Common Indications

Cytologic examination of exudates, blood film, tissue imprint, aspiration biopsy, or wet mount of hair is indicated when bacterial and fungal diseases (and occasionally rickettsial and viral diseases) are suspected.

Advantages

Cytology is inexpensive and readily available and may allow rapid confirmation and identification of an infectious agent. It assists in establishing normal flora and contaminants versus infection (e.g., interpretation of relative numbers of bacteria and yeasts in the ear canal). Cytologic examination also permits visualization of relative numbers of organisms at the time of collection (culture results may be misleading in terms of fast- or slow-growing bacteria).

Disadvantages

Infectious agents cannot always be found (e.g., ehrlichiosis, haemoplasmosis, infections with numbers of organism that are below sensitivity level of cytology). Sometimes a presumptive cytologic diagnosis must be confirmed by other methods (e.g., histopathology, culture, PCR assay), and cytology is of limited value in detecting viral inclusions except in brief viremic stages of canine distemper.

Common Indications

Culture and antimicrobial susceptibility are indicated in most suspected bacterial diseases (Box 15-1), especially when clinical syndromes have been resistant to medications. Remember: Skin and mucosal surfaces have a resident microflora (Box 15-2); therefore care must be taken to avoid contamination.

Advantages

Culture and antimicrobial susceptibility usually allows the most effective treatment to be administered.

Disadvantages

Culture requires time for agents to grow; also, some organisms are fastidious or have special culture requirements. Other disadvantages are the expense and the ease of contaminating or making inactivate cultures, rendering results worthless.

Body Cavities

The site of skin puncture should be prepared as for blood culture (see discussion in Cardiovascular System). If pyothorax or peritonitis seems likely but fluid cannot be aspirated, lavage (see Chapter 10) is indicated. Because mixed infections are common and pure anaerobic infections may occur, aerobic and anaerobic cultures should be performed.

Cardiovascular System

Blood cultures are indicated in suspected bacterial endocarditis or septicemia. A large vein prepared surgically with sequential iodine and alcohol scrubs is used for three blood culture specimens obtained during a febrile episode over a 24-hour period in dogs with suspected endocarditis. Culture of fewer than three specimens significantly decreases the chance of positive results. At least 5 ml of blood is placed directly into a transport medium that will support the growth of aerobic and anaerobic bacteria, and it is incubated at 20° C for 24 hours. Clotted blood or blood containing ethylenediaminetetraacetic acid (EDTA) or citrate are unacceptable because this decreases isolation of organisms (Bartonella spp. are exceptions; they can be cultured from EDTA tubes; see Bartonellosis sections). If a patient is critically ill and sepsis is suspected, three cultures should be obtained over 1 to 3 hours before antimicrobial therapy is instituted. Because the urinary system is a common portal of entry for bacteria into the body, urine is often cultured in patients when the source of septicemia or bacterial endocarditis is unknown.

Central Nervous System

Bacterial infection of the central nervous system (CNS) is uncommon. Even when infection occurs, low numbers of organisms make cytology and culture low-yield procedures. If increased numbers of neutrophils and increased protein are detected in CSF (see Chapter 14), however, aerobic and anaerobic bacterial culture and antimicrobial susceptibility testing are indicated. CSF samples should be placed in transport media and delivered to the laboratory as soon as possible. Aerobic and anaerobic bacterial culture should be performed when bacterial infection of the CNS is suspected.

Eye

Conjunctival culture should be performed before topical anesthesia or application of fluorescein stain by rolling a moistened sterile swab over the conjunctiva. Ocular paracentesis is necessary for intraocular culture.

