73310 CH20I

20

Infectious
Coryza
Pat J. Blackall and Masakazu Matsumoto

INTRODUCTION
Infectious coryza (IC) is an acute respiratory disease of
chickens caused by Haemophilus paragallinarum. The clinical syndrome has been described in the early literature as
roup, contagious or infectious catarrh, cold, and uncomplicated coryza (124). The disease was named infectious
coryza because it was infectious and affected primarily the
nasal passages (2).

Economic Significance
The greatest economic losses result from poor growth performance in growing birds and marked reduction (10—40%)
in egg production. The disease can have significant impact
in meat chickens. In California, two cases of infectious
coryza, one complicated by the presence of Mycoplasma synoviae, caused increased condemnations, mainly due to airsacculitis, which varied from 8.0—15% (38). In Alabama, an
infectious coryza outbreak in broilers, which was not complicated by any other disease agent, caused a condemnation
rate of 69.8%, virtually all due to airsacculitis (52).
When the disease occurs in chicken flocks in developing
countries, the added presence of other pathogens and stress
factors can result in disease outbreaks that are associated
with greater economic losses than those reported in high
health flocks in developed countries. In China, outbreaks of
infectious coryza have been associated with morbidities of
20—50% and mortalities of 5—20% (32). In Morocco, outbreaks on 10 layer farms caused egg drops that ranged from
17—41% and mortalities of 0.7—10% (80). A study of village
chickens in Thailand has shown that the most common
cause of death in chickens less than 2 months old and
those more than six months old was infectious coryza

(117). It was only in chickens that were between 2 and 6
months of age that other diseases, such as Newcastle disease and fowl cholera, killed more chickens than infectious
coryza (117). Overall, considerable evidence shows that

We would like to acknowledge the contribution of Dr. Dick
Yamamoto, who was either the sole author or a coauthor for this
chapter in the 6th through 10th editions.

infectious coryza outbreaks can have a much greater impact
in developing countries than in developed countries.

Public Health Significance
The disease is limited primarily to chickens and has no
public health significance.

HISTORY
As early as 1920, Beach (1) believed that IC was a distinct
clinical entity. The etiologic agent eluded identification
for a number of years, because the disease was often
masked in mixed infections and with fowl pox in particular. In 1932, De Blieck (36) isolated the causative agent
and named it Bacillus hemoglobinophilus coryzae gallinarum.

ETIOLOGY

Classification
Based on studies conducted during the 1930s, the
causative agent of IC was classified as H. gallinarum
because of its requirement for both X-(hemin) and V(nicotinamide adenine dinucleotide —NAD) factors for
growth (41, 111). Since 1962, however, Page (87) and others (18, 49, 84, 94) have found that all isolates recovered
from cases of IC required only the V-factor for growth.

This led to the proposal and general acceptance of a new
species, H. paragallinarum (134), for organisms requiring
only the V-factor. H. gallinarum and H. paragallinarum are
identical in all other growth characteristics and diseaseproducing potential (94). These observations, in addition
to the apparent abrupt change in the X-factor requirement of all isolates recovered worldwide since 1962, have
led some workers to question the validity of tests used by
earlier workers in classifying their isolates as H. gallinarum
(94). Indeed, it has been suggested that the early descriptions of the causative agent of IC as an X- and V-factor—
dependent organism were incorrect (21).
More recently, V-factor independent isolates of H.
paragallinarum have been recovered from chickens with
coryza in South Africa (25, 53, 79). Thus, it is apparent

691


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BACTERIAL DISEASES

that classification of hemophili based strictly on in vitro
growth factor requirements may be misleading, as suggested by Kilian and Biberstein (64).

Morphology and Staining
H. paragallinarum is a gram-negative nonmotile bacterium. In 24-hour cultures, it appears as short rods or
coccobacilli 1—3 mm in length and 0.4—0.8 mm in width,
with a tendency for filament formation. A capsule may be
demonstrated in virulent strains (46, 108). The organism

undergoes degeneration within 48—60 hours, showing
fragments and ill-defined forms. Subcultures to fresh
medium at this stage will again yield the typical rodshaped morphology. Bacilli may occur singly, in pairs, or
as short chains (111).

Growth Requirements
The reduced form of NAD (NADH; 1.56—25 µg/mL
medium) (87, 98) or its oxidized form (20—100 µg/mL)
(102) is necessary for the in vitro growth of most isolates of
H. paragallinarum. The exceptions are the isolates described
in South Africa, which are NAD independent (25, 53, 79).
Sodium chloride (NaCl) (1.0—1.5%) (98) is essential for
growth. Chicken serum (1%) is required by some strains
(46), whereas others merely show improved growth with
this supplement (18). Brain heart infusion, tryptose agar,
and chicken-meat infusion are some basal media to which
supplements are added (46, 69, 102). More complex media
are used to obtain dense growth of organisms for characterization studies (4, 93, 94). The pH of various media varies
from 6.9—7.6. A number of bacterial species excrete V-factor
that will support growth of H. paragallinarum (87).
The determination of the growth factor requirements
of the avian haemophili is not an easy process. Commercial growth factor disks used for this purpose may yield a
high percentage of cultures that falsely appear to be both
X- and V-factor dependent (15). The brand of disks and
the medium to be used should be checked carefully for
their suitability. For well-equipped laboratories, the porphyrin test (63) is recommended for X-factor testing. For
classical X- and V-factor testing, the use of purified hemin
and NAD as supplements to otherwise complete media
may also be considered.
The organism is commonly grown in an atmosphere of

