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Transcript
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
Applied Veterinary Bacteriology and Mycology:
Identification of aerobic and facultative
anaerobic bacteria
Chapter 5: Small Gram-negative rods and coccobacilli
Authors: Drs. M.M. Henton & J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION ...........................................................................................................................................2
Table 5.1: Phenotypic characteristics separating selected genera of veterinary importance in
the family Pasteurellaceae .............................................................................................................2
Table 5.2: Histophilus and Haemophilus species of veterinary importance in production
animals, dogs and poultry ..............................................................................................................3
Table 5.3: Identification of Haemophilus and Taylorella species of veterinary importance ...........4
Table 5.4: Actinobacillus species of veterinary importance in production animals and dogs ........6
Table 5.5: Differential characteristics of the species belonging to the genus Actinobacillus .........7
Table 5.6: Important Pasteurella and Mannheimia species in animals .........................................9
Table 5.7: Identification of bacteria belonging to the genus Pasteurella. (All are non-motile,
oxidase positive, nitrate positive and ferment glucose) ...............................................................11
Table 5.8: Differential characteristics of the species belonging to the genus Pasteurella and
some related species (Lonepinella koalarum and Phocoenobacter uteri are catalase and
xylose negative ............................................................................................................................12
Table 5.9: Differential characteristic of Mannheimia species.......................................................13
Table 5.10: Identification characteristics of Gallibacterium species ............................................15
REFERENCES ............................................................................................................................................18
1|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
INTRODUCTION
The genera Haemophilus, Actinobacillus, Mannheimia and Pasteurella are currently classified in the
family Pasteurellaceae. These species are major causes of respiratory and systemic diseases in
humans, other mammals and birds. They exist as mucosal parasites, but are capable of being primary
or opportunistic pathogens depending on the carrier status and health of the host.
Recently there have been many changes in the taxonomy of this group that now contains seven
genera of veterinary importance: Actinobacillus, Avibacterium, Haemophilus, Histophilus, Mannheimia,
Pasteurella and Bibersteinia. Important differential characteristics of genera in this family are included
in Table 5.1.
D
+
+
+
+d
-
-
D
+
+
+
D
D
D
D
D
+
D
-
D
D
+
D
+
D
+
D
+
D
D
D
-C
+
D
-
-
-
Pasteurella
Haemophilus
Histophilus
Haemolysis
Capnophilia
Yellowish pigmentation
V-factor dependence
X-factor dependence
Catalase
Oxidase
Urease
Voges–Proskauer (37 °C)
Indole
Ornithine decarboxylase
L-Arabinose
D-Fructose
D-Galactose
Meso-Inositol
Maltose
Mannitol
D-Mannose
D-Melibiose
D-Sorbitol
Sucrose
Trehalose
ONPF (-fucosidase)
ONPG
PNPG
Actinobacillus
Table 5.1: Phenotypic characteristics separating selected genera of veterinary
importance in the family Pasteurellaceae
D
+
D
D
+b
D
+
+
D
D
+
D
+
D
-
+, only positive reactions, -, only negative reactions; D, positive or negative; NK, not known.
a
Deviating strains occur.
b
P. avium is indole negative
c
Aesculin, amygdalin, arbutin, cellobiose, salicin and NPG (-glucosidase)
d
Bisgaard taxon 9 late positive
2|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
Haemophilus/ Histophilus
There have been a number of taxonomic changes to organisms within the genus Haemophilus in
recent years. The species Histophilus somni now includes the bovine commensal and opportunistic
pathogen Haemophilus somnus and the ovine organisms Histophilus ovis and Haemophilus agni. A
list of Haemophilus and Histophilus species causing disease in animals are included in Table 5.2.
Table 5.2: Histophilus and Haemophilus species of veterinary importance in
production animals, dogs and poultry
Species
Hosts
Cattle (previously
Haemophilus
somnus)
H. somni
Sheep (previously
Haemophilus agni
and Histophilus
ovis)
Disease
Septicaemia
Infarcts in the brain (Thrombotic
meningoencephalitis)
Respiratory infections
Genital tract infections
Epididymitis
Pneumonia
Mastitis
Polyarthritis
Meningitis
Septicaemia
Polyserositis, meningitis (Glasser’s
disease)
Infectious coryza in chickens and
respiratory infection in turkeys
H. parasuis
Pigs
A. paragallinarum
Poultry
H. haemoglobinophilus
Dogs
Balanoposthitis, vaginitis
H. paracuniculus
Rabbits
Mucoid enteritis
Speciation of Haemophilus in the clinical laboratory has been primarily based on the requirement for
haemin (a porphyrin also known as X factor) or NAD (also known as V factor or coenzyme I). The
methods currently used for the determination of these growth factor requirements rely on the
supplements being added to defined agar media or impregnated in disks or strips placed on agar
plates.
Unlike human pathogens in this genus, no animal isolate is dependent on both factors. They are all
dependent on only one factor with the exception of Histophilus somni , which is X and V factor
independent. The X factor is present in adequate amounts in blood agar. In chocolate agar both
factors are present by virtue of being released into the medium following haemolysis during heating of
the blood. The V factor is also produced by an organism such as Staphylococcus aureus. When the
latter is grown together with Haemophilus spp. on blood agar, the resultant enhanced growth of
Haemophilus is referred to as satellitism. In veterinary laboratories, routine isolation on chocolate
agar in an atmosphere of 5-10% CO2 (or candle jar) at 35-37°C for 3-4 days is satisfactory. Selective
media can be used when one specifically suspects the presence of Haemophilus species.
