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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
Applied Veterinary Bacteriology and
Mycology: Identification of aerobic and
facultative anaerobic bacteria
Chapter 6: Aerobic Gram-positive filamentous bacteria
Author: Dr. M.M. Henton
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
Table of CONTENTS.................................................................................................................................... 1
INTRODUCTION........................................................................................................................................... 2
Table 6.1: Appearance of pathogenic actinobacteria in exudates or tissue smears/sections ....... 3
Table 6.2: Gram-positive bacteria showing slight branching ......................................................... 4
Table 6.3: Gram-positive bacteria showing extensive branching (A= aerobic, F= facultative
anaerobe) ...................................................................................................................................... 6
Dermatophilus Congolensis ..................................................................................................................... 7
Nocardia ................................................................................................................................................. 10
Table 6.4: Hydrolysis and selected biochemical profiles of pathogenic species of Nocardia .... 12
Table 6.5: Differentiation of the causative agents of canine nocardiosis and canine
actinomycosis .............................................................................................................................. 13
Streptomyces ......................................................................................................................................... 13
Table 6.6: Identification of Actinomyces spp. (usually glucose positive) ..................................... 14
REFERENCES ............................................................................................................................................ 15
Table 6.7: Differential properties of A. kentuckyensis, A. lexingtonensis and A. pretoriensis
(agents having caused abortion in mares) compared with previously described species of the
genus Amycolatopsis ................................................................................................................... 16
APPENDIX 1 ............................................................................................................................................... 17
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
INTRODUCTION
The class Actinobacteria comprises a heterologous group of bacteria that have the ability to form Grampositive, branching filaments of less than 1 µm in diameter. Fungi are eukaryotes and their hyphae
(filaments) are greater than 1 µm in width. The main animal pathogens in the class Actinobacteria are in
the genera Actinomyces, Actinobaculum Mycobacterium, Corynebacerium, Arcanobacterium,
Rhodococcus, Nocardia and Dermatophilus. Nocardia is closely related to the genera Corynebacterium,
Mycobacterium and Rhodococcus, all 4 of which belong to the family Corynebacteriaceae.
Some
members of the Actinobacteria are not invasive but inhalation of their spores can cause allergic
pulmonary disease in man and horses and possibly other domestic animals fed or exposed to mouldy
hay in the same way as some fungi.
The general characteristics of the genera Actinomyces, Nocardia, Rhodococcus and Dermatophilus as
well as genera that can be easily confused with them are presented in Tables 6.1, 6.2 and 6.3
Morphological identification
Distinguishing pathogenic Nocardia and Actinomyces from common contaminants such as Streptomyces,
Agromyces and Oerskovia is difficult, particularly in a veterinary laboratory, as many similar contaminants
occur in soil or compost and from animal skin. Identifying unique members such as Dermatophilus is
relatively easy, but the others require specialized techniques such as high-pressure liquid
chromatography analysis to identify cell wall amino acids, sugars and mycolic acids. These techniques
are unavailable to the normal clinical laboratory.
The best approach is to examine the morphology very carefully on a variety of growth media and at
different stages of growth, in the same way that fungi are identified. Both the colonies and stained
smears should be examined. Liquid cultures may also be examined, but the entire tangled mass of
growth may only be present as a pellicle, deposit or flakes, too dense to visualize adequately.
An ordinary light microscope is required, preferably with a long working distance lens, to prevent fogging.
Colonies may be examined directly, at all stages of growth, by placing the closed Petri dish containing a
clear medium on the stage. If a pathogen is suspected, it is better to examine the dish upside down,
through the medium. A rich medium, such as blood or serum agar, is not recommended as this allows
this group of bacteria to grow rapidly and atypical growth may occur. Spores may be absent, the culture
may lack an aerial mycelium or rapid fragmentation may occur. Rapid growth often results in a leathery
colony, difficult to examine. A poor medium, such as tap water agar, oatmeal agar or inorganic salts agar
is required for the more robust species, but nutrient agar may be sufficiently poor for Actinomyces spp.
The bacteria grow slowly on these (7 - 30 days) and therefore the agars should be thickly poured to avoid
desiccation. Growth is more typical on poor media, and it is easier to prepare smears, as the growth is
more butyrous. The disadvantage is that the slower rate of growth takes far more time.
It is important to plan several simultaneous examinations or tests, so that sequential steps are kept to a
minimum.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
The first step is to make sure that the culture is pure. A tangled mass of filaments can easily carry a
contaminant with it. This is best done on a rich medium, streaking to obtain single colonies. Inoculate
pure cultures on a transparent minimal agar in a cross-hatch pattern.
