Chapter 6 Infectious Coryza 1 6 INFECTIOUS CORYZA Pat J.

Blackall SUMMARY Infectious coryza, an acute upper respiratory tract disease of chickens, is caused by the bacterium Avibacterium paragallinarum. The main impact of the disease is a drop in egg production. Other manifestations such as airsacculitis in broilers, swollen head-like syndrome, arthritis, and septicemia are either unusual or probably due to complications associated with other infectious agents. Agent Identification. Diagnosis of infectious coryza is preferably made by the isolation and identification, by biochemical properties, of the bacterium. A polymerase chain reaction (PCR) test, which can be applied either to suspect colonies or directly to samples from chickens, is now available. The PCR test is particular suited for those laboratories that lack suitable expertise and experience in the growth and phenotypic identification of Av. paragallinarum or where samples are transported for long periods to the laboratory. Although most isolates of Av. paragallinarum are dependent upon V factor for growth in artificial media (meaning they show the traditional satellitic growth), some isolates are V factor-independent. This variation in growth factor requirements, along with the existence of nonpathogenic V factor-dependent organisms, increases the need for biochemical identification or the use of the PCR test. Serotyping of isolates is important to guide the use of vaccines. Serologic Detection in the Host. A range of serologic tests to detect antibodies has been described with hemagglutination-inhibition tests now being the most widely used. INTRODUCTION Infectious coryza is an acute respiratory disease of chickens. The clinical syndrome has been recognized since the 1930s and has been described in the early literature as roup, contagious or infectious catarrh, and uncomplicated coryza (6). Early workers identified the causative agent as Haemophilus gallinarum, an organism that required both X (hemin) and V (nicotinamide adenine dinucleotide; NAD) factors for growth in vitro. However, from the 1960s to the 1980s, all isolates of the disease-producing agent have been shown to require only V factor and have been termed Haemophilus paragallinarum (6). V factor-independent H. paragallinarum isolates have been encountered in the Republic of South Africa (21) and Mexico (15). Most recently, a polyphasic taxonomic study has shown that the Haemophilus paragallinarum is not a member of the genus Haemophilus (2). Thus, H. paragallinarum was allocated to a new genus Avibacterium along with several other chicken associated members of the family Pasteurellaceae (2). Hence, the causal agent of infectious coryza is now called Avibacterium paragallinarum, an organism that can be either V factor-dependent or -independent. This text will use the new terminology of Av. paragallinarum even if the primary publications being cited used the older terminologies. The disease occurs worldwide and causes economic losses due to an increased number of culls and a marked reduction from 10% to more than 40% in egg production, particularly on multiage farms. The disease is essentially limited to chickens and does not threaten public health. CLINICAL DISEASE Infectious coryza may occur in growing chickens and layers. The most common clinical signs are nasal discharge, facial swelling, lacrimation, anorexia, and diarrhea. Decreased feed and water consumption retards growth in young stock and reduces egg production in laying flocks (6). In layers, the disease can have a much greater impact than the relatively simple scenario described above. As an example, an outbreak of the disease in older layer birds in California, which was not associated with any other pathogen, caused a total mortality of 48% and a drop in egg production from 75% to 15.7% over a 3 week period (8). 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 a swollen head like syndrome and increased condemnations, mainly due to airsacculitis, that varied from 8.0% to 15% (14). 