Gastrointestinal Tract

Primary bacterial gastroenteritis occasionally occurs. Salmonella spp., Campylobacter spp., C. perfringens, and Escherichia coli are agents that can be involved. These organisms can also be isolated from normal animals, however. Salmonella spp. and Campylobacter spp. can cause small or mixed bowel diarrhea; C. perfringens is usually associated with large bowel diarrhea. Approximately 2 to 3 g of fresh feces should be submitted to the laboratory for optimal results. If delayed transport of feces to the laboratory is expected, the clinician should consult the laboratory for appropriate transport media. Because these organisms have special culture requirements, the laboratory must be notified of the suspected pathogen. A positive culture for C. perfringens does not prove it was the cause of disease because not all C. perfringens produce enterotoxin. Conversely, not all enterotoxin-positive animals have diarrhea. Thus culture or PCR for C. perfringens should be combined with enterotoxin measurement (See Chapter 9).

Genitourinary Tract

Urine obtained by cystocentesis is preferred for urine culture. If a patient is severely thrombocytopenic (<50,000/µl), or if cystocentesis cannot be performed, catheterization or a midstream-voided sample is acceptable (quantitative culture is needed). Isolation of bacteria should always be assessed concurrently with the urine sediment. Rarely, difficult-to-diagnose urinary tract infections require maceration and culture of a bladder wall biopsy specimen. Calculi should be crushed with a sterile mortar and pestle and cultured. Culture for Mycoplasma spp. or Candida spp. should be considered if pyuria is identified in the absence of calculi, masses, and aerobic bacteria. Leptospire spp. infection should also be considered in these canine patients.

Culture of the third fraction of an ejaculate (preferred) or prostatic massage is recommended for prostatic culture. Culture of the second fraction of an ejaculate is recommended for testicular culture. Culture of prostatic or testicular material retrieved by aspiration or biopsy can also be performed. Prostatic massage and closed prostatic aspiration or biopsy should be avoided in dogs with suspected prostatic abscesses. Obtaining distal urethral specimens for quantitative culture before and after ejaculation may help avoid confusion caused by urethral contamination. Anaerobic culture of urine or prostatic fluid is rarely useful.

Integument and Ear

In superficial pyoderma, hair is clipped from the surrounding area, but disinfection is not attempted. A pustule is ruptured with a sterile fine-gauge needle, and a swab of pus is cultured. In deep pyoderma, hair surrounding the lesion is clipped and the area is disinfected with an antiseptic. The lesion is squeezed to express exudate, which is collected on a swab. Gloves should be worn.

For culture of ears, a sterile otoscope cone is inserted to the level of the horizontal canal and the ear is swabbed through the cone. When middle-ear infection is suspected, the animal is anesthetized and material for culture is retrieved by myringotomy by penetration of the tympanum with a sterile CSF needle placed through a sterile otoscope cone.

Musculoskeletal System

No normal flora exists in musculoskeletal tissues. Dogs with radiographic evidence of discospondylitis should be evaluated for Brucella canis and Bartonella spp. infections (see Diagnostic Tests for Select Bacterial Infections). Intervertebral joints can be cultured after fluoroscopically guided aspiration or when decompressive spinal surgery is required. Most cases of discospondylitis develop after hematogenous spread of bacteria from an extravertebral source. Blood and urine are commonly cultured from patients with discospondylitis; Staphylococcus spp. are commonly involved.

Dogs or cats with suppurative arthritis (with or without cytologic visualization of bacteria) should have the synovial fluid cultured for aerobes and Mycoplasma spp. (see Chapter 10). Likelihood of positive culture results increases if the synovial fluid contains degenerative neutrophils. L-form bacteria usually cannot be grown from joint fluid via routine culture techniques. Synovial biopsy for culture plus histopathologic evaluation for L-form bacteria is more sensitive than only culture of fluid. Borrelia burgdorferi is almost never isolated by routine culture from joints of dogs with Lyme disease. Use of PCR assays on synovial fluid to amplify DNA of Mycoplasma spp., B. burgdorferi, A. phagocytophilum, and Ehrlichia spp. may prove to be an effective way of documenting the presence of the organisms in the joints of affected animals, but objective data are lacking.

