Evaluation of inactivated infectious coryza vaccines in chickens challenged by serovar B strains of Haemophilus paragallinarum

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1 Avian Pathology ISSN: (Print) 65- (Online) Journal homepage: Evaluation of inactivated infectious coryza vaccines in chickens challenged by serovar B strains of Haemophilus paragallinarum H. R. Terzolo, V. E. Sandoval & F. Gonzalez Pondal To cite this article: H. R. Terzolo, V. E. Sandoval & F. Gonzalez Pondal (17) Evaluation of inactivated infectious coryza vaccines in chickens challenged by serovar B strains of Haemophilus paragallinarum, Avian Pathology, 6:, 65-76, DOI:.0/ To link to this article: Published online: 1 Nov 007. Submit your article to this journal Article views: 11 Citing articles: View citing articles Full Terms & Conditions of access and use can be found at

2 Avian Pathology (17) 6, Evaluation of inactivated infectious coryza vaccines in chickens challenged by serovar B strains of Haemophilus paragallinarum H. R. TERZOLO 1, V. E. SANDOVAL & F. GONZALEZ PONDAL l Instituto National de Tecnologia Agropecuaria (INTA), Facultad de Ciencias Agrarias, Universidad National de Mar del Plata (UNMDP), Unidad Integrada INTA-UNMDP: Departamento de Production Animal, CC 76, 760 Balcarce, Provincia de Buenos Aires, and Laboratorios Pilar S.A. (LAPSA), Ruta y Uruguay, 16 Pilar, Provincia de Buenos Aires, Argentina SUMMARY Four monovalent experimental vaccines (VI, V, V and V7) containing an Argentinean serovar B strain (H) of Haemophilus paragallinarum and three different commercial vaccines, either bivalent (V and V5) containing serovars A and C, or trivalent (V6) containing serovars A, B and C were administered by subcutaneous or intramuscular routes as a single or double dose (at -week intervals) to chickens of between 6 and weeks. Three to 7 weeks after the last vaccination, vaccinated and non-vaccinated chickens were challenged by intrasinus inoculation with Argentinean serovar B strains of H. paragallinarum. When the vaccinated chickens were exposed to a severe challenge with the vaccinal strain (H) some experimental vaccines protected, whereas all commercial vaccines failed to protect. The experimental vaccines manufactured in broth (V, V and V7) protected more effectively than the vaccine produced in chicken embryos (VI). Failure of the commercial trivalent vaccine V6 to protect may be related to the method of manufacture. Vaccine V7 protected against challenge from either the vaccinal strain (H) or three Argentinean serovar B strains (H6, Hll and HI). These results confirm the necessity of including serovar B regional strains in the formulation of local vaccines. INTRODUCTION Infectious coryza is a respiratory disease of chickens (Gallus gallus) caused by Haemophilus paragallinarum (Yamamoto, 11). The disease is acute and spreads rapidly, producing important economic losses, particularly a marked decrease in egg production in breeders and layers (Yamamoto, 11). This disease may also produce economic losses in broilers due to airsacculitis condemnations, dissecting fibrinopurulent cellulitis of the head and wattles (Droual et al., ), and severe 'Corresponding author: H. R. Terzolo, Departamento de Produccion Animal, INTA EEA Balcarce, CC 76, 760 Balcarce, Argentina. Fax: Received May 16; Accepted 1 September /7/ Houghton Trust Ltd

3 66 H. R. TERZOLO ETAL. coryza outbreaks involving septicaemia and arthritis (Sandoval et al, ). Some of these lesions could lead to confusion with chronic fowl cholera and swollenhead syndrome (Droual et al,, Sandoval et al, ). The most widely used serological classification scheme for H. paragallinarum is the Page scheme (Page, 16). By means of a plate agglutination test three different serovars, termed A, B and C are recognized (Page, 16). The same three serovars can also be recognized using a haemagglutination inhibition test that is more reliable than the former plate agglutination test (Blackall et al, ; Jacobs et al, 1). Several studies established that inactivated infectious coryza vaccines either manufactured with aluminium hydroxide gel (Davis et al, 176; Kume et al, a; Matsumoto & Yamamoto, 171; Reid & Blackall, 17) or mineral oil (Blackall et