D. Tufts University Schools of Medicine and

Veterinary Medicine Boston, MA

The Veterinary Clinician Although the task of the veterinary clinician is the same as for a human clinician, diagnosing and treating disease, there are many differences between these two groups. The average veterinary clinician works in a two-person practice, has some “in house” laboratory capabilities, and uses commercial laboratories for much of the diagnostic work not performed at their own practice. The average dollar value of this laboratory work is approximately $500/ month per clinician at current market prices. Unlike human diagnostic laboratories , veterinary diagnostic laboratories are not restricted by mandatory license procedures in most states. This means that the quality of the work performed varies greatly, depending on the laboratory used. In addition, the veterinary clinician does not have a large group of specialists to refer difficult or unusual cases to. Currently, there are 27 veterinary schools in the United States, each with a diagnostic reference laboratory of some sort. Perhaps 150 to 200 “centers” in the country represent large multi-person practices with coverage in areas such as radiology,

infectious diseases, cardiovascular diseases and specialized orthopedic sur- gery. In short, the X0,000 veterinary clinicians in practice in this country must rely to a much greater extent on their own skills and resources for the diagnosis and treatment of disease than do their human clinician counterparts.

Veterinary clinicians often see their patients when they are much more de- bilitated than most humans seeking primary medical help, since medical care is dependent on a second party (the pet owner or farmer) becoming aware of the symptoms of disease. In general, because there is no health care insur- ance, the cost of providing medical care is a central issue for even minor health care problems. The average veterinary clinician who owns his/her own practice makes approximately $45 ,OOO/ year. Much of the care provided is on an “outpatient” basis, with hospital- ization reserved only for surgical, crit- ical care, and emergency situations.

Because cost is a central issue to the practice of veterinary medicine, veterinary clinicians tend to rely on fewer diagnostic procedures and less costly tests than clinicians treating human patients. Many tests that would be or- dered routinely for a human patient are considered optional by veterinary clinicians. In addition, even such routine sampling procedures as obtaining a urine sample, culturing a wound, or drawing blood can become nonroutine or require anesthesia depending on the animal species. Imagine trying to draw blood from a large dog who does not

cooperate or understand the need for sticking a needle into one of its veins!

Perhaps the greatest challenge for the veterinary clinician is the need to have detailed knowledge about the anatomy, physiology, normal microflora, and common diseases of different animal species. Normal chemistry and hematologic values are often age, strain, and species specific. Certain drugs can be used in some species and not in others. The spectrum of disease causing agents is different, depending on whether one is working with cats, dogs, horses, or ruminant domestic animal species. In addition, veterinary clinicians need to know which infectious agents are likely to be spread to humans from their animal friends.

In This Issue

Veterinary CIinicaI Microbiology: Part1 . . . . . . . . . . . . . . . . . . . . ...153 The provision of microbiology laboratory services for animals re- quires specialized knowledge

How NCCLS Antimicrobial Susceptibility Testing Standards Are Developed . . . . . . . . . . . . . ...157 Understanding the consensus process

Name Changes and New Organisms . . . . . . . . . . . . . . . . ...160

CMNEEJ 12(20)153- 160,19!90 Elsevier 01%-4399/!m$0.00 + 02.20

S p e c i e s Varia t ion In the New England area the three most common animal species seen in veterinary practice are dogs, cats, and horses. Although there are some agri- cultural interests in cattle, poultry, and swine, the average veterinary clinician usually confines his/her practice to either " smal l animal" which includes dogs and cats, or a mixed practice which generally includes horses and

perhaps dairy cattle as well. Exotic animal practices are generally confined to veterinary schools or zoos, although some practices will see exotic birds, since these are not uncommon as pets. For laboratories accepting samples from veterinary practices, it is important to have some knowledge about the diver- sity of species with regard to laboratory values and commonly encountered diseases. Listed below are some of the areas where species variation occurs.

Normal Serum Chemistry Values A listing of the commonly employed serum chemistry tests and the normal values for our instrumentation (Hitachi 737 system) follow in Table la & b. Note that the normal ranges are quite different for dogs, cats, and horses. For example, dogs generally have amylase values that are considerably higher than those encountered in human specimens. Often, the normal value is higher than the range for automated chemistry equipment standardized for measuring the same values in humans. As with all serum chemistry systems, normal values are instrument dependent. In addition, certain tests commonly used with human serum chemistry profiles are not very useful for animals, such as uric acid. The quality of the serum samples obtained from animals often varies greatly. He- molysis is much more common and may interfere with certain test results. Some species, such as the horse, have a much higher concentration of fibrin