In osteomyelitis, culture of fistulous tracts is less sensitive than culture of affected bone. Culture for infectious myositis is seldom performed unless suspicion for an anaerobic infection (e.g., Clostridium spp.) is based on foul odor, subcutaneous (SC) emphysema, or empyema. The clinician can better evaluate for other infectious myopathies (e.g., toxoplasmosis, leptospirosis) using serologic testing or PCR assays.

Respiratory System

Lower airway specimens are best obtained by transtracheal aspiration or bronchoalveolar lavage during bronchoscopy. Fine-needle pulmonary aspiration biopsy can be used but carries more risk (see Chapter 11). Bacteria can be isolated from the trachea in some clinically healthy dogs. These bacteria are probably transient; common isolates are listed in Box 15-2. Because many organisms isolated from normal dogs have also been associated with lower respiratory tract inflammation, all transtracheal aspiration samples should be evaluated by culture, antimicrobial susceptibility, and cytology. With cytology, the clinician should look for squamous cells coated with bacteria (which indicate oropharyngeal contamination) (see Figure 11-12). Bacteria should not be considered significant unless accompanied by neutrophilic inflammation. Mycoplasma spp. have been isolated in pure culture from lower airways of patients with clinical signs of respiratory disease.^18,71 Culture for Mycoplasma spp. should be performed on all transtracheal aspiration samples; these samples need to be transported to the laboratory in Amies medium or modified Stuart bacterial transport medium. Mycoplasma spp. culture should be specifically requested. Amplification of Mycoplasma spp. DNA from airway secretions by PCR assay can also be used to document infection.

Nasal specimens are best obtained from nasal lavage or core biopsy, or by passing a swab through a sterile otoscope cone (see Chapter 11). The clinician can best obtain pharyngeal specimens using a guarded swab taken during pharyngoscopy. Nasal and pharyngeal cultures can be difficult to interpret because of extensive normal flora in the nasal cavity and nasopharynx (see Box 15-2).

Analysis

Blood agar plates grow most routine bacterial pathogens. A biplate containing blood agar and MacConkey agar is frequently used. The common anaerobic culture medium is thioglycolate. The decision to perform in-office testing instead of using a commercial laboratory is based on case load, available equipment, and expertise.

Sensitivity Testing

Sensitivity testing gives an in vitro estimation of suitability of a given concentration of an antimicrobial agent. Two techniques are used: (1) the dilution test and (2) the disk diffusion test.

Dilution Test

This test is quantitative and determines the least amount of antimicrobial agent needed to prevent growth of a microorganism (minimum inhibitory concentration [MIC]). Quantitative susceptibility testing is indicated when antimicrobial dosing schedules need to be monitored closely (e.g., gentamicin) or when disk test results are inapplicable, equivocal, or unreliable (e.g., slow-growing organisms, confirmation of susceptibility to polymyxins, confirmation of susceptibility or resistance to given doses of aminoglycosides). Other indications include anaerobes and testing for synergism or antagonism between antimicrobials.

Advantages

The dilution test may be effective even though disk diffusion techniques suggest otherwise (e.g., antibiotics concentrated in urine).

Disadvantages

Disadvantages of the dilution test include expense, inability to perform the test in the office, and need to determine if required concentrations of a certain antibiotic are feasible. Ideally, blood concentrations of drugs should be more than 4 times the MIC and urine concentrations 10 to 20 times the MIC. MIC sensitivity for topically administered antimicrobials is seldom determined because these methods are based on blood or urine concentrations.

Disk Diffusion Test

This is the most widely used method in clinical practice (i.e., Kirby-Bauer technique). A zone of inhibition of bacterial growth is noted around a disk containing a fixed amount of antibiotic. The procedure is qualitative and allocates organisms to the sensitive (susceptible), intermediate (indeterminate), or resistant category.

Advantages

Advantages of the disk diffusion test are its simplicity and suitability for most routine cultures, that it can be performed in the office, and its applicability for rapidly growing organisms (e.g., Enterobacteriaceae, Staphylococcus aureus).