al, 1; Coetzee et al, 1; Jacobs et al, 1) can protect chickens against the disease. Such vaccines provide typespecific immunity within Page serovars, but no cross-protection across serovars (Blackall & Reid, 17; Kume et al, b; Rimler et al, 177). Therefore, inactivated vaccines provide protection only against those serovars present in the vaccine. Rimler & Davis (177) reported that serovars A, B, and C represent three immunotypes. However, other studies performed with Page serovar B strains stated that these strains only had the common antigens of the species and were therefore considered variants of serovar A and C strains that had lost their type specific antigen (Kume et al, b, Sawata et al, ). More recently, it has been conclusively shown that serovar B strains are truly a distinct serovar and are typically pathogenic (Yamaguchi et al,, 11; Jacobs et al, 1). Since serovar B is a major serovar in Argentina (Terzolo et al, 1; Sandoval et al, ), vaccines must contain this serovar to be effective in that region. Outbreaks of infectious coryza in Argentina are frequently complicated with other infectious agents and can be persistent or recurrent in nature (Sandoval et al, ). These outbreaks are frequently associated with high mortality and affect a wide region of Argentina (Sandoval et al, ). Some of these outbreaks occur in flocks vaccinated against the disease. Terzolo et al. (1) and Sandoval et al. () suggested that vaccine failures occurred because the commercial vaccines did not include local isolates of the three serovars of H. paragallinarum present in the target population. Recently, the inclusion of these local isolates in some commercial vaccines controlled the disease in farms where coryza outbreaks used to be recurrent (V. E. Sandoval & H. R. Terzolo, unpublished data). The purpose of this study was to compare and evaluate the protection conferred by different monovalent experimental vaccines containing an Argentinean serovar B strain and commercial vaccines. Vaccines were compared in their ability to protect chickens against challenge with the same Argentinean serovar B strain used to manufacture the experimental vaccines. Cross protection between this vaccinal strain and three different Argentinean serovar B strains was also evaluated.

4 Chickens EVALUATION OF INFECTIOUS CORYZA VACCINES 67 MATERIALS AND METHODS Vaccines were evaluated using male White-Rock chickens reared in isolation and kept under quarantine. Chickens were negative by the rapid serum plate test for Mycoplasma gallisepticum and M. synoviae. Water free from coliform bacteria, and balanced food free from antibiotics and drugs were provided ad libitum. Animals did not receive any treatment or vaccination against any disease other than infectious coryza. Bacteria Four Argentinean serovar B strains of H. paragallinarum were used for the challenge exposure. The strains (H6, H, Hll and HI) were isolated from the infra-orbital sinus of hens affected by acute infectious coryza, with each strain being isolated from a different flock. Strains were stored frozen at 16 C as described (Terzolo et al., 17). They were cultured on Columbia blood agar base plus 7% equine lysed blood (CLBA) and incubated as described (Terzolo et al., 1). Experimental vaccines Four vaccines, VI, V, V and V7 were produced. These vaccines were monovalent and included the Argentinean serovar B strain H. In all batches of vaccines, 0.01% (w/v) of thimerosal was added as the inactivating agent and 0% (v/v) of aluminium hydroxide gel, containing % (w/v) of aluminium oxide, was used as the adjuvant. To prepare vaccine VI, strain H was grown overnight in tryptose phosphate broth and 0. ml of this broth was inoculated into the yolk sacs of 7-day-old chicken embryos. After 1 h incubation at 7 C the embryos died and the contents were harvested, pooled, inactivated, homogenized and bacteriologically enumerated (Miles & Misra, 1) on duplicate CLBA plates. The pooled yolk contained 5 X colony-forming units (CFU) of if. paragallinarum per ml and after addition of the aluminium hydroxide gel the final antigen concentration vaccine VI was diluted to X CFU/ml. Vaccines V, V and V7 were grown in modified Casman broth (Rimler et al., 175) supplemented according to Reid & Blackall (17). Vaccine V was produced in a fermentor as described by Davis et al. (176). Vaccines V and V7 were produced in a 50-1 fermentor. The number of bacteria was estimated by comparing the turbidity produced by the bacterial growth with the Me Farland nephelometer standards (Finegold & Martin, 1). The antigen concentration was adjusted before incorporation of the adjuvant. In vaccines V and V the antigen was adjusted to McFarland tube No. and after addition of the aluminium hydroxide gel both vaccines contained approximately. X cells of