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% to 50% and mortalities of 5% to 20% (13). In India, outbreaks of infectious coryza are often complicated with fowl cholera and can result in high mortalities e.g. one farm experienced 50% mortality in a 14,000 bird layer flock (33). Arthritis and septicemia, possibly complicated by the presence of other pathogens, have been reported in broiler and layer flocks in South America (27). Infectious coryza can also be a problem in the village production system e.g. there are reports of outbreaks in such chickens in Thailand (32) and Indonesia (24). Overall, there is considerable evidence that infectious coryza outbreaks can have a much greater impact in developing countries than in developed countries. SAMPLE COLLECTION Two to three acutely diseased chickens should be killed and the skin over their sinuses seared with a heated spatula. The skin is then incised with a sterile scalpel blade, and a sterile cotton swab is inserted into the sinus cavity. Typically, in the early phase of the disease, Av. paragallinarum is found in pure culture in the sinus. Swabs of the trachea and air sacs may be taken, although the organism is less frequently isolated from these areas. Avibacterium paragallinarum is a fragile organism that does not survive for more than 5 hr outside of birds. Sinus swabs held in Ames Transport medium (without charcoal) can yield positive cultures for up to 8 days at transport temperatures of either 250C or room temperature (9). Live bird sampling is also possible. In this technique, gentle milking pressure is exerted on the sinus area and mucus forced from the nostril. The expressed mucus should be sampled by a bacteriologic loop, with care being taken to avoid touching the surface of beak or nostril. A swab of the expressed mucus is also the optimal sample for the Av. paragallinarum PCR. Swabs collected by this method will still yield a PCR positive reaction after storage in glycerol-enriched phosphate buffered saline for 180 days at either 4 C or -20 C (12). PREFERRED CULTURE MEDIA AND SUBSTRATES Artificial Media Blood agar is commonly used for the isolation of Av. paragallinarum. The medium is prepared from a dehydrated base such as Bacto-tryptose-blood-agar base (Difco, Detroit, Mich.) and is enriched with 5% erythrocytes, which may be from any animal. The inoculum is streaked onto the blood agar plate in the conventional manner, after which the plate is cross-streaked with a nurse culture. Blood agar is deficient in V factor, and the role of the nurse culture is to excrete excess V factor to support the growth of Av. paragallinarum. Although several bacterial species are possible nurse cultures, it is recommended that Staphylococcus hyicus, a normal inhabitant of the skin of chickens, be used. Avibacterium paragallinarum is typically grown in the presence of 5% CO2 at 37 C. A convenient procedure is to use candle jars if CO2 incubators are not available.An alternative isolation (and maintenance) medium that is particularly suited to laboratories in developing countries has been described by Terzolo et al. (31). This medium consists of Columbia blood agar base (Becton Dickinson Microbiology Systems, Sparks, Md.) with 7% lysed equine blood. The lysed equine blood is prepared by holding fresh equine blood at 56 C for 40 min with occasional stirring. The lysed blood can be stored at -20 C. A selective version of the medium, which should only be used in parallel with the nonselective medium, contains bacitracin (5 U/ml), cloxacillin (5 g/ml), and vancomycin (25 g/ml). The plates are incubated under a microaerophilic atmosphere. Several complex media have been described that support growth of avian hemophili. Such media, although not suitable for isolation due to problems with overgrowth by contaminants, are particularly useful for characterization tests following initial isolation. Two media that have proven very useful are Haemophilus maintenance medium (HMM) and supplemented test medium agar (TM/SN), originally described by Rimler et al. (25, 26). HMM base consists of 1% polypeptone (BBL), 1% biosate peptone (BBL), 0.24% beef extract, 0.005% para-aminobenzoic acid, 0.005% nicotinamide, 0.1% starch, 0.05% glucose, 0.9% NaCl, 0.23% leptospira base EllinghausenMcCullaughJohnson