Figure 6.1: Cross-hatch streak plate. View directly under microscope fitted with a long working distance
objective (x25 and x40). Mature hyphae with spores should be looked for in the angles of the streaks.
Table 6.1: Appearance of pathogenic actinobacteria in exudates or tissue
smears/sections
Genus
Streptomyces
Appearance in exudates (pus) and tissue
Granules are rare, but when present are
small (25-150um), white to yellowish,
lobated, sometimes clubbed. Colonies
form a loose mycelium consisting of
Gram-positive and partially acid-fast
branching filaments, which often
fragment into coccoid elements. With
Gram’s stain the filaments often stain
irregularly giving a beaded appearance.
Does not stain with H & E stain.
Actinomyces
Pathogenic strains form yellow, brown
or black granules 1 - 2 mm in diameter,
which stain with H & E, often breaking
up into parallel bands. Branched
filaments do not fragment. Aerial
mycelium characterized by medium to
long chains of conidia, often in spirals or
whorls, which are sometimes acid-fast.
A mixture of small cocci, rods and pear-
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
shaped, Gram-positive bacteria.
Rhodococcus
No granules are present. Diphtheroids
(Gram-positive rods, with traces of
branching). The rods often break up into
coccoid cells. No aerial mycelium
present.
Dermatophilus
Organism commonly found on the under
surface of skin crusts, where it is
typically seen as irregular branched
filaments which divide both
longitudinally and transversely forming
packets of coccoid cells. They are not
acid-fast. Best stain is Giemsa, but
Gram’s stain is satisfactory.
+
d
S
+
+
+
-
+
d
S
+
+
+/(-)
+/(-)
S
d
+/(-)
+
-/(+)
-
+
d
-
Arcanobacterium
+
-/(+)
-/(+)
S
+/(-)
d
+
d
d
-(d)
-
An/CO
Oerskovia
F
Agromyces
F
Rhodococcus
F
Mycobacterium
Propionibacterium
F
Bifidobacterium
Arachia
Aerobic (A)/ Facultative (F)
Anaerobic (An)
Anaerobic, better growth
Catalase
Acid-fast
Gelatine
Motile
Penicillin
Pigment
Nitrate
Urea
O/F glucose
Aesculin
Starch
Casein
Sabouraud’s dextrose
Growth at 55°C
ONPG
Rothia
TEST
Actinomyces
Table 6.2: Gram-positive bacteria showing slight branching
A
A
A
F
F
D
+
+
(+)
R
D
D
D
D
S
+
d
d
-
R
(+)
-
+
+
D
S
+
+
+
+
D
S
+
D
D
d
2
-
-
-
+
S
d
+
d
d
-
d
d
-
d
+
+
-
+
+
Set several sterile coverslips into the agar at angles so that they can be examined at different times
without disturbing the growth near the others (Figure 6.2).
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
Figure 6.2: Inclined coverslips for observing actinobacterial morphology. Inoculate agar plate with
coverslips inserted at an angle. After incubation withdraw coverslips and mount, upper surface down, in a
water-containing wetting agent.
Alternatively, agar blocks on sterile slides covered by sterile coverslips may be inoculated on the sides of
the blocks. The slide is placed in a moist chamber and incubated. It can be regularly examined
microscopically without disturbing the growth (Figure 6.3).
A lipid-rich medium such as egg yolk agar, milk, serum or glycerol is also inoculated for acid-fast staining.
Thioglycollate with gelatine (5%) is useful for determining whether the culture grows well anaerobically
and determines motility and gelatine liquefaction as well. An antibiogram differentiates between members
of this group and fungi. Penicillin is included as a distinguishing test.