3|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
Histophilus somni (formerly Haemophilus somnus, H. agni and Histophilus ovis)
H. somni is an opportunistic pathogen of cattle. It is a common component of the normal urogenital
and respiratory flora and sporadically causes thromboembolic meningoencephalitis, abortion, infertility
and pneumonia. It is often the only bacterial isolate from the many reported cases of necrotic
laryngitis, necrotic tracheitis, fibrinous bronchopneumonia and fibrinous pleuritis in cattle. There are
probably many subtypes of H. somni.
This organism is a nutritionally demanding, moderately slow growing, capnophilic, Gram-negative
bacillus. Colonies on blood agar are unremarkable. H. somni does not require X or V factors for
growth. A selective growth medium for H. somni has been described. It incorporates vancomycin (5
g/ml), neomycin (5 g/ml), sodium azide (50 g/ml), nystatin (100 g/ml) and cycloheximide (100 g/ml)
into 5% horse blood agar. Addition of thiamine monophosphate (1 g/ml) also enhances growth of the
organism.
When attempting to isolate H. somni from cases of suspected thromboembolic meningoencephalitis,
brain tissue that includes visible lesions should be homogenised in sterile saline at the ratio of 5 mg
tissue in 20 ml saline. Spread aliquots of 0.1 ml over the surface of blood agar. H. somni is considered
to be homologous to Haemophilus agni and Histophilus ovis both found in sheep and goats. There are
slight biochemical differences between them e.g. H. agni is usually urease positive and indole
negative. Histophilus somni may be weakly indole positive and when scraped together the colonies
are usually yellow. Actinobacillus seminis is also very similar to H. somni. Both are sucrose negative,
which
distinguishes
them
from
the
rest
of
the
community
found
in
the
Haemophilus/Pasteurella/Actinobacillus (HPA) group, but A. seminis is grey when the colonies are
scraped together. Differential characteristics are summarized in Table 5.3.
Requirement for X factor
Requirement for V factor
Growth on MacConkey
Catalase
Oxidase
Capnophilic
Haemolysis
Indole
Urease
Nitrate reduction
Glucose (acid)
Aesculin hydrolysis
ONPG
Phosphatase
Ornithine decarboxylase
Gas from glucose
4|Page
+
+
+w
+
+
d
+
-
+
+
(+)
+
+
+
-
+
+
+
+
+
-d
+
-
+
d
d
+
+
+
+
d
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
d
-
+
+
+
+
+
+
+
+
+
-
T. equigenitalis
H. paracuniculus
H.
haemoglobinophilus
Avibacterium.
paragallinarum
A. pleuropneumoniae
H. parasuis
Test
H. agni
H. somnus
Table 5.3: Identification of Haemophilus and Taylorella species of veterinary
importance
+
+
+
-
+
-
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
Acid production from:
L-arabinose
Cellobiose
Dulcitol
Galactose
m-Inositol
Lactose
Maltose
Mannitol
Mannose
Melibiose
Raffinose
Salicin
Sorbitol
Sucrose
Trehalose
D-Xylose
d
d
d
d
+
+
+
+
+
+
+/w
+/w
+/w
+/w
+/w
+
d
d
+
+
+
-
+/w
d
+
+
+
d
+
+
(+)
+
+
+
+
d
+
+
+
+
+
+
+
-
Haemophilus parasuis
H. parasuis is considered to be part of the normal flora of the respiratory passages of conventionally
raised pigs. It causes Glässer’s disease, characterized by polyserositis and meningitis. The disease
only occurs sporadically, mainly in pigs two to four weeks of age, subjected to stressful environmental
conditions. A selective medium for H. parasuis consists of chocolate agar containing 1.5 g/ml
lincomycin, 5 g/ml bacitracin and 0.1 g/ml crystal violet. The specimen material should be spread with
a sterile swab to completely cover the agar surface. Haemophilus parasuis grows weakly and may
take 2 to 3 days to become apparent. Actinobacillus pleuropneumoniae in comparison grows rapidly (1
day), is CAMP and urease positive.
Tayorella Equigenitalis
This organism was formerly known as Haemophilus equigenitalis. It is an important cause of genital
tract infections in horses and is either venereally transmitted or via assisted reproduction (artificial
insemination). Swabs of the cervix, clitoral fossa and urethra can be examined in mares, and the
urethral fossa and penile sheath (lamina interna) in stallions
For primary isolation agar bases such as Oxoid blood agar base No. 2, or Columbia agar (BBL) can
be used with the addition of sodium sulphate 200 mg/L and L-cysteine HCl 100 mg/L. Chocolated
blood (preferably sheep blood) are added at 10% concentration. Some plates should be prepared with
streptomycin at 200 g/ml media and some without. One of each should be used to examine swab
specimens. Plates are incubated in air with 5-10% CO2 for up to 6 days before discarding them.
Colonies are small pinpoint to pin head in size.
The minimum profile of tests to be carried out on suspicious colonies is:
5|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
Motility
non-motile in Robertson's cooked meat medium
Oxidase
positive (rapid and strong reaction)
Catalase
positive
Urease
negative
Phosphatase
positive
Actinobacillus
Actinobacillus spp. are commensals on the mucous membranes of their hosts. They do not penetrate
intact skin or healthy mucosa, but are responsible for endogenous infections following trauma. These
organisms are aerobic, Gram-negative coccobacilli. Members of this genus that are of veterinary
importance are listed in Table 5.4. Note that A. muris has only been found in rodents and A.
actinomycetum-comitans and A. hominis in humans.