Harris (EMJH) (Difco), and 2% Noble agar (Difco). Immediately before being poured, this medium is supplemented with 0.0025% reduced NAD and 1% chicken serum. TM/SN base consists of 1% biosate peptone (BBL), 1% NaCl, 0.1% starch, 0.05% glucose, and 1.5% Noble agar (Difco) and is supplemented with 5% oleic albumin complex, 1% chicken serum, 0.0005% thiamine, and 0.0025% reduced NAD. A modified version of TM/SN which consists of brain heart infusion agar that is supplemented with 5% oleic albumin complex, 1% chicken serum, 0.0005% thiamine, and 0.0025% reduced NAD has proven to be as suitable as the original formula given above. Broth versions of HMM and TM/SN are prepared by omission of the agar. Chicken Embryos Avian hemophili can be propagated in 5-to-7-day-old chicken embryos, commonly by inoculation via the yolk sac route. After overnight incubation, large numbers of hemophili are present in the yolk, which then can be harvested and preserved by freezing at -70 C (or lower) or by lyophilization. Chicken Inoculation Another efficient diagnostic procedure is to inoculate suspect exudate into the infraorbital sinus of two or three susceptible chickens (preferably 4 wk old or more). The appearance of the typical clinical signs of infectious coryza in 24-48 hr is diagnostic. On occasion, if the number of viable organisms is low, particularly in chronic cases, the incubation period may be delayed for up to 1 wk. In such cases, a second passage may be required to produce the typical rapid onset of clinical signs. If the exudate is heavily contaminated with extraneous bacteria, the chicken inoculation test can be more reliable than culture. In such instances, most of the extraneous bacteria will be cleared by the host defense mechanisms, whereas Av. paragallinarum will colonize and produce typical disease. AGENT IDENTIFICATION Background Avibacterium paragallinarum is not the only growth factor-dependent organism that can be isolated from chickens. Nonpathogenic avian hemophili have been recognized since the 1930s. The organisms have been the subject of several taxonomic changes being initially named as Haemophilus avium (16) and then as Pasteurella volantium, Pasteurella avium, and Pasteurella species A (22). In a recent polyphasic study, these non-pathogenic organisms have been transferred to the genus Avibacterium as Av. avium, Av. volantium and Avibacterium species A (2). The final member of the new genus is Av. (Pasteurella) gallinarum. A range of other hemophili, none of which are yet assigned to named species, have been isolated from birds other than chickens. The identification of these other hemophili will not be discussed further here. Morphology Examination of a direct smear of the exudate by Gram stain can be useful for initial assessment of the microbial flora. The finding of gram-negative rods in direct smears, however, is not sufficient grounds for definitive diagnosis and must be followed by culture. After incubation of blood agar plates for 2448 hr, V factor-dependent isolates of Av. paragallinarum produce tiny dewdrop colonies up to 0.3 mm in diameter adjacent to the nurse culture. The colonies become smaller with increasing distance from the nurse culture. For the satellitic growth to be obvious, cultures must be examined within 2448 hr. The V factor-independent Av. paragallinarum isolates produce small colonies (12 mm) that do not show satellitic growth. Colonies of Av. avium, Av. volantium, Avibacterium species A show satellitic growth and are typically much bigger than V factor-dependent Av. paragallinarum. Some isolates of Av. volantium may produce a yellowish pigment. Growth Requirements Most avian hemophili have a requirement for V factor but not for X factor (6). The determination of the growth factor requirements of these organisms is not an easy process. The use of some brands of commercial growth factor discs on media such as brainheart infusion