5% carbon dioxide; however, carbon dioxide is not an
essential requirement, because the organism is able to grow
under reduced oxygen tension or anaerobically (41, 87).
The minimal and maximal temperatures of growth are
25 and 45°C, respectively, the optimal range being 34—
42°C. The organism is commonly grown at 37—38°C.

Colony Morphology
Tiny dewdrop, nonhemolytic colonies up to 0.3 mm in
diameter develop on suitable media. In obliquely transmitted light, mucoid (smooth) iridescent, rough noniridescent, and other intermediate colony forms have been
observed (48, 94, 107, 103).

Biochemical Properties
The ability to reduce nitrate to nitrite and ferment glucose
without the formation of gas is common to all the avian
haemophili. Oxidase activity, the presence of the enzyme
alkaline phosphatase, and a failure to produce indole or
hydrolyse urea or gelatin are also uniform characteristics
(7). Considerable confusion surrounds the carbohydrate
fermentation patterns of the avian haemophili. Much of
the variability recorded in the literature may be due to the
use of different basal media. False-negative results are
associated mainly with poor growth and can be a significant problem (4). In general, recent studies have used a
medium consisting of a phenol red broth containing 1%
(w/v) NaCl, 25 µg/mL NADH, 1% (v/v) chicken serum,
and 1% (w/v) carbohydrate. For routine identification, the
use of the phenol red broth just described and a dense
inoculum is a most suitable approach for determining carbohydrate fermentation patterns. Alternatively, agarbased methods (4, 115) may be used.
A range of organisms that superficially resemble H.
paragallinarum can be found in chickens. In particular,

organisms once known as Haemophilus avium are common in chickens and are regarded as nonpathogenic (51).
Based on DNA hybridization studies, isolates of H. avium
were found to be comprised of at least three DNA homology groups (81). They have been named Pasteurella avium,
P. volantium, and Pasteurella species A. Not all isolates of H.
avium, however, can be assigned to these three new taxa
solely on the basis of phenotypic properties (6). Table 20.1
presents those properties that allow a full identification of
the avian haemophili. The failure of H. paragallinarum to
ferment either galactose or trehalose and its lack of catalase clearly separates this organism from the other avian
haemophili. The properties shown in the table for H. paragallinarum have been found to be typical of isolates from
Argentina, Australia, Brazil, China, Germany, Indonesia,
Japan, Kenya, Malaysia, and the United States (18, 20, 32,
51, 61, 67, 84, 91, 94, 115, 131). The main characteristics
that differentiate the NAD-independent from the NADdependent H. paragallinarum are that the former does not
have ß-galactosidase activity and does not ferment maltose (79).

Susceptibility to Chemical and Physical Agents
H. paragallinarum is a delicate organism that is inactivated
rather rapidly outside the host. Infectious exudate suspended in tap water is inactivated in 4 hours at ambient
temperature; when suspended in saline, the exudate is infectious for at least 24 hours at 22°C. Exudate or tissue remains
infectious when held at 37°C for 24 hours and, on occasion,
up to 48 hours; at 4°C, exudate remains infectious for several days. At temperatures of 45—55°C, hemophili are killed
within 2—10 minutes. Infectious embryonic fluids treated
with 0.25% formalin are inactivated within 24 hours at 6°C,
but the organism survives for several days under similar
conditions when treated with thimerosal, 1:10,000 (125).
The organism may be maintained on blood agar plates
by weekly passages. Young cultures maintained in a “can-

CHAPTER 20
Table 20.1.
Property

693

INFECTIOUS CORYZA

Differential tests for the avian haemophili.
Hemophilus paragallinarium

H. avium

Pasteurella avium

P. volantium

Pasteurella species A

Ϫ
Ϫ
Ϫ
ϩ

Yellow V
ϩ
ϩ
V

Ϫ

ϩ
ϩ
Ϫ

Yellow U
ϩ
ϩ
ϩ

Ϫ
ϩ
ϩ
V

Ϫ
Ϫ
ϩ
ϩ
V
V
Ϫ

V
ϩ
V
V
V
ϩ
ϩ

Ϫ
ϩ
Ϫ
Ϫ
Ϫ
ϩ
ϩ

Ϫ
ϩ
ϩ
ϩ
V
ϩ
ϩ

ϩ
ϩ
V
V
Ϫ
ϩ
ϩ

Pigment
Catalase
Growth in air
ONPG
Acid from
Arabinose

Galactose
Maltose
Mannitol
Sorbital
Sucrose
Trehalose

Susceptibility to Chemical and Physical Agents
U = usually; V = variable; ϩ = positive; Ϫ = negative.