5 6 H. R. TERZOLO ETAL. H. paragallinanim per ml. Vaccine V7 was adjusted to McFarland tube No. 6 and diluted to approximately 1. X cells/ml when the adjuvant system was added. Commercial vaccines Commercial vaccines in use in Argentina when outbreaks of infectious coryza were frequent, were also included in this study. Vaccines V and V5 were imported from USA and Germany, respectively. Both were bivalent, being based on strains W (serovar A) and Modesto (serovar C) (Rimler et al, 177) and contained mineral oil as adjuvant. Vaccine V6 was a commercial Argentinean product based on strains of serovars A, B, and C (no further details about the origin of strains were provided with the vaccine). According to a manufacturer's report vaccine V6 was grown in supplemented chicken infusion broth with constant agitation and controlled CO atmosphere; formalin was used as inactivating agent and aluminium hydroxide gel as adjuvant. Inocula and challenge exposure Strains were removed from storage, thawed, and cultured on CLBA with and without antibiotics (Terzolo et al, 1). Characterization tests, incubation, and maintenance of haemophili were performed as reported (Terzolo et al., 1). After days incubation, growth of CLBA plates was subcultured onto -ml CBLA slopes in screw-capped Universal bottles. Following overnight incubation, ml of Balcarce broth was added to each CLBA slope and the biphasic medium was further incubated for 0 h. Balcarce broth includes the following ingredients: proteose-peptone No. (Difco, USA) 1 g, tryptose (Difco, USA) 1 g, meat extract 0. g, dextrose 0.05 g, soluble starch 0.1 g, sodium chloride 0.5 g and distilled water 0 ml. After autoclaving the broth was supplemented with 5 fig NADH/ml and 1% (v/v) inactivated horse serum. Final ph of the biphasic medium was set at 7.. H. paragallinanim grown in Balcarce broth was used directly to inoculate the chickens. Ten-fold dilutions were produced and bacterial enumeration was performed on duplicate CLBA with and without antibiotics by the surface viable count method of Miles & Misra (1). The counts were carried out in 0 /A drops and the CFU given to each bird were calculated. Three to 7 weeks after vaccination, vaccinated and unvaccinated chickens were inoculated into the infra-orbital right sinus with 0. ml of the inoculum described above. Most birds received around 7 CFU (exact doses given in Tables 1 and ). To evaluate innocuousness of Balcarce broth, another four 5-week-old and four -week-old chickens were similarly inoculated with 0. ml of the sterile broth. Vaccination and experimental design All chickens were vaccinated with a needle and syringe either by the subcutaneous route in the neck behind the head or by the intramuscular route into the breast

6 EVALUATION OF INFECTIOUS CORYZA VACCINES 6 Table 1. Efficacy of monovalent experimental and bivalent or trivalent commercial vaccines in chickens subjected to intrasinusal challenge of 0. ml of broth containing 5.5 X W 7 CFU of H. paragallinarum per dose of Argentinean serovar B, strain H No. of chickens with Vaccine (serovar, dose) n Clinical signs Mucus in sinuses Haemophili in sinuses Protection rate c Trial I a VI (B, doses of 1 ml) VI (B, doses of ml) V (B, 1 dose of 1 ml) V (B, doses of 1 ml) V (B, doses of ml) V (A & C, doses) V6 (A, B & C, doses) None Trial II b V (B, 1 dose of 1 ml) V (B, doses of 1 ml) V5 (A & C, doses) None * 70** 11 0 ** ** 0 0 Differences between vaccinated and non-vaccinated chickens are significant as determined by Fisher exact test (* P< 0.05 and ** P< 0.01). " Vaccinated at 6 and weeks, and challenged at 15 weeks of age. h Vaccinated at 7 and weeks, and challenged