Table 5.4: Actinobacillus species of veterinary importance in production animals and
dogs
Species
A. lignieresii
Hosts
Wooden tongue
Pyogranulomas
Sheep
Pyogranulomas
pigs
foals
Granulomatous mastitis
Neonatal foal septicaemia
Arthritis
mares
Abortion
Septicaemia
Pigs
Arthritis
dogs
pigs
Opportunistic infections
Pneumonia
Septicaemia
Fibrinous pleuropneumonia
Epididymitis
A. equuli
A. suis
A. pleuropneumoniae
A. seminis
Disease
Cattle
pigs
rams
They can all be isolated on blood agar and incubated aerobically for 24 hours at 37°C. Growth on
MacConkey agar may take longer than 24 hours. Members of the genus Actinobacillus typically
produce sticky colonies, grow well on MacConkey agar, and hydrolyze urea. The differential
characteristics of the species belonging to the genus Actinobacillus are included in Table 5.5.
6|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
(+)
+
+
+
+
-
-
-
+
+, L
-
d
+
-
+
d
+
-b
-
+
(+)
+
(+)
+
-
-
-
-
-
-
d
-
+
-
+
d
-
+
-
+
+, L
-
+
d
+
+
-
d
-
+
+
-
-
(+)
-
-
(+)w
-
+
(-)
(+)
-
A. suis
A. seminis
(+)
(+)
+
(+)
-
A. rossii
d
d
+
+
+
A. lignieresii
+
+
+
-
(+)
+
+
+
A. hominis
d
(+)
+
d
+
d
(+)
+
d
+
A. equuli subsp.
haemolyticus
+
+
+
+
A. equuli subsp.
equuli
A. arthrittidis
+
A. pleuropneumoniae
+a
+
+
+
A. muris
Catalase
Oxidase
Nitrate reduction
ONPG reaction
Phosphatase
Ornithine
decarboxylase
Indole production
Urease
Aesculin hydrolysis
NAD requirement
Growth on
MacConkey agarc
Beta-haemolysis
(sheep cells)
Fermentation f
Gas production from
glucosed
Acid production from:
L-arabinose
Arbutin
Cellobiose
Dulcitol
Galactose
m-Inositol
Lactose
Maltose
Mannitol
Mannose
Melibiose
Raffinose
Salicin
Sorbitol
Sucrose
Trehalose
D-Xylose
G+C content (mol%
range)
A. capsulatus
Test
A. acetomycetumcomitans
Table 5.5: Differential characteristics of the species belonging to the genus
Actinobacillus
-
-
-
+
-
-
-
+
d
-
+
+
NF
+
+
+
+
+
+
+
W, L
+
(+)
-
-
-
-
-
-
d
-
-
+
+,L
d,w
d,w
+
+
+
+,L
+
+
+
+
-
NT
+,w
d
+
+
+
d
+
+
+
+
(+)
+
(-)
(+)
d
(-)
(-)
(+)
(-)
(+)
d, L
d,L
d
d,L
d,L
-
(+)
+
+
(+)
+
+
+
+
+
+
+
+
+
46.9
42
41.9
43.7
40.5
+
+
(+)
+
(-)
d
42.747.1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
42.4
d
(+)
+
+
+
(-)
+
+
+
+
+
+
+
+
+
+
4042.9
(-)
+
+
+
(-)
+
+
(+)
(-)
+
NT
+
+
+
+
+,L
+
d
+
+
+
40.9
(-)
+
d,L
+
+
+
d
(-)
+
+
41.843.2
+, ≥90% of the strains are positive; (+), 80 – 89% of the strains are positive; d, 21 to 79% of the strains are positive; (-), 1120% of the strains are positive; -, ≤10% of the strains are positive; w, weak reaction; L, late reaction; NT, not tested.
b
Positive according to Piechulla et al, and negative according to Sneath and Stevens.
c
Results for A. pleuropneumonia is on media supplemented with NAD
d
In Hugh-Leifson medium containing glucose, with incubation for 48 h at 37°C.
a
Actinobacillus lignieresii
This organism is a normal Gram-negative inhabitant of the oral cavity, and is the recognized cause of
“wooden tongue” and local infections of the pharyngeal region, nostrils and neck of cattle. In sheep
and goats, skin or lung lesions may be found. Drainage from these lesions tends to be non-putrid and
watery. Tissues and pus from lesions may contain firm granules 1-3mm in diameter. They represent
7|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
the so-called “sulphur” granules. Pus diluted with water and swirled around in a Petri dish may show
up the small granules. Staining of crushed granules usually reveals masses of Gram-negative
bacteria. In histological sections of granulomatous tissues, the masses of organisms are surrounded
by Gram-negative club-shaped bodies.
Actinobacillus lignieresii is often difficult to isolate and only a few colonies may be present. It dies
rapidly in the laboratory.
Actinobacillus rossii
This organism has been isolated from pigs and is a small Gram-negative rod. It produces smooth,
grey colonies on blood agar incubated aerobically and anaerobically at 37 °C, but not at 42°C or at
25°C. It does not grow on MacConkey or Simmon’s citrate agar.
Actinobacillus suis
It is always strongly beta-haemolytic and can be distinguished from A. rossii on biochemical tests.