TABLE la. Veterinary reference values- -serum chemis t r ies

Assay Canine Feline Equine Units

Glucose by GLUHK 65-120 70 - 120 80 - 110 mg/dl BUN 6 - 24 17- 30 8 - 20 mg/dl Creatinine 0.4-1.4 0 .6-1.6 1.2-1.9 mg/dl

Total protein 5 .2- 7.2 5.3 - 7.2 5.3 - 7.7 g/dl Albumin 2.4-4.3 2.6-3.9 2.4-3.6 g/dl Calcium 9.5-12.0 9.4-11.2 11.3-13.5 mg/dl

Phosphorous 3 .3- 6.8 4.0 - 7.0 2.8 - 5.2 mg/dl Alkaline phosphatase 20 - 200 20- 220 140 - 395 U/L GGT 1.2-10.0 0-10.0 8.0-20.0 IU/L

SGOT/AST 10-40 8-35 174-380 U/L LDH 30-190 35- 280 170 - 370 U/L Cholesterol 110- 314 90-150 59-120 mg/dl

Total bilirubin 0.04-0.40 0.08-0.30 0.2-3.0 mg/dl SGPT/ALT 10-70 10-130 3-20 U/L Amylase 400-1400 - - g/dl

CPK 20 - 200 20 - ! 60 10- 350 U/L CO2 17- 24 17 - 24 24 - 36 mEq/L Triglycerides - 20- 100 - mg/dl

Direct bilirubin 0 - 0.30 0-0.30 0-1 .0 mg/dl Uric acid 0 -2 .0 0-1 .0 0 .9- I. 1 mg/dl Sodium (by ISE) 140-151 143-153 130-144 mEq/L

Potassium (by ISE) 3.4-5.4 3.5-5.2 2.8-4.8 mEq/L Chloride (by ISE) 105-120 108-128 100-115 mEq/L Lipase 0 - 200 0 - 100 - IU/L

Calculated Values

Globulins 0 .9-4.0 1.5-4.0 1.7-4.5 g/dl A/G 0.53-3.50 0.56-2.6 0.45-2.1 - AGAP 5 - 30 5 - 30 5 - 30 g/dl B/CR 4.2-60.0 10.6-50.0 4.2-16.6 -

than human serum samples. This can lead to serious instrumentation problems unless the sample is filtered before being analyzed.

Normal Hematology Values Included in Table 1 is a listing of the normal hematologic values for dogs, cats, and horses. Although most automated hematology instruments can count red and white blood cells from

the common animal species, the size of the cells may be different. This often means that the threshold settings for the instrument must be changed, depending on the species evaluated. Addit ionally, calculated hematocrits and hemoglobin values may be misleading. A spun he- matocrit is recommended for certain animal species.

Differential counts can represent a real problem for individuals who have

NOTE. No responsibility is assumed by. the Pu.blisher .for any injury a~l/.or dmnal~e to .person,, or property, as a matter of pmduc_ts, liability., neglise .m:¢ or _otherwi.~,_o,t from any .u~ or ope.raa~ of any methods, p..~uets, ins~cuons or ,d~s.cont~rwa m ~ ~ n~.*. ~o s u ~ . ~ t .or ~ s i v a ~. emy~ ~ out t~. . , .m t~ ~ s j .~m~!, risk is jnstifa~d. Because of raptd advances m the .m¢~l. teal sciences, we recommena tlmt tla¢ ~ t vcnncatmn ol magnoses aria orug aosages snoum ~¢ numc. utscusstons, tews and recommendations as to medical procedures, choice of drugs and drug dosages are the re.sponsththty of the authors.

Clinical Microbiology Newsletter (ISSN 0196-4399) is issued twice monthly in one indexed volume by Elsevier Science Publishing Co., 655 Avenue of the Americas, New York, NY 10010. Subscription lmices per year:. $98.00 including postage and handling in the United States, Canada, and M ex.tco.. Add $43.00 for p o ~ e m ~e.rnst of ~ , .w~ld:. S c c ~ - ¢ l ~ postage paid at New York, Is/Y, and at additional malting offices. Postmaster: send address changes to Clinical Micromotogy Newsletter, l~lsevler ~¢tcnce l'uollsrung UO., me., o3~ Avenue of the Americas, New York, NY 10010.