agar or susceptibility test agar can result in a high percentage of cultures that falsely appear to be both X and V factor-dependent. The combination of Oxoid growth factor discs (Unipath, Ltd., distr. Unipath, Ogdensburg, N.Y.) and TM/S (TM/SN without added NAD) has been shown to be suitable for growth factor testing (5). Some isolates of avian hemophili may have such lowered V factor requirements that the serum must be omitted from TM/S. Alternative tests such as the porphyrin test (18) for X factor testing or the use of purified hemin (X factor) and NAD (V factor) as supplements to otherwise complete media are possible but are generally too complex for diagnostic laboratories. V factor-independent isolates of Av. paragallinarum have been recognized to date in the Republic of South Africa (21) and Mexico (15). Hence, diagnostic bacteriologists need to be aware of the possibility of such variants emerging in other areas. Typically, Av. paragallinarum isolates fail to grow in air, although some strains will develop this ability on subculturing. Av. avium, Av. volantium and Avibacterium species A all grow vigorously in air. Physicochemical Properties The ability to reduce nitrate to nitrite and ferment glucose without the formation of gas is common to all of the avian hemophili. Oxidase activity, the presence of the enzyme alkaline phosphatase, and a failure to produce indole or hydrolyse urea or gelatin are also uniform characteristics. Av. paragallinarum is catalase-negative, whereas all other members of the genus Avibacterium including the hemophilic species of Av. avium, Av. volantium and Avibacterium species are catalase-positive (2). Carbohydrate fermentation patterns of the avian hemophili are generally determined using a phenol red broth (Difco) containing 1% NaCl, 0.0025% NADH, 1% chicken serum, and 1% carbohydrate. For routine identification, the use of this broth and a dense inoculation (i.e., the modified reference technique of Blackall (1)), is a most suitable approach for determining fermentation patterns. For larger studies, a replica plating technique is more suitable (1). Table 6.1 presents those properties that allow full identification of the avian hemophili. The properties of all members of the genus Avibacterium are presented. The failure of Av. paragallinarum to ferment either galactose or trehalose clearly separates it from all other members of the genus, both hemophilic and non-hemophilic. Serotyping Tests Two different, but inter related, serotyping schemes for Av. paragallinarum have been used mainly the Page (23) and the Kume (19) schemes. The most widely recognized and applied scheme is the Page scheme, which recognizes three serovars (A, B, and C) of Av. paragallinarum. Although the original scheme was based on an agglutination test, the Page serovars are best recognized by a hemagglutination-inhibition (HI) test. The antigen for this HI test can be produced by either of two methods. In the first method, whole bacterial cells are harvested from an overnight broth and stored at 4 C in 1/100 of the initial broth volume for at least 3 days (3). Alternatively, the antigen can be produced by the technique originally used in the Kume scheme (19). In this method, cells of Av. paragallinarum are harvested, washed in phosphate-buffered saline (PBS) (pH 7.0), resuspended in 0.5 M KSCN/0.425 M NaCl to a density equivalent to a MacFarland nephelometer tube number 5 (Difco), held at 4 C for 2 hr with agitation, and then sonicated. The antigen is washed three times in PBS and resuspended in PBS with 0.01% merthiolate to a density equivalent to a MacFarland nephelometer tube number 5. With either antigen type, the HI test should be performed with glutaraldehyde-fixed chicken erythrocytes. The erythrocytes are prepared by collecting fresh chicken blood into an equal volume of Alsevers solution. The suspension is centrifuged and the erythrocytes are washed three times in 0.15 M NaCl. The erythrocytes are then suspended to 1% (v/v) in a glutaraldehydesalts solution and held at 4 C for 30 min. The glutaraldehydesalts solution is prepared by diluting 25% glutaraldehyde to 1% in a solution containing one volume of 0.15 M Na2HPO4 (pH 8.2), nine volumes of 0.15 M NaCl, and five volumes of distilled water. The fixed erythrocytes are collected by centrifugation, washed five times in 0.15 M NaCl and five times in