Figure 6.3: Slide culture. Thin agar block, cut from poured plate, is placed on a sterile microscope slide and
inoculated, and a sterile coverslip is applied. After incubation in a moist chamber, view slide culture directly
on microscope stage when it should be possible to see the aerial mycelium (A) and the substrate mycelium
(B) within the agar.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
TEST
Nocardia
Streptomyces
Dermatophilus
Actinomadura
Nocardiopsis
Thermomonospora
Saccharomonospora
Saccharopolyspora
Table 6.3: Gram-positive bacteria showing extensive branching (A= aerobic, F=
facultative anaerobe)
Aerobic (A)/ Facultative (F)
A
A
F
A
A
A
A
A
Anaerobic, better growth
-
-
-
-
-
-
-
-
Catalase
+
+
+
+
+
+
+
+
Acid-fast
(+)
- (Spores +)
-
-
-
-
-
-
Gelatine
d
+
+
+
+
+
+
Motile
-
-
+ (spores)
-
-
-
-
-
Penicillin
R
R
S
R
R
S
S
R
Pigment
d
d
+
+
+
+
+
+
Nitrate
+/(-)
+
-
+
d
-
-
d
Urea
+/(-)
d
+
d
+
+
+
+
O/F glucose
+
-
Aesculin
+
+
Starch
d
+
+
d
+
Casein
-/(+)
+
+
+
+
Sabouraud’s dextrose
+
+
-
+
Growth at 55°C
-
-
-
ONPG
+
+
-
d
+
+
+
d
+
+
+
+
+
+
+
-
+
+
+
-
+
-
-
-
Examine the colonies regularly microscopically (once or twice a week if slow-growing, daily if rapid
growing). Either place the agar block slide under the microscope and examine under high dry power
without staining, or remove one of the cover slips angled in the agar. The growth adhering to the cover
slip may be stained if required. Some Actinomyces spp. only branch at a very early stage (spider
colonies) when still so small that they are not visible to the naked eye.
Note any branching, whether most of the branches are right angled or oblique, and whether all the growth
is on or in the agar, or whether an aerial mycelium is formed (Figs. 6.4 and 6.5).
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
Figure 6.4: Streptomyces, Actinomadura, Actinomyces and Nocardia: coccoid, diphteroid fragments of
filaments; aerial mycelium fragments to form unsheathed arthrospore-like units.
Figure 6.5: Agromyces, Dermatophilus, Actinomyces and Rhodococcus species have a rapidly fragmenting
substrate mycelium; the segments become rounded and the colony usually consists of a mass of coccoid
elements.
This is first and best seen in the angles of growth on the cross-hatched plate. Members of this group can
undergo a rod-coccus or rod-coccus-hyphus cycle which means that they are usually seen as cocci
during the resting stage.
Once placed on fresh medium, outgrowths occur so that they resemble rods which may develop into
rudimentary hyphae which fragment again. When nutrients are depleted, cocci are again formed. The
rate of growth as well as the individual isolate determines at which stage each change occurs. As this
rate is difficult to predict, regular examinations will show changes as they occur. Aerial mycelia or
filaments are darker and more retractile than those growing in the agar and may bear spores, the shape,
number and size of which should be noted. As soon as growth is noted on the lipid rich medium, modified
acid fast staining can be done as well as the catalase test.
These few tests should either be sufficient to identify the isolate or indicate whether it is a potential
pathogen or contaminant. Further tests which are helpful in this group are nitrate, urease, acid production
on oxidation and fermentation medium, aesculin, starch or casein hydrolysis, growth on Sabouraud's
agar, growth at 55°C and ONP.
Dermatophilus Congolensis
Dermatophilus congolensis is the agent of cutaneous streptothricosis and Sencobo disease in cattle and
lumpy wool and strawberry foot rot in sheep. Other domestic animals and humans may also be affected.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
The disease is important in countries in southern, central and West Africa, the Caribbean and Israel. The
infection is characterized by the formation of thick crusts which come away with a tuft of hair, leaving a
moist depressed area with bleeding points from capillaries. Morbidity can be high, but mortality is low.
The position of the lesions in the skin varies with predisposing conditions such as water maceration and
heavy infestation with Ambylomma ticks.
Laboratory diagnosis
1.
Specimen collection
Scabs, biopsies or exudates collected from unopened pustules are best. Exudates should be submitted
to the laboratory in sterile tubes or on swabs moistened with sterile water. Biopsy specimens should be
submitted in sterile screw cap bottles without preservative. The samples should be refrigerated if
culturing is to be delayed. Scabs, scrapings and crusts may be transported dry, in sterile tubes or jars
and stored at ambient temperatures.
2.
Direct microscopy of smears
Actively growing organisms are found on the undersides of the scab and in the epidermis at the edge of
the lesion. Collect scabs from different sites because the organisms may be scanty and difficult to
demonstrate. D. congolensis survives for months, even years, in dry scabs but is soon killed in wet scabs
by contaminating bacteria. Make a thick smear from the underside of the scab, if necessary soaking in a
bit of distilled water or saline. Stain with 10% Giemsa for 30 minutes, DiffQuik or methylene blue (1% for
30 seconds). Although Gram’s stain can be used, both the cells of D. congolensis and debris absorb the
crystal violet-iodine stain avidly staining too darkly. A modification is to leave the crystal violet on the
smear for only 2-3 seconds, after which the morphology of the bacterium is easier to see. Giemsa and
Grocott methenamine-silver stains are best for paraffin sections, which often reveal the micro-organism
abundantly in hair follicles, accompanied by large numbers of eosinophilic leukocytes.