154 0196-4399/90/$0.00 + 02.20 © 1990 Elsevier Science Publishing Co., Inc. Clinical Microbiology Newsletter 12:20,1990

TABLE lb. Veterinary hematology reference values

Equine Test Units Canine Feline (hot blooded breeds)

Range Avg Range Avg Range Avg

RBC x 106/cmm 5.5-8.5 6.8 5.0-10.0 7.5 6.8-12.9 9.0 WBC × 103/cmm 6.0-17.0 11.5 5.5-19.5 12.5 5.4-14.3 9.0 HCT (%) 37.0-55.0 45.5 28.0-45.0 37.0 32.0-53.0 41.0 Hgb g/dl 12.0-18.0 15.0 8.0-15.0 12.0 11.0-19.0 14.4 MCV fl 60.0-77.0 70.0 39.0-55.0 45.0 37.0-58.5 45.3 MCH pg 19.5-24.5 22.8 12.5-17.5 1 5 . 5 12.3-19.7 15.9 MCHC g / d l 32.0-36.0 34.0 30.0-36.0 33.2 31.0-38.6 35.2 Reticulocytes (%) 0.0-3.0 1.6 0.4-3.2 1.2 not seen 0

Differential Neutrophil-Sega (%) 60-77 70 35-75 59 22-72 53 Bands (%) 0-3 0.8 0-3 0.5 0- 8 4 Lymphocytes (%) 12 - 30 20 20- 55 32 17- 68 39 Monocytes (%) 3-10 5.2 1-4 3 0-14 4 Eosinophils (%) 2-10 4 2-12 5.5 0-10 3 Basophils (%) rare 0 rare 0 0-4 1 Platelet x 105/cram 2-5 3.0 2-7 4.5 1-3.5 2.25 TSP g/dl 6.0-8.0 6.0-8.0 5.8-8.7

little experience with veterinary samples. Although the cells may ap- pear similar, many of the staining char- acteristics and morphologic markers are different. It would be wise for laboratories handling veterinary samples to send one technologist for training either to a local veterinary school or a veterinary clinician skilled in hematology before attempting to perform differential counts.

Normal Microflora Microbiologically, knowing something about the normal microflora of the various animal species being evaluated is essential. Dogs, cats, and horses each have organisms present normally which are rarely recovered from humans. Conversely, many organisms commonly encountered in humans are rarely isolated from animals. Because most animals are furbearing, the normal skin flora is quite different than that encountered in humans. Moreover, obtaining a superficial swab of the skin is usually an opportunity to contaminate a sample with normal microflora. Listed below are some of the organisms considered to be normal microflora of the various animal species.

Skin: dogs, cats, and horses Micrococcus Staphylococcus epidermidis

Staphylococcus intermedius Staphylococcus aureus Acinetobacter Clostridium sp. Streptococcus sp. Gram-negative rods Diphtheroids Fungi--Aspergiilus, Cladosporium,

Fusarium, Alternaria Oral cavity: dogs, cats, and horses

Staphylococcus sp. Streptococcus sp. Neisseria sp. Lactobacillus sp. Pseudomonas sp. Pasteurella sp. (dogs and cats) Clostridium sp. Bacillus sp. Corynebacterium sp. Actinomyces sp. (horses) Gram-negative enteric bacilli (dogs

and cats)

Intestinal Tract Intestinal microflora is similar to humans, but Bacteroides fragilis is not commonly found. Virtually any microbial species can be isolated. Of particular importance is the finding of C. perfringens in ruminant animals, because this can lead to a variety of enteric disease situations. The ruminant animal represents a special situa- tion and diseases such as Johne's

disease (Mycobacterium) are rarely found in animals other than ruminants.

Anatomy

The anatomic features of the various animal species are often important considerations when dealing with microbiologic problems. As stated above, monogastric animals (dogs, cats, horses) are distinctly different both physiologically and anatomically from ruminant animals such as sheep, cattle, and goats. The f'mding of C. perfringens in a dog or cat would not be considered very important; however, if this organism is isolated from a goat with GI distress, the interpretation would be entirely different. Similarly, finding Gram-negative organisms in the GI tract of dogs, cats, or horses would have no special significance, but the finding of these organisms in the bursa of a seed-eating bird could indicate disease.

Because animals (including dogs and cats) often rely on their teeth as a pro- tective measure, many puncture wounds caused by bites and/or fights result in infections. In cats, the rapid healing rate of the skin often results in a bite wound becoming infected with organisms which are resident in the mouth of other cats. Given this situa- tion, it is not surprising to find Paste-

Clinical Microbiology Newsletter 12:20,1990 © 1990 Elsevier Science Publishing Co., Inc. 0196-4399/90/$0.00 + 02.20 155

uella multocida in many cat bite infections.

Species Specific Diseases As stated above, each animal species has pathogens which are uniquely its own. A few examples of such pathogens are listed below.