Disadvantages

This test is not suitable for slow-growing organisms and anaerobes. In addition, there is inaccuracy in predicting susceptibility of poorly diffusing antibiotics (e.g., polymyxins), and factors that influence the test (e.g., pH and thickness of the medium, concentration of organisms, incubation time) must be standardized. It is imperative that proper procedures be followed to avoid errors in diagnosis.

Artifacts

Artifacts result from improper sample collection (i.e., wrong sample, contamination), improper sample transport, failure to notify the laboratory of suspected pathogens (e.g., Salmonella spp., anaerobic bacteria, Campylobacter spp., Mycoplasma spp.), recent antibiotic treatment, and culture for a secondary rather than a slow-growing primary pathogen (i.e., insufficient duration of culture). Failure to grow fastidious anaerobes may be caused by short, seemingly insignificant exposure to oxygen or failure to use prereduced culture media.

Interpretation

Recognizing normal flora (see Box 15-1) is necessary for correct interpretation. Preliminary identification is expected in 18 to 24 hours, and antibiotic sensitivity is reported in 36 to 48 hours. Most aerobic and facultative organisms are identified within 5 days; identification of anaerobic organisms or Mycoplasma spp. may require an additional 2 to 3 days.

Bacterial pathogens commonly isolated from various body systems are listed in Box 15-2. The overlap between resident and pathogenic organisms should be noted.

Staphylococcus pseudintermedius is the major pathogen isolated from the skin of dogs with pyoderma. Gram-negative organisms are likely to be contaminants in superficial pyoderma and secondary to S. pseudintermedius in deep pyoderma.

Primary bacterial rhinitis is rare in dogs and cats but can result from infection with Bordetella bronchiseptica, Mycoplasma spp., and Chlamydophila felis (cats). Primary bacterial pneumonia can result from B. bronchiseptica or Mycoplasma spp., whereas other organisms are usually secondary to viral infections or aspiration.

Bacterial growth from urine obtained by cystocentesis is significant because the bladder is normally sterile. Urine cultures, however, are best interpreted in conjunction with a urinalysis. If growth occurs despite absence of significant pyuria (see Chapter 7), sample contamination, improper sample transport, or diseases causing immune suppression (e.g., hyperadrenocorticism, diabetes mellitus, FIV infection) must be considered. In quantitative culture of urine obtained by catheterization or midstream voiding, greater than or equal to 100,000 colonies/ml is significant. Lower concentrations may be significant in chronic infections or in females. In samples of prostatic fluid obtained by ejaculation, infection is diagnosed if the specimen contains greater than or equal to 100 times more bacteria than the urethral sample.³⁴ Culture of prostatic aspirates may be more accurate.

Blood cultures can be difficult to interpret. False-positive results are caused by contamination with normal cutaneous microflora, including Corynebacterium spp., Bacillus spp., coagulase-negative staphylococci, anaerobic diphtheroids, streptococci, and Clostridium spp. Isolation of the same organism from two or more cultures strongly suggests that it is pathogenic, whereas growth in only one culture is less certain unless it is a pathogenic bacterium unlikely to be a contaminant.

CSF and synovial fluid are normally sterile; any growth in an aseptically obtained sample is significant.

Occasional Indications

To date, most cats with clinical bartonellosis have been infected with Bartonella henselae. However, B. clarridgeaie, B. koehlerae, and B. quintana have been identified in some clinically ill cats.^13,14 Bartonella henselae, B. clarridgeaie, and B. koehlerae are transmitted among cats by Ctenocephalides felis and so cats with a history of flea infestation are more likely to be infected.²⁰ Most cats exposed to Bartonella spp. maintain subclinical infections; however, fever, uveitis, lymphadenopathy, endocarditis, myocarditis, hematuria, and hyperglobulinemia have been documented convincingly in experimentally infected or naturally exposed cats. Results have been equivocal for a link between Bartonella spp. infection and gingivitis or stomatitis.²³ Bartonella testing may be indicated in cats with one or more of these clinical syndromes for which another explanation is not readily apparent.