The appearance of D. congolensis is unique, in that a presumptive diagnosis can be made by the
examination of glass smears by light microscopy. A search must be made for the typical chains of blue
cocci (zoospores) in two, four or eight parallel rows resulting in a “tram-track” or “cat’s paws” appearance.
Single chains or single cocci resemble other common cocci and cannot be distinguished from them.
Typical chains can be hard to find even from lesions which look active and it is advisable to search
several smears before declaring them negative. If the flakes of scab are treated too roughly when the
smears are made, the filaments will disintegrate and only Gram-positive cocci (zoospores) will be seen.
Table 6.1 and Fig 6.6 show the morphology of D. congolensis.
3.
Culture
Uncontaminated specimens can be directly streaked onto appropriate media. It is best to use the
underside of scabs or crusts or exudates. As scab material often contains many contaminants, Haalstra’s
method for the primary isolation of D. congolensis is recommended:
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
1.
Make a suspension of scab material by grinding with sterile sand and distilled water. Place the
material in 2ml of distilled water in a sterile bijou bottle (or any other suitable bottle) for 3.5 hours at
room temperature.
2.
Place the container with lid removed, in a candle jar at room temperature for 15 minutes.
3.
The motile zoospores are chemotactically attracted to the carbon dioxide-enhanced atmosphere in
4.
Remove a loop full of fluid from the surface and streak on to a blood agar plate.
5.
Incubate in a candle jar at 37°C for 48 to 72 hours. Occasionally five days are required for colony
the candle jar and move to the surface of the distilled water.
formation.
The blood agar plate often shows a mixed growth of skin bacteria with a few D. congolensis colonies.
Colonies are very characteristic, white or yellow (2 - 5 days), raised with an irregular rough surface
(confluent growth may appear mucoid). In addition, they show a narrow zone of beta-haemolysis, pit the
medium and are hard to lift off the surface. No growth occurs on Sabouraud’s dextrose agar. The betahaemolysis is best seen on horse blood agar. These bacteria unlike Nocardia species will grow
anaerobically. Dermatophilus congolensis colonies often produce short aerial hyphae when cultured in a
candle-jar.
Microscopic appearance
Smears from colonies do not show the characteristic “tram-track” appearance seen on direct microscopy.
Gram’s stain reveals a mycelium of Gram-positive branching filaments, some of which are broken down
transversely and longitudinally into chains of cocci i.e. parallel rows of cocci. If only cocci are seen and
D. congolensis is suspected, cultures should be examined at an earlier stage for hyphae.
Biochemical reactions
As the colony morphology and microscopic appearance is characteristic, biochemical tests are usually
not performed. D. congolensis is catalase negative, urease negative and gelatin positive and produces
weak acid from glucose, fructose and maltose. It is indole negative, does not reduce nitrate and does not
attack sucrose, salicin, xylose, lactose, sorbitol, mannitol or dulcitol. It hydrolyzes starch but not xanthine
or tyrosine.
4.
Serological diagnosis
A highly specific immuno-diffusion test has been developed to detect circulating antibodies in chronically
infected cattle but is not used as a routine diagnostic test.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
Fig. 6.6: Developmental cycle of Dermatophilus congolensis
Nocardia
Nocardia infections occur sporadically in domestic and wild animals. They are important because lesions
resemble those of tuberculosis and because they cause tuberculin sensitivity. Nocardia asteroides is a
cause of a purulent thoracic effusion and deep-seated abscesses in cats and dogs and abortions, jaw
abscesses and mastitis in cattle. In other animals various purulent infections are found.
precautions used for Mycobacterium infections should be followed when handling samples.
Safety
The nocardiae are aerobic, catalase-positive, Gram-positive, non-motile, branching filamentous bacteria
that fragment into irregularly shaped rods and cocci. Aerial hyphae are produced. With the exception of
N. amarae, all nocardiae are resistant to lysozyme. As the cell wall contains mycolic acids, these bacteria
often stain partially acid-fast. The taxonomy of Nocardia is rapidly changing at present.
Laboratory diagnosis
1.