Actinobacillus pleuropneumonia
Pneumonic lung specimens are usually cultured on blood agar with a S. aureus streak or on chocolate
horse blood agar as rare strains are V factor dependent. The selectivity of the medium is enhanced
with the addition of bacitracin (500 units/ml), and cloxacillin (5 l/ml). Plates should be incubated for 24
hours aerobically or in an atmosphere of 5-10% CO2 (or a candle jar) at 37 °C. Colonies on chocolate
agar are opaque and reach a diameter of 1-2mm within 48 hours. Two types of colonies often are
visible: a round, hard, waxy type and a flatter, soft, glistening type. The haemolytic activity of this
organism is characteristic of this species. The intensity of the haemolysis varies not only according to
the type of erythrocyte used but also with the different serotypes.
Mannitol fermentation and positive urease reaction distinguish A. pleuropneumoniae from other
respiratory pathogens of swine such as Haemophilus parasuis
Actinobacillus seminis
Actinobacillus actinomycetum-comitans mentioned in older texts should be considered to be A.
seminis.
This organism is a highly pleomorphic, Gram-negative rod that does not grow on MacConkey agar, is
not haemolytic, and does not require CO2 for growth. Colonies on blood agar are small (less than 1
mm), clear and glistening.
Actinobacillus seminis should be distinguished from other Gram-negative pleomorphic rods that can
be isolated from the semen of rams such as Histophilus ovis, Histophilus somni and Haemophilus
agni. These three organisms cannot be distinguished on the basis of DNA studies, and should
therefore be regarded as the same. They differ from A. seminis by the production of a yellow pigment,
enhancement of growth in air with 10% CO2, are catalase negative and indole positive.
8|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
Actinobacillus equuli
Bacterial septicaemia in foals usually occurs during the first week of life and incurs a high mortality
rate. Various pathogens can cause bacterial septicaemia in foals, also referred to as sleepy foal
disease. They include A. equuli, E. coli, Streptococcus equi subsp. zooepidemicus, Salmonella
Typhimurium, Klebsiella pneumoniae and S. aureus.
Mannheimia/ Pasteurella/Gallibacterium
The genus Pasteurella is widely parasitic on mammals and birds. Under conditions of stress, these
organisms can become invasive and play a significant role in the pathogenesis of a variety of
infections in animals, including pneumonia, sinusitis, abortion, mastitis and septicaemia. Table 5.6
lists the most important species that can be isolated from ruminants, horses, pigs, companion animals,
rabbits and birds. Colonies from mammals are small, grey and glistening, or larger, shiny colonies with
a brown centre. Poultry strains are tiny beta-haemolytic colonies similar to Streptococcus. Pasteurella
species are small, Gram-negative rods or cocco-bacilli. Clinical material should routinely be inoculated
on blood agar and MacConkey agar. Colonies of all species are usually visible after 24 hours.
Table 5.6: Important Pasteurella and Mannheimia species in animals
Species
Hosts
Pneumonia
Sheep
Pneumonia
Gangrenous mastitis “Bluebag”
Septicaemia in lambs under 3 months of
age
Pigs
Sheep
Pneumonia
Septicaemia in lamds 5-12 months old
Cattle
Pneumonia (primary or secondary)
Occasional but severe mastitis
Sheep/goats
Pneumonia
Mastitis
Pigs
Pneumonia (usually secondary)
Rabbits
“Snuffles”, pleuropneumonia,
abscesses, otitis media, conjunctivitis
and genital infections
Poultry
Cattle and Water Buffalo
Fowl cholera (primary infection)
Haemorrhagic septicaemia (type B in
SE Asia and type E in Africa)
Mannheimia haemolytica
B. trehalosi
P. multocida type A
P. multocida types B and E
P. multocida type D
P. multocida type F
P. pneumotropica
P. canis
P. dagmatis
P. stomatis
P. caballi
9|Page
Disease
Cattle
Pigs
Turkeys mainly
Dogs and cats
Rodents
Dogs (humans)
Dogs and cats
Dogs and cats
Horses
Nasopharynx of carrier animals
Atrophic rhinitis
Role as a pathogen is unclear
Normal nasopharyngeal micro flora
Pneumonia and abscesses
Commensal in the nasopharynx
Commensal in oral cavity
From dog bite injuries in humans
Commensal in oral cavity
From dog bite injuries in humans
Commensal in respiratory tract
Respiratory infections including
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
P. aerogenes
P. anatis
P. gallinarum
P. avium, P. langaa & P. volantium
P. testudinis
P. granulomatis
Pigs
Ducks
Poultry
Chickens
Turtles, tortoises
Cattle
pneumonia
Commensa; in digestive tract
Intestinal commensal
Commensal in respiratory mucosa.
Occasional mild respiratory infections.
Commensal in the respiratory tract
Abscessation
Fibrogranulomatous disease
The differential characteristics of the species belonging to the genus Pasteurella only are given in
Table 5.7 and that of Pasteurella and related species in Table 5.8.
Mannheimia haemolytica and Pasteurella trehalosi
Pasteurella haemolytica biotype A has been divided into the pathogenic Mannheimia haemolytica,
Mannheimia granulomatis and Mannheimia variegata and the non-pathogenic Mannheimia glucosida.
The former Pasteurella haemolytica biotype T has been allocated to a new species known as
Pasteurella trehalosi. Of these, M. haemolytica and P. trehalosi have the greatest veterinary
significance. Poultry strains of P. haemolytica retained their name. They are small Gram-positive
cocco-bacilli that grow both aerobically and as facultative anaerobes. Mannheimia haemolytica
produces acid in arabinose, and P. trehalosi acid in trehalose. The fermentation reaction should be
allowed to continue for up to 10 days. On blood agar, M. haemolytica strains produce small, grey
colonies with distinct zones of haemolysis. The larger slightly yellow/brown colonies of P. trehalosi are
less uniformly haemolytic.
