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 months old and those more than six months old was infectious coryza (117) It was only in chickens that were between and 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 691 692 SECTION II 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 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 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 SECTION II 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 months after intranasal inoculation Essential changes in 696 SECTION II 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 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— 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 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 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 days of storage at 4°C or Ϫ20°C (31) 698 SECTION II BACTERIAL DISEASES 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 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 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 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 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