dle jar” will remain viable for 2 weeks at 4°C. Chicken
embryos 6—7 days old may be inoculated with single
colonies or broth cultures via the yolk sac; yolk from
embryos dead in 12—48 hours will contain a large number
of organisms that may be frozen and stored at Ϫ20 to
Ϫ70°C or lyophilized (124). A good suspension medium
for lyophilization of H. paragallinarum from agar cultures
is used at the Animal Research Institute and contains 6%
sodium glutamate and 6% bacteriological peptone (filter
sterilized). After any storage, whether frozen or
lyophilized, revival should include inoculation of a suitable liquid growth medium (egg inoculation is even better) as well as an agar medium.

Strain Classification
Antigenicity. Page (87, 88) classified his organisms of H.
paragallinarum with the plate agglutination test using
whole cells and chicken antisera into serovars A, B, and C.
Although Page’s serovar A strain 0083 and B strain 0222
are available today, all the serovar C strains were lost during the mid-1960s. Matsumoto and Yamamoto (73) isolated strain Modesto, which was later classified as a strain
of serovar C by Rimler et al. (96). It is also possible to use a
hemagglutination inhibition (HI) test to serotype isolates

by the Page scheme (11). This HI test uses fixed chicken
erythrocytes and results in fewer nontypable isolates than
the original agglutination technology (11) and is now the
recommended technique when performing serotyping by
the Page scheme.
The distribution of Page serovars differs from country
to country. Page serovar A has been reported in China
(32) and Malaysia (129); serovar C in Taiwan (71);
serovars A and B in Germany (47); serovars A and C in
Australia (10); and serovars A, B and C in Argentina (115),
Brazil (20), Indonesia (91, 114), Mexico (42), the Philippines (82), South Africa (26), Spain (89), and the United
States (87, 88).
Another method of assigning isolates of H. paragallinarum to a Page serovar is based on the use of a panel of
monoclonal antibodies developed by workers in Japan
(23), but the technique is available only in a few laborato-

ries due to the limited availability of the monoclonal antibodies. Other sets of MABs have been described but either
lack serovar-specificity (28, 133) or detect only Page
serovar A (112).
There have been suggestions that Page serovar B is not
a true serovar, but rather consists of variants of serovar A
or C that have lost their type-specific antigen (69, 108).
Recent studies, however, have shown conclusively that
Page serovar B is a true serovar (119).
Kume et al. (66) proposed an alternative serologic classification based on an HI test using potassium thiocyanate-treated and -sonicated cells, rabbit hyperimmune
serums, and glutaraldehyde-fixed chicken erythrocytes. In
the original study, Kume et al. (66) recognized three
serogroups and seven serovars. The terminology of the
Kume scheme has been altered so that the Kume
serogroups match the Page serovars of A, B, and C (13).

Thus, the nine currently recognized Kume serovars are A1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4 (13). Some
Kume serovars seem to be unique in terms of geographic
origin—serovar A-3 has been found only in Brazil, serovar
C-3 only in South Africa, and serovars A-4 and C-4 only in
Australia (13, 40, 66). Many isolates that were nontypable
in the Page scheme by agglutination tests were typed easily using the Kume scheme (40). Fernández et al. (43) have
recently reported the presence of Kume serovars A-1, A-2,
B-1, and C-2 in isolates of H. paragallinarum from Mexican
chickens.
The Kume scheme has not been widely applied, as it is
technically demanding to perform. Hence, only a few laboratories are able to perform the serotyping on a routine
basis.
Other serological tests described in the literature
include an agar-gel precipitin (AGP) test (50) and a serum
bactericidal test (105). Neither of these tests has been
widely used.

Immunogenicity or Protective Characteristics. Infectious coryza is relatively unique among common bacterial
infections in that a bacterin (inactivated whole cell vaccine) is protective against the disease when the bacterin is


694

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BACTERIAL DISEASES

adequately prepared. From the early days of bacterin production, it was obvious that protection was limited (73).
Later studies confirmed a correlation between Page
serovars and immunotype specificity (19, 69, 96). Chickens vaccinated with a bacterin prepared from one serovar

