5       Laboratory diagnosis of bacterial disease

 

Laboratory investigation of bacterial disease is necessary for the identification of the aetiological agent and sometimes for determining the antimicrobial susceptibility of bacterial pathogens. A full clinical history, including the age and sex of the species affected together with the number of animals involved and any treatment administered, should accompany the specimens. A tentative clinical diagnosis should be included.

Care should be taken in the selection, collection and submission of specimens to the laboratory. Ideally, specimens should be obtained from live animals before administration of antimicrobial therapy. Samples from dead animals should be collected, if possible, before putrefactive changes occur. Procedures which minimize contamination should be used during specimen collection. Samples must be submitted in separate leak-proof containers, including secondary and tertiary packaging if submitted by post or courier. Each container should be labelled with the identity of the animal, the type of specimen and the date of collection.

If bacteria are present in large numbers, examination of stained smears prepared directly from clinical specimens may indicate the presence of pathogens; cultural and biochemical characteristics and immunological and molecular methods are used for specific identification. Staining methods routinely used in diagnostic bacteriology are presented in Table 5.1. The Gram staining method is employed for the majority of pathogens. The Ziehl–Neelsen stain is used to detect acid-fast bacteria. Coxiella burnetii, Brucella species, Nocardia species and chlamydiae can be demonstrated in smears using the modified Ziehl–Neelsen stain. The fluorescent antibody staining method gives rapid specific identification of bacterial pathogens in smears and cryostat tissue sections.

Table 5.1 Routine staining methods for bacteria.

Method	Comments
Gram stain	Widely used for the routine staining of bacteria in smears. Gram-positive bacteria are stained blue by the crystal violet which is retained in their cell walls despite decolorization. In contrast, Gram-negative bacteria, which do not retain the crystal violet, are counterstained red
Giemsa	Useful for demonstrating Dermatophilus congolensis, rickettsiae and Borrelia species, which stain blue
Dilute carbol fuchsin	Especially useful for recognizing Campylobacter species, Brachyspira species and Fusobacterium species, which stain red
Polychrome methylene blue	Used for the identification of Bacillus anthracis in blood smears. The organisms stain blue with distinctive pink capsules
Ziehl–Neelsen stain	Hot concentrated carbol fuchsin which penetrates mycobacterial cell walls is retained after acid-alcohol decolorization. The red- staining bacteria are described as acid-fast or Ziehl–Neelsen positive
Modified Ziehl–Neelsen stain	Unlike the Ziehl–Neelsen stain, this method employs dilute carbol fuchsin with decolorization by acetic acid

The culture medium, atmospheric conditions and other requirements essential for bacterial isolation are determined by the characteristics of the suspected bacterium. Routine isolation of many pathogens involves inoculation of blood agar and MacConkey agar plates followed by incubation for 24–48 hours. Media used in diagnostic bacteriology are indicated in Table 5.2.

Table 5.2 Laboratory media commonly used for the isolation and presumptive identification of bacterial pathogens.

Medium	Comments
Nutrient agar	A basic medium on which non-fastidious bacteria can grow. Suitable for demonstrating colonial morphology and pigment production; also used for viable counting methods
Blood agar	An enriched medium which supports the growth of most pathogenic bacteria and is used for their primary isolation. Allows the recognition of haemolysin produced by bacteria
MacConkey agar	A selective medium containing bile which is especially useful for isolation of enterobacteria and some other Gram-negative bacteria. Allows differentiation of lactose fermenters and non-lactose fermenters. Colonies of lactose fermenters and the surrounding medium are pink
Selenite broth, Rappaport–Vassiliadis broth	Selective enrichment media used for the isolation of salmonellae from samples containing other Gram-negative enteric organisms
Edwards medium	A blood agar-based selective medium used for the isolation and recognition of streptococci
Chocolate agar	Heat-treated blood agar which supplies special growth requirements (X and V factors) for the isolation of Haemophilus species and for the culture of Taylorella equigenitalis
Brilliant green agar	An indicator medium for the presumptive identification of Salmonella species. Salmonella colonies and surrounding medium have a pink colour

Plates should be inoculated using a streaking technique which facilitates growth of isolated colonies. This is an essential step for the identification of pathogens in clinical specimens which may contain microbial contaminants. In addition, clinical specimens from sites normally populated with commensal organisms, such as the upper respiratory or gastrointestinal tract, may yield a mixed flora on culture. Identification of the suspected pathogen may involve quantification to establish the dominant organism in the specimen and/or testing for virulence attributes to prove pathogenicity. Definitive identification of a potential pathogen involves subculture of an isolated colony to obtain a pure growth which can then be subjected to biochemical or other tests. Morphological characteristics and biochemical tests allow presumptive identification of a bacterial pathogen. Biochemical tests relate to the catabolic activities of bacteria and an indicator system is usually employed to demonstrate utilization of a particular substrate.

Immunological techniques such as fluorescent antibody staining can be used for identifying bacterial pathogens. Serotyping is based on the immunological identification of surface antigens on pathogens such as Escherichia coli and Pasteurella multocida.

The fact that a particular bacteriophage (phage) is specific for a limited number of susceptible strains of bacteria allows differentiation by phage typing. This method is commonly used to differentiate isolates of Staphylococcus aureus and also serotypes of Salmonella Typhimurium.

Selected molecular techniques which can be used for the detection and typing of pathogenic bacteria are described in Chapters 4 and 6.