Dogs Kennel cough, caused by Bordetella

bronchiseptica Brucella canis Lyme disease--caused by Borrelia

burgdorferi Chronic pyoderma---Staphylococcus

intermedius Heartworm--Dirofilaria immitis Canine distemper--paramyxovirus Canine parvovirus Rocky Mountain Spotted Fever

Cats Feline leukemia virus Toxoplasmosis Feline Infectious peritonitis Pasteurella multocida FIV/FTLV - - symptoms similar to

AIDS Other

C. perfringens enterotoxemia in goats, sheep, and cattle

Mastitis in cattle Rhodococcus equi in horses Actinobacillus Potomac Valley Fever--horses

Zoonmes--Diseases Common to Both Humans and Animals One of the most compelling reasons for isolating and identifying bacterial infections in animals is the potential for zoonotic spread to their human owners. Although we might not con- sider the pet cat or dog to be a signifi- cant potential risk to our own health, many diseases can be acquired from the family pet (or vice versa). Examples of these diseases are salmonellosis, staphylococcal disease, psittacosis, toxoplasmosis, campylobacteriosis, lis- teriosis, and leptospirosis.

In addition, a number of diseases can be acquired from food and fiber animals or their products and are of special concern to laboratories performing microbiologic testing of animals or animal products. These include Salmonella from poultry and

poultry products, Listeria from milk and milk products, Leptospira from infected urine, anthrax from contaminated wool and hides, and Tularemia from infected animal blood.

Importance of Proper Microbial Identification In addition to the obvious public health aspects of veterinary microbiology, it is important to identify suspect pathogens properly so that veterinary clinicians can treat legitimate infections and manage potential herd health problems. It is not adequate, for example, to simply identify a Corynebac- terium isolate to the genus level, because some species are nonpath- ogens, whereas others can cause serious health problems in an entire herd or flock. Moreover, breeders often culture their animals before allowing them to breed to prevent septic abortions and other problems that could mean the dif- ference between a successful breeding season and loss of valuable animals.

Regulatory Testing Certain tests are regulated by the USDA in veterinary medicine, due to the importance of the disease to the entire animal community. The assays for equine infectious anemia, bovine ieu- cosis and anaplasmosis can only be performed by laboratories certified to perform the tests. Similarly, some local diseases are diagnosed only by one or two laboratories because of the associated health risks. For example, in Massachusetts, these include tests for rabies, eastern equine encephalitis, and psittacosis, which are jointly performed by the Tufts Veterinary Diagnostic Laboratory and the State Laboratory Institute in Massachusetts.

When human hospital laboratories are interested in performing some microbiologic testing for veterinary clients, several important questions should be considered. First, and most important, is the laboratory willing to make the commitment to isolate and identify pathogens found in samples obtained from a variety of animal patients? Second, is someone at the laboratory trained to interpret the results of cultures from the various animal species? This is important, not only

from the perspective of antimicrobial agent selection, but from a public health and herd health perspective as well. Finally, is the laboratory willing to give the same priority to samples from animal patients as from human patients? This is important to the veterinary clinician who may be attempting to treat a serious infection on an outpatient basis. If a human clinical laboratory wants to perform some of the routine testing from veterinary practices and refer the rest of the work, several additional considerations are necessary. Since veterinary clinicians are likely to send all work to one loca- tion, a human clinical laboratory must be prepared either to offer "full ser- vice" or subcontract for such services from another laboratory. The information provided below is similar to that we now provide for our veterinary students and clinicians and represents our views regarding sample transport, culture techniques, and interpretation of results. It is not meant to replace the many fine textbooks that deal exclu- sively with veterinary clinical microbiology.

Use of the Veterinary Diagnostic Laboratory

General Considerations When deciding on a choice of veterinary testing services, we always rec- ommend that our veterinary students visit the laboratory site. Certain information is essential, i.e., from what animal species are specimens accepted for workups, what testing services are available, what are the laboratory turn- around times for tests, what is the laboratory's results reporting protocol (are preliminary results reported or only final reports; are reports telephoned, mailed, faxed). Laboratories frequently have various transport media and kits available for use and often will reject specimens that are not submitted properly. Most of these questions should be answered in a laboratory's policy manual with accompanying fee schedule. It is also important to know whether a laboratory participates in a quality assurance program. Many veterinary laboratories are also accredited by the American Association of Veteri-

156 0196~-399/90/$0.00 + 02.20 © 1990 Elsevier Science Publishing Co., Inc. Clinical Microbiology Newsletter 12:20,1990

nary Laboratory Diagnosticians (AAVLD). Laboratories may also be certified by and participate in USDA certification programs for certain tests.