at 17 weeks of age. c Percentage of chickens with no clinical signs, no mucus in the sinus and no haemophili. muscle. All chickens vaccinated twice received two identical doses at 0-1-day intervals. Two sets of experiments were performed. The first set included two trials done separately (Trials I and II, Table 1). Both trials were designed to compare and evaluate the protection afforded by different experimental and commercial vaccines and to assess protection when the same experimental vaccine was administered once or twice or at different dosage (1 or ml). Each group consisted of to chickens. In both trials different groups of vaccinated chickens and a group of unvaccinated controls were included. All chickens were vaccinated by the intramuscular route. Commercial vaccines were administered twice according to the manufacturer's instructions. Vaccinated and non-vaccinated control chickens were challenged with H. paragallinarum serovar B, strain H. In trial I (Table 1) the experimental vaccines VI and V (serovar B), and the commercial vaccines V (serovars A and C) and V6 (serovars A, B and C) were evaluated. Chickens were vaccinated once at weeks of age or twice at 6 and weeks of age. Two groups of chickens were used to evaluate vaccine VI. Both groups were vaccinated twice, the first group with 1 ml ( X CFU) and the second with ml (1. X CFU). Three groups of chickens were used to evaluate vaccine V. In the first group chickens were vaccinated once with 1 ml (. X cells). Chickens in the second group were vaccinated twice, also with

7 70 H. R. TERZOLO ETAL. Table. Efficacy of an experimental monovalent (serovar B) vaccine (V7) containing H. paragallinarum Argentinean strain H in chickens challenged with the vaccinal strain (H) and other three different Argentinean serovar B strains (H6, Hll and HI) Challenge 1 " No. of chickens with Vaccination a (H) Serovar B strains Dose per chicken n Clinical signs Mucus in sinuses Haemophi in sinuses Protection rate (%) c Yes No Yes No Yes No Yes No H6 H6 H H Hll Hll H1 H1.1X' 5.X' 1.6X X '.X'. X '.X'. X d ** 0 e 56 d * 0 e 0 d ** 0 e 60 d * 0 e * Vaccinated at 5 and weeks of age. b Challenged intrasinusally with 0. ml of Balcarce broth at 11 weeks of age. c Percentage of chickens with no clinical signs, no mucus in the sinus and no haemophili. d>e Differences between vaccinated and non-vaccinated chickens are significant as determined by the Fisher exact test (* P< 0.0 and ** P< 0.001). 1 ml. In the third group chickens were vaccinated twice with ml (1. X cells). Another two groups were used to evaluate both commercial vaccines. The last group was left unvaccinated as a control. Six weeks after the last vaccination all chickens were challenged. In trial II the experimental vaccine V (serovar B) at a dose of. X cells/ml and the commercial vaccine V5 (serovars A and C) were evaluated. Chickens were vaccinated and challenged as indicated in Table 1. The second set of experiments was carried out to assess cross protection between Argentinean serovar B strains. Four groups of to chickens were subcutaneously vaccinated at 5 and weeks of age with the experimental vaccine V7. Another four groups were left unvaccinated as matched controls. Three weeks after the second vaccination, each of the vaccinated groups and their corresponding controls were challenged with a different serovar B strain (Table ). Clinical signs were recorded at the nd day post-challenge. Chickens were considered clinically positive if inflammation of fascial and periorbital tissues, swelling of infraorbital sinuses, conjunctivitis or nasal secretion was recorded. At the 7th day post-challenge all chickens were killed and necropsied. Both infraorbital sinuses were inspected, recording the presence of mucus and were sampled with a swab moistened in sterile brain-heart infusion broth. The swab was streaked onto CLBA plus antibiotics and incubated as previously described (Terzolo et al., 1). Plates were examined at the rd and 5th day of incubation and the presence of haemophili shown by typical colonial growth was recorded. A chicken was considered diseased if any of the following features was found: clinical signs of infectious coryza, mucus in one or both sinus cavities, H.