Specimens
Specimens should include exudates, aspirates, mastitis milk samples, tissue from granulomas and thin
sections from granulomas in 10% formalin for histopathology. Swabs are not recommended as the
transport media may make identification by smear examination difficult. If swabs must be used they
should rather be moistened with a little sterile water. Samples suspected of containing Nocardia should
not be refrigerated or placed on ice prior to being inoculated onto appropriate culture media. Some
strains of N. asteroides and N. brasiliensis rapidly lose viability when exposed to near freezing
temperatures. Blood cultures may be taken if disseminated disease is suspected. These cultures should
however be incubated for at least two weeks and are often unrewarding.
2.
Direct microscopy
Soft “sulphur” granules (Splendore-Hoepli phenomenon) are not common in exudates from N. asteroides
infections. Gram - and MZN-stained smears should be made from exudates, aspirates, granulomatous
tissue and centrifuged deposits of bovine mastitis milk. As the cell wall of Nocardia species contains
mycolic acids of intermediate size, they are able to retain carbol fuchsin when rinsed with a weak mineral
acid, then 70% of Nocardia isolates will stain partially acid-fast. Acid-fast staining is best seen when
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
cultured on a lipid-rich medium such as egg yolk or milk. Nocardiae are usually not apparent in
haematoxylin and eosin stained sections and are best visualized by the Brown and Brenn modification of
the Gram’s stain.
Nocardia appear as a mycelium of delicate, branching filaments in which staining may be uneven. The
filaments often show fragmentation into coccobacillary elements. They can resemble Mycobacterium
spp. (acid-fast stain) and Corynebacterium spp. (Gram’s stain). They are found both intra- and
extracellularly. They do not form Gram-negative clubs, which differentiates them from Actinomyces.
3.
Culture
Nocardia will not survive the decontamination techniques used to isolate mycobacteria, but they will grow
on blood agar and Sabouraud’s dextrose agar (Oxoid). Inoculate pus and tissue suspensions on blood
agar and Sabouraud, and incubate aerobically at 37°C. For contaminated samples paraffin agar or
buffered charcoal agar used for the isolation of Legionella are effective. Even though Nocardia are seen
on direct smears, they can be problematic to culture and thus plates should be kept for at least two
weeks before being discarded. During this period, the plates should be kept well sealed in order to
maintain a moist environment. This period of extended incubation can also result in the overgrowth of
contaminating bacteria or fungi, which may hide or inhibit the growth of nocardiae. Furthermore,
nocardiae may manifest extremely variable morphologies on different culture media, which may make
recognition difficult. In fact macroscopically, the colonies of Nocardia and Mycobacterium spp. can look
the same on media designed for the cultivation of Mycobacterium spp. Examination of colonies under a
stereo microscope can help one recognize the growth characteristic of nocardiae.
Some Nocardia may become physiologically and structurally altered as they grow within host tissues,
with the result that transferral of these altered organisms may be lethal for them or requires an extended
period to adapt the organisms to the new environment.
Colonies appear after three to five days, raised, white, pink or yellow, smooth or granular, irregularly
folded (cauliflower-like). On sheep or bovine blood agar, many clinical isolates of N. otitidiscaviarum, N.
brasiliensis and rarely N. asteroides initially are small with a narrow zone of alpha-haemolysis around the
colony. After 2 - 7 days, the colony widens, digs into the agar and often has a zone of beta-haemolysis
around it. However, some isolates of Nocardia spp. exhibit only alpha haemolysis and most isolates of N.
asteroides appear to be non-haemolytic on sheep blood agar.
On Sabouraud’s agar colonies are
orange, glabrous, heaped and folded to white or pink, raised and chalky with aerial hyphae.
Stained smears from growth on laboratory media will show coccoid and bacillary elements. The long,
branched filaments are usually not seen from agar preparations but are easily obtained from growth in
broth. Acid-fastness is best seen when the organism is cultured on a high lipid media such as litmus milk
or Løwenstein-Jenssen media. On Sabouraud’s dextrose agar, arthroconidia-like elements occasionally
form aerial hyphae.
4.
Identification
These bacteria are difficult to distinguish from Streptomyces species. Identification of the genus is based
on partial acid-fastness, inability to grow anaerobically, the production of aerial hyphae and the ability to
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
grow on Sabouraurd’s dextrose agar. Nocardia asteroides often forms a brown pigment on Sabouraud’s
dextrose agar. Species identification is more difficult and should be referred to a reference laboratory.
Tentative species identification can be performed by mycolic acid profiles using gas chromatography and
HPLC analysis. These tests are however difficult to standardize due to the great number of variables.