were protected against homologous challenge only. Evidence suggests that the cross-protection within Page
serovar B is only partial (120).
Only incomplete results are available on immunospecificity within the serogroups recognized by the Kume
scheme. Significant cross-protection has been shown
between Kume serovars C-1 and C-2 as well as between C2 and C-4 (8 ,19, 69).
Only one serovar, B-1, exists within serogroup B of the
Kume scheme. However, reports have been made of
undefined heterogeneity within the B serogroup. Bivalent
vaccines containing Page serovars A and C provide protection against Page serovar B strain Spross but not
against two South African isolates of Page serovar B (120).
Furthermore, only partial cross-protection exists within
various strains of Page serovar B (120). Poor vaccine protection against IC due to serovar B strains in Argentina
have been explained by antigenic differences between
field isolates and the “standard” serovar B strains in commercial vaccines from North America or Europe (116).
One report supports the genetic uniqueness of serovar B
strains isolated in Argentina (24). Vaccination/challenge
exposure studies are needed to study the antigenicity and
immunospecificity of recent serovar B isolates.
In both Argentina and Brazil, isolates of Page serovar
A are not recognized by a monoclonal antibody specific
for this serovar (20, 115). It has been speculated that
these “variant” Page serovar A isolates may be sufficiently
different from typical serovar A vaccine strains to cause
vaccine failure (115).
South African workers have suggested that Kume
serovar C-3 as well as other serovars of NAD-independent
H. paragallinarum are so antigenically different that they
are causing vaccine failure (26, 27, 54). However, it has
been shown that a commercial vaccine, specified as containing serovars A, B, and C without details of the actual
strains, provided acceptable levels of protection against

NAD-independent isolates of Page serovar A and Kume
serovar C-3 (60).
Overall, these recent results and field observations
clearly indicate the need for further vaccination/challenge studies. At this stage of our knowledge, no clear-cut
definitive publications negate the existence of cross-protection within Page serovars and Kume serogroups.
Indeed, the only publication to date, while not providing
full details of the vaccine seed strains, suggests that serological variation within a Page serovar is not a cause of
vaccine failure (60). There is no doubt that, on an ongoing basis, debate will continue on the topic of whether
commercially available trivalent vaccines, containing
serovars A, B, and C, give adequate protection if there are
significant antigenic differences between vaccine and
field strains.

Molecular Techniques. DNA fingerprinting by restriction endonuclease analysis has been shown to be a suitable typing technique with patterns being stable in vitro
and in vivo (17, 12). Restriction endonuclease analysis has
proven useful in epidemiologic studies (17). Ribotyping is
another molecular technique that has proven useful—
being used to confirm that the recent NAD-independent
H. paragallinarum isolates from South Africa are clonal in
nature (78) as well as examining the epidemiologic relationships among Chinese isolates of H. paragallinarum
(76). ERIC-PCR, a DNA fingerprinting method that uses
the polymerase chain reaction technique, has been shown
to be capable of strain typing (62). The technique of multilocus enzyme electrophoresis has been used to examine
the genetic diversity of H. paragallinarum isolates (24).
These nucleic acid techniques (including the speciesspecific PCR discussed later in this chapter) are advancing
to the stage where they offer a rapid and convenient
method for identification and typing. These techniques
are likely to replace time-consuming and cumbersome
cultural, biochemical, and serological means of identification and typing in the near future.

Pathogenicity. As a general observation, the pathogenicity of H. paragallinarum can vary according to both the
growth conditions and passage history of the isolate and
the state of the host. Some specific evidence of variation
in pathogenicity exists amongst H. paragallinarum isolates.
Yamaguchi et al. (119) found that one of four strains of H.
paragallinarum serovar B failed to produce clinical signs.
Horner et al. (54) have suggested that the NAD-independent isolates may cause airsacculitis more commonly than
the classic NAD-dependent H. paragallinarum isolates.

Virulence Factors
A range of factors has been associated with the pathogenicity of H. paragallinarum. Considerable attention has
been paid to HA antigens. In both Page serovar A and C,
mutants lacking HA activity have been used to demonstrate that the HA antigen plays a key role in colonization
(103, 123).
The capsule has also been associated with colonization
and has been suggested to be the key factor in the lesions
associated with IC (103, 110). The capsule of H. paragallinarum has been shown to protect the organism against
the bactericidal activity of normal chicken serum (106). It
has been suggested that a toxin released from capsular
organisms during in vivo multiplication was responsible
for the clinical disease (65).
H. paragallinarum can acquire iron from chicken and
turkey transferrin, suggesting that iron sequestration may
not be an adequate host defense mechanism (85). In contrast, two strains of H. avium were unable to acquire iron
from these transferrins, despite apparently having the
same receptor proteins (85).
Crude polysaccharide extracted from H. paragallinarum
is toxic to chickens and may be responsible for the toxic
signs that may follow the administration of bacterin (55).

CHAPTER 20

695

INFECTIOUS CORYZA

The role, if any, of this component in the natural occurrence of the disease is unknown.

PATHOBIOLOGY AND EPIZOOTIOLOGY

Incidence and Distribution
Infectious coryza occurs whereever chickens are raised.
The disease is a common problem in the intensive
chicken industry; significant problems have been reported
in California, southeastern United States, and most
recently in the northeastern regions of the United States.
The disease has also been reported in other, less intensive
situations. As an example, infectious coryza has been a
problem in kampung (village) chickens in Indonesia (91).