On the basis of specific antisera, 17 serotypes of M. haemolytica have been identified. Mannheimia
haemolytica includes serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13, 14; 16 and 17 and P. trehalosi includes
serotypes 3, 4, 10 & 15. Selected characteristics of M. haemolytica are compared with other
respiratory pathogens of cattle in Table 5.9.
Pasteurella multocida
P. multocida colonies are non-haemolytic and may be mucoid or non-mucoid in appearance. They do
not grow on MacConkey agar. On the basis of their capsule polysaccharides, they have been divided
in types A, B, D, E & F. In Africa, only types A, D & E have been described. Haemorrhagic
septicaemia is caused by types B and E.
Pasteurella caballi
Strains isolated from horses, but not fulfilling the characteristics of any established species within the
genus were described in 1989. Subsequent biochemical and genetic studies led to the proposed name
of P. caballi. These strains differed from other Pasteurella species in that all were aerogenic and
catalase-and oxidase negative, and some strains produced acid from myo-inositol and L-rhamnose.
They seem to have a limited host range and have only been isolated from horses. They are
sporadically isolated from horses where they are opportunistic pathogens. They also produce gas from
glucose, are urease-negative and ferment raffinose (slow), mannitol, maltose, dextrin, glucose and
xylose. They utilize ODC and don’t hydrolyze aesculin. Colonies are yellow and non-haemolytic.
10 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 5: Small Gram-negative rods and coccobacilli
P. canis and P. dagmatis
These organisms are part of the normal oral flora of dogs but are potentially pathogenic for humans
following bite wounds from dogs. When trying to isolate these bacteria from the buccal cavities of
dogs, contaminating bacteria such as Staphylococcus, Streptococcus and Corynebacterium can be
inhibited by the addition of 20 mg/litre thiostrepton to the culture media.
Other Pasteurella species
Pasteurella lymphangidis has only been found in cattle in India and Mannheimia granulomatis in cattle
in Brazil. Pasteurella mairii causes abortion and septicaemia in pigs and rarely in other animals. Table
5.8: Phenotypic separation of species and taxa within the genus Mannheimia (From Angen et al,
International Journal of Systematic Bacteriology, p. 47). Pasteurella testudinis is associated with
respiratory infections in tortoises. Pasteurella bettae and P. ureae are only found in humans.
Infections of poultry by members of the Pasteurellaceae
These are important disease causing agents in poultry, and common species infecting poultry are
listed in Table 5.7.
These bacteria can easily be confused with Riemerella anatipestifer (formerly Pasteurella
anatipestifer) and Cytophagia.
11 | P a g e
P. m gallicida
P. dagmatis
P. galinarum
P. canis
P. anatis
Pasteurella A
Pasteurella B
P. langaa
P. avium
P. volantium
Catalase
Indole
Urease
Ornithine
Glucose gas
Acid from
Dulcitol
Maltose
Mannitol
Sorbitol
Trehalose
P. m. septica
Species
P. m. multocida
Table 5.7: Identification of bacteria belonging to the genus Pasteurella. (All are nonmotile, oxidase positive, nitrate positive and ferment glucose)
+
+
+
-
+
+
+
-
+
+
+
+
+
+
+
-
+
-
+
d
+
-
+
-
+
d
+
+
+
-
-
+
d
-
+
-
+
+
d
+
+
+
+
+
-
+
+
+
+
d
+
+
d
+
+
+
+
-
+
d
+
+
d
+
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli
+
+,w
-
d
-
-
-
-
+
-
-
-
-
-
-
-
-
+
+
+
+
+
+
ONPG
d
+
-
-
+
+
+
+
+
d
-
-
-
Indole
-
-
-
Urease
-
-
-
Phosphatas
e
Ornithine
decarboxyl
ase
Aesculin
NAD
requirement
Fermentatio
n
Gas
glucose
Acid from
L-
12 | P a g e
Taxon 3
MacConkey
haemolysis
(sheep
cells)
Porphyrin
test
Nitrate
Taxon 2
+
C. cynodegmi
+
C. carnimorsus
+
C. ochraceae
+
C. sputigena
+
C..gingivalis
-
+
R. anatipestifer
-
+
Haemophilus
paragallinarum
Haemolytic
Actinobacillus-like
A.salpingingitidis.