The use of DNA probes for the identification of the Nocardia is likely to enhance identification in future.
5.
Antimicrobial susceptibilities
Presently there are no standardized methods of susceptibility testing for Nocardia adopted by the
NCCLS, as it is difficult to make a uniform suspension of these bacteria for the test and the fact that
these organisms are slow-growing. However the most common methods presently employed are the
disk-diffusion method using Mueller-Hinton agar and a broth micro dilution method using Mueller-Hinton
broth. The E-test has also proven to be useful. Antimicrobials used in the panel should include
sulfamethoxazole in combination with trimethoprim, amoxycillin-clavulanic acid, tetracycline, amikacin,
ceftriaxone, cefotaxime and imipenem.
6.
Serology
Reliable immunodiffusion tests have been established for systemic nocardiosis in dogs and nocardial
mastitis in cattle, but are not routinely used. In humans, an indirect immunofluorescence test is used to
screen patients. However because of the cross-reaction of the test with Mycobacterium spp. and other
actinobacteria, samples for culture should be taken from patients with positive test results.
7.
Interpretation and reporting of results
As Nocardia are common in the environment, but rare in clinical specimens, results should be interpreted
with consideration of clinical disease, and the presence of bacteria with typical Nocardia morphology on
exudates or tissue smears. Due to the fact that it is not always possible to isolate Nocardia spp. from
clinical specimens, a presumptive result can be reported if Gram-positive or partially acid-fast staining
branching filamentous bacteria that broke up into coccoid elements were seen in tissue or exudate
smears, particularly if the organism stained acid-fast.
Table 6.4: Hydrolysis and selected biochemical profiles of pathogenic species of
Nocardia
12 | P a g e
+
-
+
+
+
-
+
-
+/+
+/-
+
-
Glucose
Rhamnose
Acid from
(14 day)
Starch
Gelatin
+
-
Arylsulf
atase
at 45°C
Growth
Xanthine
Tyrosine
Casein
Organism
N. asteroides
N. farcinica
N. nova
N. brasiliensis
N. otitidiscaviarum
Hypoxanthine
Decomposition of:
+
+
+
+
+
-/+
+/-
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
N. transvalensis
Streptomyces spp.
+
+/-
+
+\+
-
+
-
-
+
-
Table 6.5: Differentiation of the causative agents of canine nocardiosis and canine
actinomycosis
Aetiology
Granules in exudates
Filaments MZN-positive
Fragmentation of filaments
Growth on Sabouraud’s dextrose agar
Powdery, white colonies (aerial hyphae)
Susceptibility to penicillin
Anaerobic growth
Canine nocardiosis
Nocardia asteroides
Not common
+ (70%)
+
+
+
-
Canine actinomyces
Actinomyces viscosus
Usually present
+
+
Streptomyces
The genus Streptomyces consists of numerous poorly defined species, which are aerobic, catalase
positive, Gram-positive, non-motile, extensively branched filamentous bacteria that form aerial hyphae,
with short to long chains of non-motile spores. Over 3 000 species of Streptomyces have been identified.
These soil saprophytes are rarely implicated in clinical disease, but are common laboratory
contaminants. Because they are similar in colony and microscopic appearance to Nocardia and
sometimes Actinomyces species they should be distinguished from them. Streptomyces spp. tends to
form colonies with a white powdery surface that emit a pungent musty-basement odour. The branching
filaments usually do not break up and the aerial hyphae produce conidia that are typically chained.
Streptomyces species, unlike Nocardia, do not stain acid-fast. They are also sensitive to lysozyme and
rapidly hydrolyze casein.
Actinomyces and Arcanobacterium
Actinomyces are Gram-positive, diphteroidal or branching filamentous rods. They are anaerobic or
microaerophilic commensal organisms found in the oral cavities of animals and humans. In this section
only Actinomyces spp. which grows under aerobic conditions will be discussed. Actinomyces viscosus is
the cause of localized or granulomatous abscesses or pyothorax in canines.
Arcanobacterium pyogenes was formerly called Actinomyces pyogenes and previously Corynebacterium
pyogenes
Diagnosis
Specimen collection
Specimens include pus, exudates, aspirates, tissue and scrapings from the walls of abscesses if they
have been incised. A volume of fluid or pus should be collected and submitted, if possible, rather than
just a small amount on a swab. Thin sections of granulomas in 10% formalin are useful for
histopathology.
13 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
Smear examination
“Sulphur” granules (Splendore-Hoepli bodies) are the best specimens for the visualisation of A. viscosus.