Natural and Experimental Hosts
The chicken is the natural host for H. paragallinarum. Several reports indicate that the village chickens of Asia are as
susceptible to infectious coryza as normal commercial
breeds (91, 130). Although there have been reports of IC
due to H. paragallinarum in a number of bird species other
than chickens, reviewed by Yamamoto (126), these
reports need to be interpreted carefully. As a range of
hemophilic organisms, none of which are H. paragallinarum, have been described in birds other than chickens
(37, 45, 90), only those studies that involve detailed bacteriology can be regarded as definitive proof of the presence of H. paragallinarum in birds other than chickens.

The following species are refractory to experimental infection: turkey, pigeon, sparrow, duck, crow, rabbit, guinea
pig, and mouse (124, 125).

Age of Host Most Commonly Affected. All ages are susceptible (126), but the disease is usually less severe in
juvenile birds. The incubation period is shortened, and
the course of the disease tends to be longer in mature
birds, especially hens with active egg production.

Transmission, Carriers, and Vectors
Chronic or healthy carrier birds have long been recognized as the main reservoir of infection. The application
of molecular fingerprinting techniques has confirmed the
role of carrier birds in the spread of IC (17). Infectious
coryza seems to occur most frequently in fall and winter,
although such seasonal patterns may be coincidental to
management practices (e.g., introduction of susceptible
replacement pullets onto farms where IC is present). On
farms where multiple-age groups are brooded and raised,
spread of the disease to successive age groups usually
occurs within 1—6 weeks after such birds are moved from
the brooder house to growing cages near older groups of
infected birds (33). Infectious coryza is not an egg-transmitted disease.
Whereas the sparrow could not be implicated as a vector, epidemiologic studies suggested that the organism
may be introduced onto isolated ranches by the airborne
route (127).

Incubation Period
The characteristic feature is a coryza of short incubation
that develops within 24—48 hours after inoculation of
chickens with either culture or exudate. The latter will
more consistently induce disease (94). Susceptible birds

exposed by contact to infected cases may show signs of the
disease within 24—72 hours. In the absence of a concurrent
infection, IC usually runs its course within 2—3 weeks.

Clinical Signs
The most prominent features are an acute inflammation
of the upper respiratory tract including involvement of
nasal passage and sinuses with a serous to mucoid nasal
discharge, facial edema, and conjunctivitis. Figure 20.1
illustrates the typical facial edema. Swollen wattles may
be evident, particularly in males. Rales may be heard in
birds with infection of the lower respiratory tract.
A swollen head—like syndrome associated with H. paragallinarum has been reported in broilers in the absence of
avian pneumovirus, but in the presence or absence of
other bacterial pathogens such as M. synoviae and M. gallisepticum (38, 100). Arthritis and septicemia have been
reported in broiler and layer flocks, respectively, in which
the presence of other pathogens has contributed to the
disease complex (100).
Birds may have diarrhea, and feed and water consumption usually is decreased; in growing birds, this means an
increased number of culls; and in laying flocks, this
means a reduction in egg production (10—40%). A foul
odor may be detected in flocks in which the disease has
become chronic and complicated with other bacteria.

Morbidity and Mortality. IC is usually characterized by
low mortality and high morbidity. Variations in age and
breed may influence the clinical picture (3). Complicating
factors such as poor housing, parasitism, and inadequate
nutrition may add to severity and duration of the disease.
When complicated with other diseases such as fowl pox,

infectious bronchitis, laryngotracheitis, Mycoplasma gallisepticum infection, and pasteurellosis, IC is usually more
severe and prolonged, with resulting increased mortality
(100, 124).

Pathology
Gross. H. paragallinarum produces an acute catarrhal
inflammation of mucous membranes of nasal passages
and sinuses. Frequently, a catarrhal conjunctivitis and
subcutaneous edema of face and wattles occur. Typically,
pneumonia and airsacculitis are rarely present; however,
reports of outbreaks in broilers have indicated significant
levels of condemnations (up to 69.8%) due to airsacculitis
(Fig. 20.2), even in the absence of any other recognized
viral or bacterial pathogens (38, 52).

Microscopic. Fujiwara and Konno (44) studied the
histopathologic response of chickens from 12 hours to 3
months after intranasal inoculation. Essential changes in


696

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BACTERIAL DISEASES

20.1. Chickens artificially infected with Haemophilus paragallinarum. A. Mature male with coryza and facial edema.
B. Mature female showing conjunctivitis, nasal discharge, and open-mouth breathing.

changes and cell damage leading to coryza. A dissecting

fibrinopurulent cellulitis similar to that seen in chronic
fowl cholera has been reported in broiler and layer chickens (38).