8 EVALUATION OF INFECTIOUS CORYZA VACCINES 71 paragallinarum isolated from either one or both of the infra-orbital sinuses. The protection rate was calculated and expressed as the percentage of chickens with no clinical signs, no mucus in the sinus and no haemophili. Statistical analysis Differences between vaccinated and non-vaccinated chickens and between different vaccines and vaccinal treatments were determined by the Fisher exact test by means of the software EPI INFO 6.01 (Dean et al, 11). RESULTS In the first set of trials (Table 1) the vaccine manufactured in chicken embryos (VI) did not protect against challenge with the Argentinean serotype B strain (H); not even a triple volume of vaccine ( ml) was protective. Vaccine V conferred significant protection only when two doses were administered and gave improved protection when two doses of a triple volume ( ml) were given. Vaccine V gave significant protection whether administered as a single or double dose. Fewer chickens were colonised by H. paragallinarum in the group vaccinated twice with vaccine V as compared with the group given a single dose of this vaccine. None of the commercial vaccines (V, V5 and V6) provided significant protection. All control non-vaccinated chickens were diseased indicating the pathogenicity of the challenge strain. When the different vaccines and vaccinal treatments of Trial I were compared, it was found that chickens vaccinated twice with ml of vaccine V were better protected than chickens vaccinated with vaccine VI (P< 0.0), V (P= 0.5), and V6 (P< 0.01). No significant differences between the treatments with vaccine V were found. In the second set of trials (Table ) the experimental vaccine V7 significantly protected chickens against challenge from either the vaccinal strain (H) or three different Argentinean serovar B strains. In comparison, none of the control non-vaccinated chickens were protected against challenge with any of the four serovar B strains. None of the eight chickens inoculated into the infraorbital right sinus with the sterile Balcarce broth showed any inflammatory response. DISCUSSION Serious outbreaks of infectious coryza occurred when these studies were undertaken and there was an urgent need to control the disease. Therefore, different monovalent serovar B vaccines, based on strain H, were developed and some of them used to prevent field outbreaks. Some batches of monovalent vaccines proved to be effective in controlling the field coryza outbreaks, even before the laboratory studies and trials were completed. Hence, these experimental trials were designed with a practical approach and intended not only to compare the

9 7 H. R. TERZOLO ETAL. protection afforded by experimental and commercial vaccines, but also to evaluate different techniques of vaccine manufacture, effect of revaccination and bacterial concentration, and cross-protection between Argentinean serovar B strains. These investigations demonstrated that it is possible to produce vaccines containing an Argentinean serovar B strain of H. paragallinarum that protect chickens exposed to a very severe challenge with different serovar B strains. The experimental broth grown vaccines (V, V and V7) gave significant protection while the vaccine produced in chicken embryos (VI) did not. Others have reported similar findings (Matsumoto & Yamamoto, 175; Davis et al., 176). The chicken embryo vaccine (VI) was unable to confer significant protection either given at X CFU or 1. X CFU per vaccine dose. On the other hand, increasing the number of bacterial cells from. X to 1. X cells per dose in the broth vaccine V may increase protection. Matsumoto & Yamamoto (175) established that 1 X CFU per vaccine dose is the minimum needed to confer good protection. Our results are not directly comparable as the bacterial cell concentration of the broth experimental vaccines was estimated using the Me Farland nephelometer standards, a method that considers both live and dead bacteria. When vaccines V and V were compared at one or two doses, the second dose possibly increased the protection rate (V) or diminished the number of chickens colonized by H. paragallinarum (V). Although it is known that a second dose of any killed vaccine boosts the immune response, we needed to know if, in