The pus or exudate is placed in a Petri dish and washed carefully with a little distilled water to expose the
soft greyish-white granules of A. viscosus. A granule is placed on a microscope slide in a drop of 10%
KOH and gently crushed by applying pressure on the coverslip. The characteristic clubs can be seen if
the preparation is examined under the low power objective of a microscope. To visualise the bacteria, a
Gram’s stain is made, which reveals delicate, Gram-positive, branching filaments. Occasionally short
filaments or pleomorphic diptheroidal forms may predominate.
Isolation
Material is inoculated onto blood agar plates and incubated in air with 5 – 10% CO2. (Or candle jar).
Actinomyces viscosus will usually grow in 2 - 4 days.
A. viscosus usually produces two colony forms: one is smooth, entire, convex, glistening, mucoid or soft
colony composed of diptheroidal forms; and the other consists of rough, irregular, heaped, granular and
slightly dry colonies, which yield branching filaments. Both forms are usually seen and neither is
haemolytic. It is distinguished from other Actinomyces spp. in that it grows well aerobically, and from
Nocardia or Streptomyces by its failure to grow on Sabouraud’s dextrose agar.
Biochemical tests
Table 6.6: Identification of Actinomyces spp. (usually glucose positive)
A. odontolyticus
A. howellii
A. naeslundi
A. viscosus*
A. pyogenes
A.
hordeovulneris
A. denticolens
A. hyovaginalis
A. neuii anitratus
A. neuii neuii
A. slackii
A. suimastitidis
14 | P a g e
Gelatin hydrolysis
Aesculin hydrolysis
-
-
V
V
+
+
+
-
-
-
+
-
-
V
V
+
-
-
-
-
+
+
+
+
v
+
Xylose
-
-+
v
-
Trehalose
+
-
-
Sucrose
+
+
Salicin
-
+
+
Ribose
-
+
+
v
Raffinose
-
Mannitol
-
Glycogen
-
-
Arabinose
-
Catalase
Melezitose
A israelii
Nitrate reduction
A. bowdenii
A. canis
A. catuli
M or
An
F
F
F
M or
An
M or
An
F
F
F
F
M or
An
F
F
F
F
F
F
Urease
A. bovis
Aero tolerance
Species
Fermentation of
-
-
+
+
+
+
+
(-)
+
-
+
(+)
+
+
w
V
-
+
V
-
v
-
(-)
-
-
+
+
+
-
-
V
V
-
+
(+)
+
v
+
v
+
V
V
(+)
+
w
-
-
-
v
v
-
w
v
+
+
v
+
+
+
+
-
+
+
+
?
v
-
(-)
+
v
+
+
-
-
(+)
(+)
+
+
+
v
+
+
+
+
v
+
+
v
+
-
+
(+)
+
+
+
-
-
D
-(d)
+
+
-
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
-
-
-
-
-
+
+
+
v
-
-
* Serotype 1 does not produce pyrazinamidase but serotype II does.
Susceptibility to antimicrobial agents
Strains of Actinomyces are usually sensitive to a wide range of antimicrobials, and evidence of the
development of resistance is lacking. However as Actinomyces spp. are usually components of
polymicrobial infections, and other bacteria with antimicrobial resistance may protect the actinobacteria.
These bacteria are usually sensitive to penicillins and tetracycline and usually resistant to metronidazole,
the aminoglycosides and peptide antibiotics.
Serological identification
Reference laboratories make use of serological identification to identify the different actinobacteria.
REFERENCES
1. The Actinomycetes. In: Clinical Veterinary Microbiology, Eds Quinn P J, Carter M E, Markey B and
2.
Carter G R (1994). Wolfe Publishing. 144 - 155.
Nocardioforms and aerobic actinomycetes. In: Colour Atlas and Textbook of Diagnostic Bacteriology
(4th Edition). Eds. Koneman E W, Allen S D, Janda W M, Schreckenberger, & Winn W C (1992). J B
Lippincott Company
3.