Immunity

20.2. Field infection with IC showing caseopurulent air
sac lesions.

the nasal cavity, infraorbital sinuses, and trachea consisted of sloughing, disintegration, hyperplasia of mucosal
and glandular epithelia, and edema and hyperemia with
heterophil infiltration in the tunica propria of the mucous
membranes. Pathologic changes first observed at 20 hours
reached maximum severity by 7—10 days, with subsequent
repair occurring within 14—21 days. In birds with involvement of the lower respiratory tract, acute catarrhal bronchopneumonia was observed, with heterophils and cell
debris filling the lumen of secondary and tertiary bronchi;
epithelial cells of air capillaries were swollen and showed
hyperplasia. Catarrhal inflammation of air sacs was characterized by swelling and hyperplasia of the cells, with
abundant heterophil infiltration. In addition, a pronounced infiltration of mast cells was observed in the
lamina propria of the mucous membrane of the nasal cavity (110). The products of mast cells, heterophils, and
macrophages may be responsible for the severe vascular

Chickens that have recovered from active infection possess varying degrees of immunity to reexposure. Pullets
that have experienced IC during their growing period are
generally protected against a later drop in egg production.
Resistance to reexposure among individual birds may
develop as early as 2 weeks after initial exposure by the
intrasinus route (101).
Experimentally infected chickens develop a crossserovar (Page scheme) immunity (95). In contrast, as discussed earlier, bacterins provide only serovar-specific
immunity (19, 70, 96). This suggests that cross-protective
antigens are expressed in vivo that are either not expressed

or expressed at very low levels in vitro.
The protective antigens of H. paragallinarum have not
been definitively identified. It has been suggested that the
capsule of H. paragallinarum contains protective antigens
(104). Using both a Page serovar A and C strains, a crude
polysaccharide extract was shown to provide serovar-specific protection (55).
Considerable attention has been paid to the role of HA
antigens as protective antigens. It has been long noted
that for Page serovar A organisms, a close correlation
exists between HI titer and both protection (70, 86) and
nasal clearance of the challenge organism (65) in vaccinated chickens. Purified HA antigen from a Page serovar A
organism has been shown to be protective (56). Takagi
and colleagues have shown that a monoclonal antibody
specific for the HA of Page serovar A provides passive pro-


CHAPTER 20

INFECTIOUS CORYZA

tection and that the HA antigen purified by use of this
antibody is also protective (112, 113).
Based on studies conducted to date, considerable evidence shows that the protective antigens of H. paragallinarum are surface located. The antigens implicated have
been the antigens detected during Page serotyping, HA
antigens, and some component or components of the
polysaccharide content of the cell. It seems probable that
a number of different antigens (outer-membrane proteins,
polysaccharides, lipopolysaccharides) are all likely to be
involved.

DIAGNOSIS

Isolation and Identification of Causative Agent
Although H. paragallinarum is considered to be a fastidious organism, it is not difficult to isolate, requiring simple
media and procedures. Specimens should be taken from
two or three chickens in the acute stage of the disease (1—
7 days’ incubation). The skin under the eyes is seared with
a hot iron spatula, and an incision is made into the sinus
cavity with sterile scissors. A sterile cotton swab is inserted
deep into the sinus cavity where the organism is most
often found in pure form. Tracheal and air sac exudates
also may be taken on sterile swabs. The swab is streaked
on a blood agar plate, which is then cross-streaked with a
Staphylococcus culture and incubated at 37°C in a large
screw-cap jar in which a candle is allowed to burn out
(Fig. 20.3). Staphylococcus epidermidis (87) or S. hyicus (18),
which are commonly used as “feeders,” should be
pretested because not all strains actively produce the Vfactor. Terzolo et al. (115) have reported the successful use
of an isolation medium that contains selective agents
which inhibit the growth of gram-positive bacteria. This

20.3. Satellite phenomenon. Tiny dewdrop colonies of
Haemophilus paragallinarum growing adjacent to
Staphylococcus culture (broad streak) on a blood agar
plate.

697
medium has the advantage of not using either a “feeder”
organism or additives such as NADH.
At the simplest level, IC may be diagnosed on the basis

of a history of a rapidly spreading disease in which coryza
is the main manifestation, combined with the isolation of
a catalase-negative bacterium showing satellitic growth.
At this level, the sinus exudate or culture should be inoculated into two or three normal chickens by the intrasinus
route. The production of a coryza in 24—48 hours is diagnostic; however, the incubation period may be delayed up
to 1 week if only a few organisms are present in the inoculum, such as in long-standing cases.
Better equipped laboratories should attempt a more
complete biochemical identification as described earlier.
Additional studies of this nature are essential when isolates of NAD-independent H. paragallinarum are suspected. To perform this biochemical testing, the suspect
isolates are best grown in pure culture on medium that
does not require the addition of a nurse colony. Many different media have been developed to support the growth
of H. paragallinarum (69, 86, 94, 115). The medium
described by Terzolo et al. (115) is particularly suited for
those laboratories that find the cost of such ingredients as
NADH and albumin expensive. The carbohydrate fermentation tests shown in Table 20.1 can be done in either a
phenol red broth base (94) or in an agar plate format (4).
The agar plate method can be performed in conventional
petri dishes (9 cm), allowing multiple isolates to be tested
at once, or in small petri dishes (2 cm), allowing one to
three isolates to be economically characterized. The agar
plate method (4) has also been modified to be performed
as a tube method (115).
A PCR test specific for H. paragallinarum has been
developed (30). This test is rapid (results available within
6 hours compared with days for conventional techniques)
and has been shown to recognize all H. paragallinarum isolates tested, including the NAD-independent H. paragallinarum from South Africa and the variant Page serovar A
isolates and the unusual Page serovar B isolates from
Argentina (30). The PCR, termed the HP-2 PCR, has been
validated for use on colonies on agar or on mucus
obtained from squeezing the sinus of live birds (30).