distilled water and finally resuspended to 30% (v/v) in distilled water containing 0.01% merthiolate. This stock of fixed erythrocytes is held at 4 C, and a working dilution of 1% is prepared in PBS (pH 7.0) containing bovine serum albumin (0.1%) and gelatin (0.001%) (PBS-B-G). Reports that Page serovar B is not a true serovar (28) have been discounted, and Page serovar B has been conclusively shown to be serologically distinct (34). The Kume scheme is a hemagglutination-inhibition serotyping scheme using bacterial cells that have been treated with potassium thiocyanate and then sonicated, and glutaraldehyde-fixed chicken erythrocytes. The preparation of the antigen and the chicken erythrocytes for the Kume scheme has already been described in the section on the Page serotyping scheme. The nomenclature of the Kume scheme has been changed since the original publication. Under the altered terminology the three Kume serogroups are termed A, B, and C (4). This terminology emphasizes that the Kume serogroups correspond to the Page serovars. Within the Kume serogroups, the use of absorbed antisera allows the recognition of serovars, with the nine currently recognized Kume serovars being A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4 (4). The antisera, with the exception of the antisera for B-1 and C-3, need to be absorbed to allow recognition of the Kume serogroups. The antisera to be absorbed are diluted 1 in 40 in PBS-B-G. The absorption is performed using antigens adjusted to 64 HA units and at five times the volume of the diluted serum. The adjusted antigen is centrifuged and the supernatant discarded. The pelleted antigen is resuspended in the diluted antiserum. The suspension is left at room temperature for 2 hours and then overnight at 4C. The suspension is then centrifuged and the supernatant retained as the absorbed antiserum (still at 1 in 40 dilution). Depending on the antiserum, a succession of absorptions may need to be performed. Once the antigen absorption has been completed, the sera need to be absorbed with fixed erythrocytes, using 5 times the volume of fixed erythrocytes compared with the serum. The relevant volume of erythrocytes is centrifuged and the supernatant discarded. The pelleted erythrocytes are resuspended in the diluted antiserum. The suspension is left at room temperature for 2 hours and then overnight at 4C. The suspension is then centrifuged and the supernatant retained as the absorbed antiserum (still at 1 in 40 dilution). The Kume scheme has not been widely applied as it is technically demanding to perform. Molecular Identification A polymerase chain reaction (PCR) test that is specific for Av. paragallinarum has been described (11). The test has been evaluated with a wide range of Av. paragallinarum isolates and is specific and sensitive. The test, termed the HP-2 PCR, has been shown to be suitable for use on purified DNA extracts as well as on crude colony preparations obtained from isolation plates. The HP-2 PCR can be used directly on swabs of nasal mucus obtained from the squeezing of the nostril. The test can be inhibited in the presence of blood and swabs obtained via by slicing open the infra-orbital sinus during necropsy are not optimal for use in the HP2 PCR. Direct PCR examination of swabs has been shown to outperform culture in China (10, 12). The HP-2 PCR is particularly useful in regions where both NAD-independent Av. paragallinarum and Ornithobacterium rhinotracheale are present. Molecular typing methods such as restriction endonuclease analysis (7) and ribotyping (20) have proved useful in epidemiologic studies. A PCRbased typing method ERIC-PCR has also shown a capacity to sub-type isolates of Av. paragallinarum (30). Maintenance Although the initial isolation of Av. paragallinarum from acute infectious coryza is not difficult, it is a fragile organism requiring special care for propagation and maintenance in the laboratory. Cultures can be maintained on blood agar plates by weekly passages. Cultures incubated for 2448 hr at 37 C and then stored at 4 C in a candle jar will remain viable for up to 2 wk. Cultures can be preserved by the lyophilization or freezing (at -70 C or lower) of infected yolk (see the section on chicken embryos). Storage at -70 C of a heavy suspension in the commercial bead systems now commonly available is also possible.