broilers, only one dose could confer some protection. Due to the broilers' short life and to reduce the cost of vaccination, only one dose was given to flocks exposed to natural infection. In the present work, one dose of vaccine V protected the experimentally infected chickens. Also, under field conditions, broilers were protected with a single dose given at 15 to 0 days. It was demonstrated in the first trials that the commercial vaccines failed to protect chickens challenged with Argentinean serovar B strains. The commercial vaccines we examined were in use when extensive outbreaks of the disease took place. We examined two commercial vaccines produced by different laboratories from different countries (USA and Germany). They were probably prepared by different procedures though both contained the same vaccinal strains. Trials I and II confirm that the absence of serovar B strains in vaccines V and V5 is the cause of the lack of protection observed. Yamaguchi et al. (11) reported that bivalent vaccines based on serovars A and C provided protection against challenge with serovar B strain Spross but failed to protect against South-African serovar B strains 6 and 17. They suggested that the South-African strains were immunologically distinct. Our results suggest that this may be also the case for the Argentinean serovar B strains. It is more difficult to understand why the trivalent commercial vaccine V6 also failed to protect against challenge with strain H. This may be related to the method of manufacture. Vaccine V and V6 have differences in culture media, atmosphere and inactivating agent. Blackall & Reid (17) compared vaccines for

10 EVALUATION OF INFECTIOUS CORYZA VACCINES 7 which the antigen and the adjuvant phases were the same and only the inactivating agent differed. Under these circumstances, the use of formalin, compared with thimerosal, resulted in a reduction in the efficacy of vaccines, specifically when aluminium hydroxide was used. Vaccine V6 contains formalin and aluminium hydroxide and therefore may be less efficient than experimental vaccines manufactured with thimerosal and aluminium hydroxide. The distinct nature of the Argentinean serovar B strains has already been demonstrated with multi-locus enyme electrophoresis (MEE) (Bowles et al, 1). In a study of 11 strains from around the world, they found that the Argentinean B strains were allocated to a cluster quite distinct from all other strains. Strains H6, H, Hi 1 and H1 were found to be similar to each other but different from other strains of serovar B, including reference strains and isolates from various countries of 5 continents. It remains to be proved if the genetic differences detected by MEE also represent immunological differences. In fact, the immunogenic relationships between Argentinean and serovar B strains from elsewhere should be a subject of future research. It would be interesting to perform cross protection studies between Argentinean and other serovar B strains used to manufacture commercial vaccines. In the present work it was shown that chickens vaccinated with strains H were protected against challenge with any of the other three regional strains. Therefore, these Argentinean serovar B strains are antigenically and immunogenically homogeneous. In comparing the results of these trials with those performed by a similar method (Blackall, 1s Blackall & Reid, 17; Blackall et al, 1; Jacobs et al, 1; Reid & Blackall, ), we achieved an apparently lower protection rate. As well, unlike these previous studies, we often failed to recover H. paragallinarum from chickens with frank clinical signs. These differences may be explained by the high pathogenicity of Argentinean serovar B strains and by the rapid colonisation of the infraorbital sinuses followed by quick clearance of these bacteria from the th day post-challenge onwards (V. E. Sandoval & H. R. Terzolo, unpublished data). In the present trials, chickens were killed at the 7th day post-inoculation. Clearance of the challenge organisms may have occurred, in some chickens, before that time. Another difference between our trials and the previous work was the age at challenge; in this work the chickens were older than the ones used by Blackall and colleagues (Blackall, 1; Blackall & Reid, 17; Blackall