When used directly on sinus swabs obtained from artificially infected chickens in pen trials performed in Australia, the HP-2 PCR has been shown to be the equivalent
of culture—but much more rapid (30). When used in
China, direct PCR examination of sinus swabs outperformed traditional culture when used on routine diagnostic submissions (29). The problems of poor samples,
delayed transport, and poor quality (but expensive) media
mean that culture will have a higher failure rate in developing countries than in developed countries—making the
PCR an attractive diagnostic option.
The HP-2 PCR is a robust test; sinus swabs stored for up
to 180 days at 4°C or Ϫ20°C were positive in the PCR (31).
In contrast, culture of known positive swabs failed to
detect H. paragallinarum after 3 days of storage at 4°C or
Ϫ20°C (31).


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The HP-2 PCR has proven very useful in South Africa
where the diagnosis of infectious coryza is complicated by
the presence of NAD-independent H. paragallinarum,
Ornithobacterium rhinotracheale, as well as the traditional
form of NAD-dependent H. paragallinarum (77).

Serology
No totally suitable serological test exists for the diagnosis
of infectious coryza. However, despite this absence of a
“perfect” test, serological results are often useful for retrospective/epidemiological studies in the local area. A
review of the techniques that have been used in the past

is presented by Blackall et al. (16).
At this time, the best available test methodology is the
HI test. Although a range of HI tests have been described,
three main forms of HI tests have been recognized—these
being termed simple, extracted, and treated HI tests (22).
Full details of how to perform these tests are available
elsewhere (22). In the following text, the advantages and
disadvantages of the three HI tests are briefly and critically
discussed.
The simple HI is based on whole bacterial cells of Page
serovar A H. paragallinarum and fresh chicken erythrocytes (58). Although simple to perform, this HI test can
detect antibodies only to serovar A. The test has been
widely used to both detect infected as well as vaccinated
chickens (16).
The extracted HI test is based on KSCN-extracted and
sonicated cells of H. paragallinarum and glutaraldehydefixed chicken erythrocytes (109). This extracted HI test
has been validated mainly for the detection of antibodies
to Page serovar C organisms. The test has been shown to
be capable of detecting a serovar-specific antibody
response in Page serovar C vaccinated chickens (109). A
major weakness with this assay is that, in chickens
infected with serovar C, the majority of the birds remain
seronegative (121).
The treated HI test is based on hyaluronidase-treated
whole bacterial cells of H. paragallinarum and formaldehyde-fixed chicken erythrocytes (122). The treated HI has
not been widely used or evaluated. It has been used to
detect antibodies to Page serovars A, B, and C in vaccinated chickens with only serovar A and C vaccinated
chickens yielding high titers (120). The test has been used
to screen chicken sera in Indonesia for antibodies arising
from infection with serovars A and C (114).

Vaccinated chickens with titers of 1:5 or greater in the
simple or extracted HI tests have been found to be protected against subsequent challenge (109). Enough knowledge or experience is not yet available to draw any sound
conclusions on whether there is a correlation between
titer and protection for the treated HI test.
An alternative serological test is a monoclonal antibody-based blocking ELISA, the B-ELISA (132). While having shown very good specificity and acceptable levels of
sensitivity, this test has several drawbacks. As there are
only monoclonal antibodies for Page serovar A and C, the
assay can detect only antibodies to these two serovars.

The monoclonal antibodies that form the heart of the
assays are not commercially available, limiting access to
the assays. Finally, some isolates of H. paragallinarum do
not react with the monoclonal antibodies and, thus,
infections associated with these isolates cannot be
detected with these ELISAs (132). This ELISA has not been
widely evaluated, and there is no knowledge about any
correlation between ELISA titer and protection. The
reduced sensitivity of the ELISA for serovar C infections
indicates that the test would have to be used as a flock test
only (132). A B-ELISA kit based on the preceding B-ELISA
has been developed (75). Based on pen trial data, the
serovar A B-ELISA kit had a sensitivity of 95% and a specificity of 100%. The serovar C B-ELISA kit had a sensitivity
of 73% and a specificity of 100% (75).
Overall, the serological test of choice remains either
the simple HI test (58) for either infections or vaccinations associated with serovar A, the extracted or treated
HI tests (109, 122) for vaccinations associated with
serovar C, and the treated HI test (122) for infections
associated with serovar C. There has been so little work
performed on serological assays for infections or vaccinations associated with serovar B that it is not possible to
recommend any test.