SEROLOGIC DETECTION IN THE HOST Although a range of serologic tests for the detection of antibodies to Av. paragallinarum have been described (6), only hemagglutination-inhibition (HI) tests are in widespread use. The various HI tests differ in the methods used to prepare the antigen and the type of red blood cells used. For the purposes of this overview, the three main HI tests have been termed the simple, extracted, and treated HI tests. Although most of these HI tests were originally described as tube or macroplate tests, all can also be performed in microtiter trays using appropriate volumes. The simple HI test is based on whole bacterial cells of a Page serovar A organism (17). Cells are harvested and suspended in PBS containing 0.01% merthiolate (pH 7.0). Pretreatment of the sera may be necessary to eliminate nonspecific agglutinins. A 5% suspension of chicken erythrocytes is added to the sera (1:5 proportion) and the mixture is incubated for 2 hr at room temperature and 12 hr at 4 C (29). Two-tenths milliliter of antigen (containing 20 HA units/ml) is added to 0.2 ml of serially diluted serum (initial dilution of 1:5). After incubation at room temperature for 10 min, 0.4 ml of 0.5% chicken erythrocyte suspension containing 0.02% (v/v) gelatin is added. The HI titers are determined after incubation for 3040 min at room temperature. This test has been performed mainly using antigen prepared from Av. paragallinarum strain 221. The extracted HI test is based on potassium thiocyanate-extracted and sonicated cells of Av. paragallinarum and glutaraldehyde-fixed chicken erythrocytes (29). The preparation of the antigen and the chicken erythrocytes has already been described in the section on the Page serotyping scheme. Although the methodology of the extracted HI test is as described for the simple HI test, the sera are pretreated with 10% glutaraldehyde-fixed erythrocytes to eliminate nonspecific hemagglutinins, and PBS containing 0.1% bovine serum albumin and 0.001% gelatin is used as the diluent. The extracted HI test has been used to detect antibodies in chickens receiving inactivated vaccines based on Page serovar C organisms (29). In chickens infected with a serovar C organism, the majority of the birds remain negative in this test (36). Note that, in this report, the erythrocytes were fixed in formalin instead of glutaraldehyde (36). The treated HI test is based on hyaluronidase-treated whole bacterial cells of Av. paragallinarum and formaldehyde-fixed chicken erythrocytes (37). Whole bacterial cells that have been adjusted to 10 times the optical density of 0.4 at 540 nm are treated with hyaluronidase (50 units/ml) in phosphate buffer (pH 6.0) in a waterbath at 37 C for 2 hr. The treated antigen is then washed twice in PBS and then resuspended in the original volume of PBS. The test uses the same methodology as that described for the extracted HI test except that the erythrocytes are 1.0% formaldehyde-fixed chicken erythrocytes. The diluent used in this HI test is the same as that used in the extracted HI test. All sera are pretreated with 50% (v/v) formaldehyde-fixed chicken erythrocytes (one volume of antigen with eight volumes of erythrocytes for 2 hr at 37 C with serum recovered by centrifugation and regarded as being a one in five dilution). The extracted HI test has been used to detect antibodies to Page serovars A, B, and C in vaccinated chickens (35). Vaccinated chickens with HI titers of 1:5 or greater in the HI tests based on the simple and extracted antigens have been found to be protected against subsequent challenge (29). DIFFERENTIATION FROM CLOSELY RELATED AGENTS Infection with Av. paragallinarum must be differentiated from other diseases, such as chronic respiratory disease, chronic fowl cholera, fowl pox, ornithobacteriosis (due to O. rhinotracheale), swollen head syndrome (associated with avian pneumovirus) and hypovitaminosis A, which can produce similar clinical signs. Because Av. paragallinarum often occurs in mixed infections, one should consider the possibility of other bacteria or viruses as complicating agents, particularly if mortality is high and the disease takes a prolonged course. The simple isolation of a satellitic, gram-negative organism is not sufficient to identify an isolate as Av. paragallinarum. For laboratories with limited facilities, sufficient evidence for a diagnosis of infectious coryza would be the isolation of a catalase-negative, gram-negative organism that exhibits satellitic growth and fails to grow in air, together with a history of a rapidly spreading acute coryza in a flock. The use of the PCR is recommended as the definitive