salpingitidiasalpin
gitidissalpingitidis
Taxon 14
+
+
Ornithobacterium
rhinotracheale
+,w
+
P. volantium
(+)
+
P. ureae
Oxidase
-
P. trehalosi
+/
d
P. testudinis
+
P. stomatis
+
P. pneumotropica
Gallibacterium
+
P. multocida
M. haemolytica
+
P. mairi
P. gallinarum
-
P. lymohangitidis
P. dagmatis
d
M. langaaensis
P. canis
+,w
P. caballi
+
P. bettigae
+
P.avium
P. anatis
Catalase
Test
P. aerogenes
Table 5.8: Differential characteristics of the species belonging to the genus Pasteurella and some related species (Lonepinella koalarum
and Phocoenobacter uteri are catalase and xylose negative
-
d
+
-/+
-
+
-
-
-
+
+
-
+
-
(-)
+
+
+
+
+
d
+,
w
-
d
-
d
d
-
d
+
(+)
d
-
-
-w
-
-
-
+
(+
)
+
+
+
+
+
+
+
-
-
-
d
+
+
+
+
+
+
-
-
-
-
-
d,w
+
+
-
-
-
-
+
-
-
+
+
(+
)
d
-
+
+/-
(-)
-
-
-
d
-
-
-
+
+
+
+
+
-
+
+
+
+
+
+
-
d
-
+
-
+
+
+
+
+
+
-
-
+
d
-
-
-
-
-
-
+
+
+
+
(+
)
+
+
+
-
+
-
-
+
+
+
+
-
+
+
+
+
-
-
-
-
d
(+
)
+
+
w
+
-
-
-
-
-
-
+
-
-
-
+
-
+
-
+
+
+
-
+
-
-
-
+
+
+
-
-
-
-
-
-
+
-
d
-
-
-
-
d
-
d
d
-
-
-
-
d
-
+
+
+
+
-
+
-
-
-
-
+
+
+
+
-
-
-
-
-
-
-
-
d
-
-
-
-
-
/d
-
(+
)
-
-
(+
-
-
-
+
-
+
+
+
+
+
(+
)
+
+
+
+
+
+
(+
)
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
d
-
-
(-)
-
-
(-)
-
-
-
-
-
-
-
d
d
-
+
+)
-
-
-
+
d
-
-
-
-
d
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
L
+
(+)
-
-
d,
w
+
-
+,w
-
-
-
-
-
-
-
d
-
-
-
+
-
-
-
+
-
-
-
+
-
-
+
+
d
(-)
-
d
-
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli
Arabinose
)
Arbutin
-
-
-
-
-
-
-
(-)
Cellobiose
Dulcitol
-
-
-
-
-
-
-
(-)
(-)
Galactose
+
+
+,L
-
d
+
+
+
m-Inositol
+
-
-
-
(+)
-
-
Lactose
+
+,L
-
-
(+)
-
-
Maltose
+
-
-
d
+
-
+
+
-
-
-
-
-
-
-
-
(-)
-
-
+
+
+
+
+
+
+
+
-
d
(+)
-
-
+
-
d
-
-
d
d
+,L
-
-
-
d
-
(-)
d
+
+
d
-
d
-
(-)
-
-
-
+
d
+
+
+
+
-
+
+,w
+
+
+
+
d
d
+
+
+
+
+
-
+
(+)
(-)
+
(-)
d
(-)
+
+
d
d
d
+
+
+
(+
)
+
(-)
d
+
-
d
+
+
-
(+)
-
+
(-)
d
+
(-)
-
d
+
+
-
-
+
d
Mannitol
(-)
+
-
-
+
Mannose
Melibiose
Raffinose
Rhamnose
Salicin
Sorbitol
Sucrose
+
(-)
+
+
+,w
+
+,L
-
d
-
-
Trehalose
-
+
+
-
D-Xylose
(+)
+
d
-
(+)
-
+
L
(+
)
+
L
d
-
d
-
-
-
-
-
-
-
-
-
-
-
+
+
-
-
+
+
-
d
-
-
-
-
-
(-)
(+
)
d
-
-
d
w
+
-
+
-
-
-
d
+
+
-
(+
)
+
(+
)
+
+
(+)
+
(+
)
-
(+
)
w
+
+
-
-
d
+
d
+
-
+
d
+
-
-
-
-
+
d
d
+
-
+
d
d
-
+
(+)
+
d
d
w-
+
(-)
+
(+
)
+
(-)
d
+
+
d
+
+
(-)
+
d
+
-
+
+
+
d
-
-
-
-
-
+
+
d
+
-
+
-
-
+
+
-
-
d
-
+
-
-
d
-
d
-
(+
)
+
-
d
+
-
-
-
d
-
+
+
+
+
+
+
d
+
+
-
-
-
-
-
+
+
+
+
+
d
d
-
-
+
+
d
+
+
+
-
d
+
-
+
-
-
Species
Test
Beta- haemolysis
Ornithine decarboxylase
L-arabinose
D-sorbitol
D-xylose
Maltose
13 | P a g e
Taxon
(+)
+
+w
+
M. granulomatis
M. haemolytica
Table 5.9: Differential characteristic of Mannheimia species
M. glucosida
A
B
C
D
E
F
G
H
I
(+)
+
+
+
+
+
(+)
+
+
+
+
(+)
+
+
+
+
+
(+)
+
+
+
+
+
(+)
+
+
+
+
(+)
+
+
+
(+)
+
+
+
+
(+)
+
+
+
(+)
+
+
+
+
+
+
d
d
M.
ruminalis
1
-¥
d
d
-
2
+
M.
variegans
1
d
+
+
+†
d
2
d
+
+
d
Unnamed taxa within Mannheimia
8A
8B
8C
7
9*
10§
(+)
+
+
(+)
+
+
(+)
+
d
+
+†
+
(+)
+
+
+
+
+
d
+
+
+
+
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 5: Small Gram-negative rods and coccobacilli
Dextrin
Glucosides∏
Gentiobiose
NPG (β-glucosidase)
Meso-inositol
ONPF (α-fucosidase)
ONPX (β-xylosidase)
ONPG (β-galactosidase)
Indole
D-Melibiose
Origin of isolates
No. of strains
+
d
+
d
d
BO
30
+
+
+
+
+
+w
+
+
O
34
+
+
+
+
+w
+w
+
+
O
+
+
+
+
+
+
O
+
+
+
+
+w
+
+
O
+
+
+
+
+w
+w
+
O
+
+
+
+
+w
+
+
O
+
+
+
+
+w
+w
+
O
+
+
+
+
+w
+w
+
O
+
+
+
+
d
+
O
d
d
d
+
d
w/D
BLD
28
+
O
12
d
+
BO
d
d‡
d
d
d
d
d
d
BP
51
*meso-inositol- and NPG-negative strains of [P.] haemolytica biogroup 9, the meso-inositol- and NPG-positive strains reclassified as M. glucosidal biovar I.