Differential Diagnosis
Infectious coryza must be differentiated from other diseases, such as chronic respiratory disease, chronic fowl
cholera, fowl pox, ornithobacterosis, swollen head syndrome, and A-vitaminosis, which can produce similar
clinical signs. Because H. paragallinarum infections often
occur in mixed infections, one should consider the possibility of other bacteria or viruses as complicating IC, particularly if mortality is high and the disease takes a
prolonged course (see “Pathogenicity; Morbidity and
Mortality”).

INTERVENTION STRATEGIES

Management Procedures
Because recovered carrier birds are the main source of infection, practices such as buying breeding males or started
chicks from unknown sources should be discouraged.
Only day-old chicks should be secured for replacement
purposes unless the source is known to be free of IC. Isolation rearing and housing away from old stock are desirable practices. To eliminate the agent from a farm, it is
necessary to depopulate the infected or recovered flock(s)
because birds in such flocks remain reservoirs of infection.
After cleaning and disinfection of the equipment and
houses, the premises should be allowed to remain vacant
for 2—3 weeks before restocking with clean birds.

Vaccination
Types of Vaccines. Commercial IC bacterins are widely
available. As the literature of the various factors influencing the efficacy of bacterins has been reviewed (9), only
key points are considered here. Although bacterins have


CHAPTER 20

699

INFECTIOUS CORYZA

been prepared from chicken embryos (33), broth (34), and
cell culture (118), most commercial products are currently
based on broth-grown cultures. They must contain at least
108 colony-forming units/mL to be effective (73). The following section reviews only the literature on broth-based
bacterins.
There is disagreement in the literature as to the effect
of different inactivating agents on the efficacy of bacterins. Thimerosal has been shown to be effective (19, 35,
73), as has formalin (34, 99). In three studies directly comparing formalin and thimerosal, formalin reduced the efficacy of the vaccines, although there was evidence that the
effect was adjuvant specific. The use of formalin, compared with thimerosal, resulted in a reduction of the efficacy of aluminum hydroxide—based vaccines in two
studies (19, 35) but had no such effect in a third (72). Similarly, formalin, compared with thimerosal, impaired the
efficacy of a vaccine containing chrome alum as an adjuvant (73) and another based on mineral oil (35). These
studies suggest that while vaccines containing formalin as
the inactivating agent can be protective, it is possible that
a similar vaccine containing thimerosal would be even
more efficient.
A number of adjuvants have been shown to be effective for IC bacterins, in particular, aluminum hydroxide
gel, mineral oil, and saponin (59, 14, 34, 35, 70, 72, 73,
93, 60). The report of mineral oil being less effective than
aluminum hydroxide gel (93) may result from a formulation problem rather than any inherent deficiency in the
ability of mineral oil to act as an effective adjuvant. As
with any bacterin that contains adjuvants, particularly
mineral oil, the potential adverse reaction at the site of
injection (39) should be considered when using such
products.
As inactivated IC bacterins provide protection only
against the Page serovars included in the vaccine, it is

vital that bacterins contain the serovars present in the
target population. The confirmed existence of Page
serovar B as a true serovar with full pathogenicity, as well
as its widespread occurrence, means that this serovar
must be included in inactivated bacterins in areas where
serovar B is present. However, because different strains of
serovar B provide only partial cross-protection among
themselves (120), it may be necessary to prepare an autogenous bacterin for use in areas where the B serovar is
endemic.
Because dissociation of H. paragallinarum has been
reported (107), care should be taken in selecting the
proper seed culture, media, and incubation period to
obtain the most immunogenic product.
Mixed bacterins containing inactivated infectious
bronchitis virus, Newcastle disease virus, and H. paragallinarum have been described (86, 128). A combined H.
paragallinarum—M. gallisepticum bacterin was reported to
provide protection against transient and chronic coryza
(97). However, antibody response to H. paragallinarum
was suppressed in chickens inoculated with a similar
product (74).

Field Vaccination Protocol and Regimes. Bacterins
generally are injected in birds between 10—20 weeks of
age and yield optimal results when given 3—4 weeks prior
to an expected natural outbreak. Two injections given
approximately 4 weeks apart before 20 weeks of age seem
to result in better performance of layers than a single
injection. When administered to growing birds, the bacterin reduces losses from complicated respiratory disease.
Both subcutaneous and intramuscular routes have been
effective (19, 35, 73). Injection of the bacterin into the leg

muscle gave better protection than when injected into
the breast muscle (57). The intranasal route was not effective (19). Oral delivery of an IC bacterin was effective, but
this route required 100 times as many cells as with the
parenteral route (83). Significant immunity has been
demonstrated for about 9 months following vaccination
(19, 68, 73).

TREATMENT
Various sulfonamides and antibiotics are useful in alleviating the severity and course of IC and have been reviewed
(16). It should be noted that drug resistance does develop
(5, 92), and hence the performance of antimicrobial sensitivity tests is recommended. Strains of H. paragallinarum
resistant to various antibiotics did not carry plasmids (5).
Relapse often occurs after treatment is discontinued and
the carrier state is not eliminated (125). Erythromycin
and oxytetracycline are two commonly used antibiotics.

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