test. Laboratories without access to PCR technology should determine growth factor requirements and the ability to ferment glucose, galactose, and trehalose. At this level, Av. paragallinarum can be differentiated from the other members of the genus Avibacterium. For laboratories with extensive resources, a complete phenotypic identification based on the properties shown in Table 6.1 is possible. As V factor-independent Av. paragallinarum have been isolated, diagnostic microbiologists must be aware of the fact that non-satellitic bacteria should be considered as suspect Av. paragallinarum if they show the typical biochemical properties of the species and have been obtained from chickens showing upper respiratory disease. REFERENCES 1. Blackall, P. J. An evaluation of methods for the detection of carbohydrate fermentation patterns in avian Haemophilus species. J. Microbiol. Methods 1:275-281. 1983. 2. Blackall, P. J., H. Christensen, T. Beckenham, L. L. Blackall, and M. Bisgaard. Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. International Journal of Systematic and Evolutionary Microbiology 55:353-362. 2005. 3. Blackall, P. J., L. E. Eaves, and G. Aus. Serotyping of Haemophilus paragallinarum by the Page scheme: comparison of the use of agglutination and hemagglutination-inhibition tests. Avian Dis 34:643-645. 1990. 4. Blackall, P. J., L. E. Eaves, and D. G. Rogers. Proposal of a new serovar and altered nomenclature for Haemophilus paragallinarum in the Kume hemagglutinin scheme. J. Clin. Microbiol. 28:11851187. 1990. 5. Blackall, P. J., and J. G. Farrah. An evaluation of commercial discs for the determination of the growth factor requirements of the avian haemophili. Vet. Microbiol. 10:125-131. 1985. 6. Blackall, P. J., and M. Matsumoto. Infectious coryza. In: Diseases of Poultry, 11th ed. Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. A. Swayne, eds. Iowa State University Press. pp. 691-703 7. Blackall, P. J., C. J. Morrow, A. McInnes, L. E. Eaves, and D. G. Rogers. Epidemiologic studies on infectious coryza outbreaks in northern New South Wales, Australia, using serotyping, biotyping, and chromosomal DNA restriction endonuclease analysis. Avian Dis 34:267-276. 1990. 8. Bland, M. P., A. A. Bickford, B. R. Charlton, G. C. Cooper, F. Sommer, and G. Cutler. Case Report: A severe infectious coryza infection in a multi-age layer complex in central California. In 51st Western Poultry Disease Conference/XXVII convencion anual ANECA. Peurto Vallajarta, Mexico. p. 56-57. 2002 9. Bragg, R. R., P. Jansen Van Rensburg, E. Van Heerden, and J. Albertyn. The testing and modification of a commercially available transport medium for the transportation of pure cultures of Haemophilus paragallinarum for serotyping. Onderstepoort J Vet Res 71:93-98. 2004. 10. Chen, X., Q. Chen, P. Zhang, W. Feng, and P. J. Blackall. Evaluation of a PCR test for the detection of Haemophilus paragallinarum in China. Avian Pathol. 27:296-300. 1998. 11. Chen, X., J. K. Miflin, P. Zhang, and P. J. Blackall. Development and application of DNA probes and PCR tests for Haemophilus paragallinarum. Avian Dis 40:398-407. 1996. 12. Chen, X., C. Song, Y. Gong, and P. J. Blackall. Further studies on the use of a polymerase chain reaction test for the diagnosis of infectious coryza. Avian Pathol. 27:618-624. 1998. 13. Chen, X., P. Zhang, P. J. Blackall, and W. Feng. Characterization of Haemophilus paragallinarum isolates from China. Avian Dis 37:574-576. 1993. 14. Droual, R., A. A. Bickford, B. R. Charlton, G. L. Cooper, and S. E. Channing. Infectious coryza in meat chickens in the San Joaquin Valley of California. Avian Dis 34:1009-10016. 1990. 15. Garcia, A. J., E. Angulo, P. J. Blackall, and A. M. Ortiz. The presence of nicotinamide adenine dinucleotide-independent Haemophilus paragallinarum in Mexico. Avian Dis 48:425-429. 2004.