Analysis, Artifacts, and Interpretation

Circulating antibodies are detected by immunofluorescent antibody assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blot immunoassay.^13,49 Results of these assays prove previous exposure to a Bartonella spp., but as serologic cross-reaction occurs between B. henselae, B. clarridgeaie, and B. koehlerae, assays based on B. henselae antigens cannot differentiate between the agents. Current Bartonella spp. infection is proven by culture or PCR assay results. Results of some PCR assays or genetic sequencing can be used to identify the species of Bartonella involved with the infection.⁴³ Cats can be infected with more than one Bartonella spp. concurrently. Detection of local antibody production by the eye and documentation of Bartonella spp. DNA in aqueous humor has been used to document uveitis as a result of bartonellosis.⁵¹ (See Appendix I for laboratories for infectious diseases.)

Up to 93% of healthy cats exposed to C. felis can be seropositive, and so antibody tests results alone cannot be used to prove clinical bartonellosis.⁶⁷ Between 3% and 15% of seronegative cats are bacteremic, and so antibody test results cannot be used to exclude Bartonella spp. infection from the differential list. Positive blood culture or PCR results prove current infection but do not document clinical illness. Repeated bacteremia has been detected in experimentally inoculated and naturally infected cats; therefore a single negative blood culture or PCR result does not exclude infection.⁴⁷ Because of these findings, it is currently recommended to combine serologic test results with those of blood culture or PCR assay results when evaluating clinically ill cats for bartonellosis. (See Appendix I for laboratories for infectious diseases.) If test results are positive and there is no better explanation for the cause of illness, treatment may be indicated. To date, there has been no proven clinical benefit to following Bartonella spp. assay results after positive response to therapy.

Because serologic test results do not accurately correlate with presence of bacteremia and individual culture or PCR assay results can be falsely negative, there is no indication for testing healthy cats for Bartonella spp. infection.^14,46 The Centers for Disease Control and Prevention recommends maintaining flea control and avoiding bites and scratches to avoid bartonellosis.⁴⁵ Healthy cats used for blood donors should be seronegative and culture or PCR negative and should be maintained on flea control products.⁸¹

Occasional Indications

Dogs from endemic areas or with an appropriate travel history with unexplained myocarditis, endocarditis, granulomatous lymphadenitis, cutaneous vascular disease, hemolytic anemia, polyarthritis, idiopathic effusions, granulomatous meningoencephalitis, granulomatous rhinitis, or thrombocytopenia should be considered for Bartonella vinsonii testing.^12,13 The full spectrum of B. henselae–associated illnesses in dogs has not been determined but appears to be similar to that for B. vinsonii.^13,30 Based on seroprevalence studies, rural dogs with fleas or ticks are most likely to be exposed to Bartonella spp.

Analysis, Artifacts, and Interpretation

Circulating antibodies against B. vinsonii and B. henselae are most commonly detected by IFA. Infection can be documented by culture or PCR assay result. (See Appendix I for laboratories for infectious diseases.)

Serologic cross-reactivity between B. vinsonii and B. henselae is variable; therefore individual assays for both agents are indicated. Antibodies can be detected in dogs with and without clinical signs. Seronegative test results make clinical illness caused by B. vinsonii or B. henselae less likely. However, many infected dogs have been falsely negative for Bartonella spp. antibodies. Thus the current recommendation is to combine serologic test results with PCR assay or culture. Bartonella spp. are more difficult to amplify or culture from dog blood than cat blood. The gold standard test for documentation of Bartonella spp. infection is the combination of culture on BAPGM media with PCR assay (Galaxy Diagnostic, Inc).^25,59 (See Appendix I: Select Laboratories for Infectious Disease.)

As in cats, no test result proves clinical bartonellosis in dogs, but if test results are positive and there is no better explanation for the cause of illness, treatment may be indicated.