§
Consisting of two genetically distinct groups that could not be phenotypically separated.
Arabinose-positive strains of Bisgaard taxon 18 exist but their taxonomic allocation is still undecided.
†
One strain negative.
∏
Amygdalin, arbutin, aesculin (acid), cellobiose and salicin.
‡
Most strains negative for all glucosides; the strains that ferment glucosides also ferment D-Melibiose.
B: Bovine; O: Ovine: P; Porcine; L; Leprine; D: Deer.
¥
14 | P a g e
d
d
+
+
P
+
+
w/+
O
5
+w
+
d
+
+w
B
2
+w
+
+
+w
B
3
+w
d
d
+
d
BO
7
+
+
B
3
d
d
d
d
+
w/BO
8
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter
5: Small Gram-negative rods and coccobacilli
Gallibacterium species
Gallibacterium is a newly described genus incorporating the former Pasteurella haemolytica – A.
salpingitis bacteria isolated from poultry. They are members of the family Pasteurellaceae. Colonies on
bovine blood agar are mostly strongly β-haemolytic, greyish, non-transparent, but eventually translucent at
the periphery, with a butyrous consistency, smooth and shiny, circular, raised with an entire margin and
1·0–2·0 mm in diameter after 24–48 h at 37 °C. They are catalase-, oxidase- and phosphatase-positive.
Nitrate is reduced. Porphyrin and alanine aminopeptidase tests are positive. Acid is formed without gas
from glycerol, (-) D-ribose, (+) D-xylose, (-) D-mannitol, (-) D-fructose, (+) D-galactose, (+) D-glucose, (+)
D-mannose, sucrose and raffinose. ONPG and PNPG tests are positive. Negative in symbiotic growth,
Simmons citrate, mucate-acid, malonate-base, H2S/tri-sugar iron (TSI), growth in the presence of KCN,
Voges–Proskauer at 37 °C and urease.
Negative results are also observed with arginine dehydrolase, lysine decarboxylase, ornithine
decarboxylase, phenylalanine deaminase, indole, gelatinase and Tween 20 and 80. Pigment is not
formed. Acid is not produced from m-erythritol, adonitol, (+)D-arabitol, xylitol, (-)L-xylose, dulcitol, (+)Dfructose, (+)L-rhamnose, (-)L-sorbose, cellobiose, (+)D-melibiose, (+)D-melezitose, (+)D-glycogen, inulin,
aesculin, amygdalin, arbutin, gentiobiose, salicin, (+)D-turanose or -N-CH3–glucosamid. Reactions for pnitrophenyl -D-glucopyranoside (NPG), o-nitrophenyl -L-fucopyranoside (ONPF), p-galactosidase, pnitrophenyl -D-glucopyranosiduronic acid (PGUA), -mannosidase and o-nitrophenyl -D-xylanopyranoside
(ONPX) are also negative. Variations are observed in the methyl red reaction at 37 °C, growth on
MacConkey agar and acid production from (+)L-arabinose, (-)D-arabinose, m-inositol, (-)D-sorbitol, (-)Lfucose, lactose, maltose, trehalose and dextrin. The type species is Gallibacterium anatis and the
identification characteristics are included in Table 5.10.
Table 5.10: Identification characteristics of Gallibacterium species
Character
Haemolysis
(-)D-Arabinose
(+)L-Arabinose
m-Inositol
(-)D-Sorbitol
(-)L-Fucose
Maltose
Trehalose
Dextrin
G. anatis bv.
haemolytica
G. anatis bv. anatis
Gallibacterium
genomospecies 1
Gallibacterium
genomospecies 2
+
(+)
d
d
(+)
d
d
d
d
d
+
-
+
(+)
d
+
+
+
+
+
d
d
d
d
+
d
+
Ornithobacterium rhinotracheale
This organism is a Gram-negative, pleomorphic rod that grows slowly and was isolated as recently as
1991 from cases of respiratory disease in broiler chickens in South Africa. In 1994 it was classified as a
new species. This organism is not only linked to air sacculitis and purulent pneumonia in broiler chickens,
but also in turkeys. It affects broiler chickens of 28 days and older and is associated with high mortalities
15 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter
5: Small Gram-negative rods and coccobacilli
during this period. In some cases it is characterized by a subcutaneous and oedematous swelling over the
cranium with a severe bacterial osteitis. Other clinical signs include weakness, gasping, severe dyspnoea,
mucus discharge and poor growth. It is normally resistant to all antibiotics except tetracyclines and
amoxicillin.
O. rhinotracheale grows well on 5 % sheep blood agar incubated for 48 hours at 5 to 10 % CO2 at 37 º C.
They appear as grey to grey-white colonies. After primary isolation the size of the colonies may vary from
1 to 3 mm.
Seven serotypes, namely A to G have been demonstrated with agar gel precipitation tests (AGP). There
appears to be some cross reactions between serotypes A, B and E and some strains do not fit into the
present system. An ELISA has been developed that can detect the presence of antibodies in chickens.
Pasteurella serotyping using the haemagglutination method
Preparation of the Pasteurella multocida extracts
1.
Inoculate a lawn of P. multocida on blood agar a day before the extraction is to be done and incubate
overnight at 37°C.
2.
Set the water bath to 60°C about 1½ hours before required.
3.