The veterinary diagnostic laboratory is not a "black box" through which any sort of specimen can be submitted and from which a meaningful result and solution to a diagnostic problem can be obtained. The specimens that are re- ceived by the laboratory are the primary limiting factor for the diagnostician. Specimens should be properly chosen, collected, identified, submitted, and shipped. Consulting the laboratory policy manual and calling the laboratory if any questions arise about choice

Editorial

How NCCLS Antimicrobial Susceptibility Testing Standards Are Developed

James H. Jorgensen, Ph.D. Department of Pathology University of Texas

Health Science Center San Antonio, TX 78284

The documents published by the Na- tional Committee for Clinical Labora- tory Standards (NCCLS) are widely used in clinical microbiology laboratories, in the pharmaceutical industry, and are often cited by regulatory agencies on issues of standardization and quality control of antimicrobial agent testing. However, relatively few people understand how the NCCLS guidelines are developed. This article will describe the multidisciplinary consensus process that leads to all NCCLS standards and guidelines.

The NCCLS was founded in 1968 as a forum for issues involving clinical laboratory testing. Since that time the organization has grown immensely in size and scope. It serves presently as the principal organization for estab- lishing clinical laboratory testing standards in the United States. Many individuals probably think of the NCCLS only as a source of guidelines relating to antimicrobial susceptibility

of sample or transport method should be a routine consideration. The laboratory needs as detailed a history as possible, including demographic information, treatment administered (espe- cially antimicrobial agents), vaccination status, clinical signs, duration, and ra- pidity of disease progression. In the case of a herd, flock, colony, or kennel problem, morbidity and mortality information is also useful. In the final analysis, diagnosis and therapy depend on clinical judgement, as well as information that is obtained from physical examination, client history, and laboratory tests that are performed.

Legal considerations also are in-

testing. However, the NCCLS encom- passes virtually all fields of clinical laboratory practice, e.g., clinical chemistry, toxicology, hematology, immunology, and general topics, such as guidelines for protection of laboratory workers from occupational hazards, writing of procedure manuals, and most recently the development of standards for alternate site laboratory testing. Even within microbiology, the NCCLS has developed standards (documents) on culture media quality control practices, testing of fetal bovine serum used for cell culture, as well as several aspects of antimicrobial susceptibility testing. The membership of the organization is comprised of sustaining, professional, governmental, and indus- trial members and corresponding members (principally hospitals and teaching institutions), and a growing list of international corresponding members.

The NCCLS is organized as a series of "Area" committees (e.g., the Area Committee on Microbiology) under the NCCLS Board of Directors. The Area Committees oversee the work of a number of "subcommittees" within their discipline. Thus, the Antimicro- bial Susceptibility Testing (AST) Sub- committee is but one part of the overall organization of the NCCLS. Neverthe- less, the AST Subcommittee produces some of the most widely used documents published by the NCCLS, and has perhaps one of the broadest influ-

volved with testing veterinary specimens. Occasionally, the need arises for a "chain-of-evidence" for diagnostic specimens, particularly in the case of sudden death, suspect toxicology problems, and postsurgical and postvaccination deaths. Diagnostic laboratory accession forms are considered legal documents provided by the laboratory for use by the veterinarian to record veterinary hospital information, patient demographics, clinical signs, differential diagnosis, and tests re- quested.

Part H is continued in 12(21). Selected references also in 12(21).

ences on testing practices of clinical microbiology laboratories, the diagnostic product and instrument manufac- turing industry, and the pharmaceutical industry. These publications include documents describing disk diffusion testing of aerobic bacteria (1), dilution testing of aerobic bacteria (2), and dilution test methods specifically for an- aerobic bacteria (3). Each document contains a detailed description of meth- odology for performing reproducible susceptibility tests (the text of the documents) and a series of tables that list quality control values for numerous antimicrobial agents and guidelines for interpreting the results of tests on clinical isolates. All of these documents are updated frequently (often yearly) as new information becomes available or problems are recognized.

Like all NCCLS endeavers, the AST Subcommittee includes a balanced rep- resentation of voting (and advisory) members from the ranks of clinical laboratories, the federal government, and industry (Table 1). In addition, the AST Subcommittee includes a fourth category of practicing infectious disease clinicians. The various NCCLS subcommittees' efforts are supported by a capable staff of laboratory- and publications-oriented professionals in the NCCLS administrative office in Villa- nova, Pennsylvania.

The AST Subcommittee accom- plishes the majority of its work during semiannual meetings held in the winter

Clinical Microbiology Newsletter 12:20,1990 © 1990 Elsevier Science Publishing Co., Inc. 0196-4399/90/$0.00 + 02.20 157

veterinary clinical microbiology: part i

Documents