et al, 1; Reid & Blackall,, 17). When experimentally challenged, older chickens (1 weeks old) have been shown to produce greater signs of infectious coryza than younger birds ( to weeks old) along with a shorter incubation period (Yamamoto, 11). The field outbreaks caused by serovar B strains in Argentina were very severe. Also, these strains produced septicaemia and purulent tarsal arthritis, new features of the disease that had never been described before (Sandoval et al, ). The occurrence of these outbreaks in breeder broilers, layer hens, and broilers, with some outbreaks occurring in vaccinated flocks (Sandoval et al, ; Terzolo et al, 1) combined with the findings of this study, demonstrate the importance of including local serovar B strains in vaccines. The epidemiolog-

11 7 H. R. TERZOLO ETAL. ical data available in Argentina with such local strains in vaccines shows that usage based on an aluminium hydroxide adjuvant vaccine resulted on effective control on layer, broiler breeder and broiler farms where previously coryza outbreaks used to be recurrent. Not only were the typical infectious coryza outbreaks in laying hens controlled, but also the mortality and respiratory symptoms associated with facial or cranial cellulitis, septicaemia and arthritis in broilers. ACKNOWLEDGEMENTS This work was supported by the 'Convenio de Vinculacion Tecnologica INTA- LAPSA'. The technical assistance of Miss M. E. Altuna, Miss R. C. Malena, Miss C. G. Morsella and Mr E. Alias of the Animal Production Department, INTA EEA Balcarce, Argentina, is gratefully acknowledged. We are indebted to Vet. H. Fernandez of the Faculty of Veterinary Sciences of Tandil, Argentina, for his collaboration in some autopsies. The critical reading of this manuscript by Dr P. J. Blackall of the Department of Primary Industries, Animal Research Institute, Queensland, Australia, is also gratefully acknowledged. REFERENCES Blackall, P.J. (1). Further comparison of adjuvants for an inactivated infectious coryza vaccine. Avian Diseases,, 1-5. Blackall, P.J. & Reid, G.G. (17). Further efficacy studies on inactivated, aluminum-hydroxideadsorbed vaccines against infectious coryza. Avian Diseases, 1, Blackall, P.J., Eaves, L.E. & Aus, G. (). Serotyping of Haemophilus paragallinarum by the Page scheme: comparison of the use of agglutination and hemagglutination-inhibition test. Avian Diseases,, Blackall, P.J., Eaves, L.E., Rogers, D.G. & Firth, G. (1). An evaluation of inactivated infectious coryza vaccines containing a double-emulsion adjuvant system. Avian Diseases, 6, Bowles, R.E., Blackall, P.J., Terzolo, H.R. & Sandoval, V.E. (1). An assessment of the genetic diversity of Australian and overseas isolates of Haemophilus paragauinarum by multiloccus enzyme electrophoresis. XIIth World Poultry Congress, Sydney, 16-1 August 1 (p. ). Coetzee, L., Strydom, G.S. & Rogers, E.J. (1). The value of oil-adjuvant vaccines in the control of Haemophilus paragauinarum infection (infectious coryza) in egg producing birds in South Africa. Developments in Biological Standardisation, 51, 16-. Davis, R.B., Rimler, R.B. & Shorts, E.B., Jr, (176). Efficacy studies on Haemophilus gauinarum bacterin preparations. American Journal of Veterinary Research, 7, 1-. Dean, A.G., Dean, J.A., Burton, AJ. & Diker, R.C. (11). Epi Info: a general-purpose microcomputer program for public health information systems. American Journal of Preventive Medicine, 7, Droual, R., Bickford, A.A., Charlton, B.R., Cooper, G.L. & Channing, S.E. (). Infectious coryza in meat chickens in the San Joaquin Valley of California. Avian Diseases,, Finegold, S.M. & Martin, W.J. (1). Bailey and Scott's diagnostic microbiology, 6th edn. London: C.V. Mosby Co. Jacobs, A.A.C., Cuenen, W. & Storm, P.K. (1). Efficacy of a trivalent Haemophilus paragallinarum vaccine compared to bivalent vaccines. Veterinary Microbiology,, -. Kume, K., Sawata, A. & Nakase, Y. (a). Relationship between protective activity and antigen structure of Haemophilus paragallinarum serotypes 1 and. American Journal of Veterinary Research, Kume, K., Sawata, A. & Nakase, Y. (b). Immunologic relationship between Page's and Sawata's serotype strains of Haemophilus paragauinarum. American Journal of Veterinary Research, 1,