16. Hinz, K.-H., and C. Kunjara. Haemophilus avium, a new species from chickens. Int. J. Syst. Bacteriol. 27:324-329. 1977. 17. Iritani, Y., G. Sugimori, and K. Katagiri. Serologic response to Haemophilus gallinarum in artifically infected and vaccinated chickens. Avian Dis 21:1-8. 1977. 18. Kilian, M. A rapid method for the differentiation of Haemophilus strains. Acta. Pathol. Microbiol. Immunol. Scand. Sect. B 82:835-842. 1974. 19. Kume, K., A. Sawata, T. Nakai, and M. Matsumoto. Serological classification of Haemophilus paragallinarum with a hemagglutinin system. J. Clin. Microbiol. 17:958-964. 1983. 20. Miflin, J. K., R. F. Horner, P. J. Blackall, X. Chen, G. C. Bishop, C. J. Morrow, T. Yamaguchi, and Y. Iritani. Phenotypic and molecular characterization of V-factor (NAD)-independent Haemophilus paragallinarum. Avian Dis 39:304-308. 1995. 21. Mouahid, M., M. Bisgaard, A. J. Morley, R. Mutters, and W. Mannheim. Occurrence of V-factor (NAD) independent strains of Haemophilus paragallinarum. Vet. Microbiol. 31:363-368. 1992. 22. Mutters, R., K. Piechulla, K.-H. Hinz, and W. Mannheim. Pasteurella avium (Hinz and Kunjara 1977) comb. nov. and Pasteurella volantium sp. nov. Int. J. Syst. Bacteriol. 35:5-9. 1985. 23. Page, L. A. Haemophilus infections in chickens. 1. Characteristics of 12 Haemophilus isolates recovered from diseased chickens. Am. J. Vet. Res. 23:85-95. 1962. 24. Poernomo, S., Sutarma, M. Rafiee, and P. J. Blackall. Characterization of isolates of Haemophilus paragallinarum from Indonesia. Aust. Vet. J. 78:759-762. 2000. 25. Rimler, R. B. Studies of the pathogenic avian haemophili. Avian Dis 23:1006-1018. 1979. 26. Rimler, R. B., E. B. Shotts Jr, J. Brown, and R. B. Davis. The effect of sodium chloride and NADH on the growth of six strains of Haemophilus species pathogenic to chickens. J. Gen. Microbiol. 98:349-354. 1977. 27. Sandoval, V. E., H. R. Terzolo, and P. J. Blackall. Complicated infectious coryza cases in Argentina. Avian Dis 38:672-678. 1994. 28. Sawata, A., K. Kume, and Y. Nakase. Biologic and serologic relationships between Page's and Sawata's serotypes of Haemophilus paragallinarum. Am. J. Vet. Res. 41:1901-1904. 1980. 29. Sawata, A., K. Kume, and Y. Nakase. Hemagglutinin of Haemophilus paragallinarum serotype 2 organisms: occurrence and immunologic properties of hemagglutinin. Am. J. Vet. Res. 43:13111314. 1982. 30. Soriano, V. E., G. Tellez, B. M. Hargis, L. Newberry, C. Salgado-Miranda, and J. C. Vazquez. Typing of Haemophilus paragallinarum strains by using enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Avian Dis 48:890-895. 2004. 31. Terzolo, H. R., F. A. Paolicchi, V. E. Sandoval, P. J. Blackall, T. Yamaguchi, and Y. Iritani. Characterization of isolates of Haemophilus paragallinarum from Argentina. Avian Dis 37:310-314. 1993. 32. Thitisak, W., O. Janviriyasopak, R. S. Morris, S. Srihakim, and R. V. Kruedener. Causes of death found in an epidemiological study of native chickens in Thai villages. Proc. 5th. Inter. Sym. Vet. Epidemiol. Economics pp. 200-202. 1988. 33. Tongaonkar, S., S. Deshmukh, and P. Blackall. Characterisation of Indian isolates of Haemophilus paragallinarum. In 51st Western Poultry Disease Conference/XXVII convencion anual ANECA. Peurto Vallajarta, Mexico. p. 58. 2002 34. Yamaguchi, T., P. J. Blackall, S. Takigami, Y. Iritani, and Y. Hayashi. Pathogenicity and serovar-specific hemagglutinating antigens of Haemophilus paragallinarum serovar B strains. Avian Dis 34:964-968. 1990. 35. Yamaguchi, T., P. J. Blackall, S. Takigami, Y. Iritani, and Y. Hayashi. Immunogenicity of Haemophilus paragallinarum serovar B strains. Avian Dis 35:965-968. 1991. 36. Yamaguchi, T., Y. Iritani, and Y. Hayashi. Serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum. Avian Dis 32:308-312. 1988.

37. Yamaguchi, T., Y. Iritani, and Y. Hayashi. Hemagglutinating activity and immunological properties of Haemophilus paragallinarum field isolates in Japan. Avian Dis 33:511-515. 1989.
Table 6.1. Distinguishing properties of the species within the genera Avibacterium.A Property Avibacterium gallinarum Avibacterium paragallinaru m V + V + Avibacterium volantium + + + + V + + V + + Avibacterium avium + + + + + Avibacterium sp. A. + + + + + V V + V

Catalase +B Symbiotic growth Growth in Air + Acid from L-arabinose D-galactose + V Lactose D-mannitol + Maltose D-sorbitol + Trehalose ONPG V