Veterinary Microbiology and Microbial Disease, (2011). Quinn, P.J., Markey, B.K., Leonard, F.C.,
FitzPatrick, E.S., Fanning, S., Hartigan, P.J. Wiley-Blackwell. ISBN 978-1-4051-5823-7
15 | P a g e
Xylose
-
Trehalose
+
Sucrose
-
Salicin
-
Ribose
-
Raffinose
v
Mannitol
An
Glycogen
-
Arabinose
+
Melezitose
Gelatin hydrolysis
-
Nitrate reduction
Aesculin hydrolysis
F
Urease
Catalase
A. vaccimaxillae
Actinobaculum
suis
Aero tolerance
Species
Fermentation of
+
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
A. kentuckyensis
A. lexingtonensis
A. pretoriensis
A. mediterranei
A. alba
A. albidoflavus
A. azurea
A. coloradensis
A. eurytherma
A. fastidiosa
A. japon
A. methanolica
A. rubida
A. sacchari
A. sulphurea
A. thermoflava
Decomposition of:
Allantoin
Casein
Aesculin
Gelatin
Hypoxanthine
Starch
Urea
Xanthine
Production of:
Soluble pigments
Nitrate reductase
Acid from:
Adonitol
Arabinose
Cellobiose
Dextrin
Erythritol
Fructose
Galactose
Inositol
Lactose
Maltose
Mannitol
Melibiose
Methyl -Dglucoside
Raffinose
Rhamnose
Salicin
Sorbitol
Sucrose
Trehalose
Xylose
Growth in/at:
5 % NaCl
45 °C
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
w
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
w
+
-
+
+
-
+
+
+
+
+
+
+
+
w
+
+
-
+
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
-
-
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
w
+
+
+
+
w
+
-
w
+
w
w
w
w
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
+
+
-
w
+
-
+
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
+
+
+
+
+
+
w
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
w
w
w
-
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
w
-
+
+
+
+
-
w
+
w
+
+
-
+
+
+
-
-
-
-
-
-
+
+
-
+
-
-
+
-
+
+, Positive; -, negative; W, weak reaction
16 | P a g e
A. orientalis subsp.
orientalis
Table 6.7: Differential properties of A. kentuckyensis, A. lexingtonensis and A.
pretoriensis (agents having caused abortion in mares) compared with previously
described species of the genus Amycolatopsis
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
APPENDIX 1
Media and tests used in the identification of aerobic/aero-tolerant Grampositive branching filamentous bacteria
1.
Casein agar
3. Allow medium to cool almost to solidification
4. Add 0,4g xanthine or 0,4g tyrosine to 100ml
Method
of basal medium.
A
B
Skim milk
10g
Distilled water
90ml
Agar
3g
Distilled water
97ml
1. Autoclave A and B separately at 121°C for
10 minutes
2. Allow to cool to 50°C
5. Mix well and pour into Petri dishes.
6. Incubate at room temperature for 4 weeks
and examine daily for clearing of medium
below and around the colony.
Interpretation
Clearing below and around the colony indicates
a positive result.
3. Combine and pour into Petri dishes
Xanthine
Interpretation
Positive:
N.
Otitidiscaviae
Streptomyces spp.
If casein is hydrolyzed a clear zone will develop
around the inoculum
and
some
Negative: N. asteroides, N. farcinica, N. Nova
& N. brasiliensis
Positive: Streptomyces spp., N. braziliensis, T.
Tyrosine
pyogenes
Negative: N. asteroides, N. otitidiscaviae, N.
Positive: N. brasiliensis & Streptomyces spp.
nova, N. farcinica
2.
Tyrosine or xanthine agar
Method
Negative: N. asteroides, N farcinica, N. Nova &
N. Otitidiscaviae
3.
Basal medium pH 7.0
Lysozyme broth
Method:
Beef extract
3g
Peptone
5
Agar
15g
Peptone
5g
Distilled water
1 000ml
Glycerol
70ml
Distilled water
1 000ml
Glycerol broth (basal medium)
1. Heat to bring ingredients into solution.
Divide into 2 equal aliquots and sterilize by
2. Place 100ml aliquots into 250ml flasks.
Sterilize by autoclaving at 121°C for 10
minutes
17 | P a g e
autoclaving at 121°C for 15 min. One aliquot will
serve as control. Lysozyme solution
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 6: Aerobic Gram-positive filamentous bacteria
Lysozyme
100mg
0,01N HCl
100ml
Sterilize by filtration.
1. To 95ml glycerol broth add 5ml lysozyme
solution
2. Mix and tube aseptically in 5ml aliquots.
3. All broths and lysozyme solution can be
5. Set up a control set of broths by inoculating
a tube with lysozyme and one without with
Streptomyces spp.
6. Incubate all tubes at room temperature until
control tubes show good growth, at which
time the tests may be read and discarded.
Interpretation
Growth in lysozyme tube: Nocardia
stored in a refrigerator.
4. Place several fragments of culture into a
tube containing lysozyme and one without
lysozyme (control).
18 | P a g e
No growth in lysozyme tube: Streptomyces