Just before the water bath has reached 60°C, scrape the bacterial growth off the two blood agar
plates into a thick-walled centrifuge tube containing 5 ml of PBS pH 6.0. (NB put the left over PBS in
the refrigerator)
4.
Seal tube with a cloth plug and place the inoculated tube in the water bath at 60°C for 30 minutes.
5.
Cool slightly.
6.
Add 0.2 ml hyaluronidase (in freezer) to the tube.
7.
Place the extracts in the 37°C incubator for 4 hours. Shake every hour or place on a shaker.
8.
Centrifuge for 1 hour at 2000 rpm.
9.
Collect the supernatant with a Pasteur pipette, taking care not to collect bacteria and place it in a
clean MacCartney bottle.
10. Adjust the pH to 7.0 by adding drops of 4 % sodium hydroxide (NaOH). Check with a pH meter.
11. Label and place the extract in a thick-walled centrifuge tube and store in a beaker in the freezer until
required.
Preparation of the Mannheimia haemolytica extracts
1.
Inoculate a lawn of M. haemolytica on blood agar a day before the extraction is to be done and
incubate overnight at 37°C.
2.
Switch on and set the water bath to 56°C about 1½ hours before required.
3.
Just before the water bath has reached 56°C, scrape the bacterial growth off the two blood agar
plates into a thick-walled centrifuge tube containing 5 ml of normal saline.
4.
Seal tube with a cloth plug and place the inoculated tube in the water bath at 56°C for 30 minutes.
16 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter
5: Small Gram-negative rods and coccobacilli
5.
Cool the tubes and centrifuge for 1 hour at 2000 rpm.
6.
Collect the supernatant with a Pasteur pipette, taking care not to collect bacteria and place it in a
clean MacCartney bottle.
7.
Label and place the extract in a thick-walled centrifuge tube and store in a beaker in the freezer until
required.
Preparation of guinea pig red blood cells
Day before typing
1.
Bleed a guinea pig from the heart using a heparin containing (green cap) vacutainer tube and 21G
venoject needle.
2.
Mix blood well by inverting it 10 times.
3.
Divide the blood equally into two different thick-walled centrifuge tubes.
4.
Centrifuge at 3000 rpm for 10 minutes.
5.
Remove the plasma (clear supernatant) using a Pasteur pipette.
6.
Add Normal saline and mix well by inverting the tube gently 10 times. Make sure that the red blood
cells do not haemolyse.
7.
Centrifuge at 3000 rpm for 10 minutes.
8.
9.
Repeat steps 5 to 7 twice more.
Remove the saline supernatant and place the blood cells in the refrigerator.
Day of typing
1.
Label and add the following to the appropriate size measuring cylinder dependent on the number of
strains to be tested.
1 strain = 0.125 ml healthy rabbit serum (HRS) + 25 ml normal saline
2 strains = 0.25 ml HRS + 50 ml normal saline
4 strains = 0.5 ml HRS + 100 ml normal saline
2.
8 strains = 1 ml HRS + 200 ml normal saline
Remove the P. multocida and M. haemolytica extracts from the freezer and place in a water bath to
defrost.
3.
Once defrosted add 0.2 ml of the washed guinea pig red blood cells to each extract using a 1 ml
syringe and pink needle (19G).
4.
5.
Mix well and place in an incubator set to 37°C for 90 minutes.
Using a multi-channel pipette, place 100μl of saline in each well of 96-well “U” plates (one per P.
multocida and two per M. haemolytica extract)
6.
Add 100 μl of the respective antiserum to the first wells labelled on the first plate 1 – 12 and on the
second plate 13 – 17 (for M. haemolytica) and wells on one plate labelled A, B, C, D and E for P.
multocida).
17 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter
5: Small Gram-negative rods and coccobacilli
7.
Using the multi-channel pipette and starting at row “A” and continuing until row “H” make serial twofold dilutions of the antiserum. Discarding the last volume in the pipette and rinsing the tips three
times in saline.
8.
Stack the plates on top of one another to reduce evaporation.
9.
Remove the extract together with red blood cells from the incubator.
10. Centrifuge the extract and red blood cells at 2000 rpm for 5 minutes.
11. Remove the supernatant (clear fluid).
12. Fill the tube will Normal saline and mix well.
13. Repeat steps 10 to 12, 2 more times.
14. Remove the supernatant (clear fluid).
15. Add 25 ml saline and HRS mixture to each 50 ml labelled beaker (one per strain to be tested)
16. Mix the washed red blood cells from step 14 with the saline and HRS mixture and mix well.
17. Place the mixture made in step 16 into a white container and using a multi-channel pipette add 100 μl
of it into each well allocated for that strain.
18. Rinse out the tips three times in saline before adding a new strain.
19. Place the plates next to each other on white paper and leave for 1 to 2 hours then read.
Agglutination is noted by the presence of an even suspension of red cells in a well and non-agglutination
by a pellet at the bottom of a well.
REFERENCES
1.
ANGEN, O., MUTTERS, R., CAUGANT, D.A., OLSEN, J.E., & BISGAARD, M., 1999. Taxonomic
relationships of the [Pasteurella] haemolytica complex as evaluated by DNA-DNA hybridizations
and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov.,
mannheimia granulomatis comb. nov., Mannheimia glucosidal sp. nov., mannheimia ruminalis sp.
nov., and mannheimia varigena sp. nov. International Journal of Systematic Bacteriology, 49:67-86.
2.
PIECHULLA, K., MUTTERS, R., BURBACH, S., KLUSSMEIER, R., POHL, S. & MANNHEIM, W.
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