12 EVALUATION OF INFECTIOUS CORYZA VACCINES 75 Matsumoto, M. & Yamamoto, R. (171). A broth bacterin against infectious coryza: immunogenicity of various preparations. Avian Diseases, 15, Matsumoto, M. & Yamamoto, R. (175). Protective quality of an aluminum hydroxide-absorbed broth bacterin against infectious coryza. American Journal of Veterinary Research, 6, Miles, A.A. & Misra, S.S. (1). The estimation of the bactericidal power of the blood. Journal of Hygiene, Cambridge,, 7-7. Page, L.A. (16). Haemophilus infections in chickens. I. Characteristics of 1 Haemophilus isolates recovered from diseased chickens. American Journal of Veterinary Research,, 5-5. Reid, G.G. & Blackall, P.J. (). Pathogenicity of Australian isolates of Haemophilus paragallinarum and Haemophilus avium in chickens. Veterinary Microbiology,, 77-. Reid, G.G. & Blackall, P.J. (17). Comparison of adjuvants for an inactivated infectious coryza vaccine. Avian Diseases, 1, 5-6. Rimler, R.B. & Davis, R.B. (177). Infectious coryza: in vivo growth of Haemophilus gallinarum as a determinant for cross protection. American Journal of Veterinary Research,, Rimler, R.B., Shorts, E.B., Jr & Davis, R.B. (175). A growth medium for the production of a bacterin for immunization against infectious coryza. Avian Diseases, 1, 1-. Rimler, R.B., Davis, R.B. & Page, L.A. (177). Infectious coryza: cross-protection studies, using seven strains of Haemophilus gauinarum. American Journal of Veterinary Research,, Sandoval, V.E., Terzolo, H.R. & Blackall, P.J. (). Complicated infectious coryza outbreaks in Argentina. Avian Diseases,, Sawata, A., Kume, K. & Nakase, Y. (). Biologic and sérologie relationships between Page and Sawata's serotypes of Haemophilus paragallinarum. American Journal of Veterinary Research, 1, 1-. Terzolo, H.R., Lawson, G.H.K., Angus, K.W. & Snodgrass, D.R. (17). Enteric campylobacter infection in gnotobiotic calves and lambs. Research in Veterinary Science,, Terzolo, H.R., Paolicchi, F.A., Sandoval, V.E., Blackall, P.J., Yamaguchi, T. & Iritani, Y. (1). Characterization of isolates of Haemophilus paragallinarum from Argentina. Avian Diseases, 7, -. Yamaguchi, T., Blackall, P.J., Takagami, S., Iritani, Y. & Hayashi, Y. (). Pathogenicity and serovar-specific hemagglutinating antigens of Haemophilus paragauinarum serovar B strains. Avian Diseases,, 6-6. Yamaguchi, T., Blackall, P.J., Takagami, S., Iritani, Y. & Hayashi, Y. (11). Immunogenicity of Haemophilus paragallinarum serovar B strains. Avian Diseases, 5, Yamamoto, R. (11). Infectious coryza. In B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid & H. W. Yoder, Jr (Eds.) Diseases of poultry, th edn (pp ). Ames: Iowa State University Press. RÉSUMÉ Evaluation du vaccin inactivé du coryza infectieux chez des poulets éprouvés par le sérovar B de Haemophilus paragauinarum Quatre vaccins expérimentaux monovalents (Vl 5 V, V et V7) contenant le sérovar B argentin de la souche H d'haemophilus paragallinarum et trois vaccins commerciaux, soit bivalents (V et V5) contenant les sérovars A et C, soit trivalent (V6) contenant les sérovars A, B, et C, ont été administrés par voie sous cutanée ou intramusculaire à dose simple ou double dose (à trois semaines d'intervalle) à des poulets âgés de 6 à semaines. Trois à sept semaines après la dernière vaccination, les poulets vaccinés ou non, ont été éprouvés par voie intrasinusale avec différentes souches appartenant au sérovar B argentin d'haemophilus paragauinarum. Lorsque les poulets vaccinés ont subi une épreuve sévère avec la souche (H) quelques vaccins expérimentaux ont induit une protection, ce qui n'a pas été le cas des vaccins commerciaux. Les vaccins expérimentaux préparés à partir de bouillon (V, V et V7) ont entraîné une protection supérieure à ceux préparés sur embryons de poulets (V1). L'absence de protection observée après l'administration du vaccin commercial trivalent V6 doit être due à la méthode de fabrication. Le vaccin V7 a entraîné une protection aussi bien vis à vis de la souche H que des souches argentines appartenant au serovar B (H6, Hll et H1). Ces résultats confirment la nécessité d'inclure les souches régionales de sérovar B dans la formulation des vaccins utilisés dans la région.

13 76 H. R. TERZOLO ETAL. ZUSAMMENFASSUNG Beurteilung von Totvakzinen gegen den ansteckenden Hühnerschnupfen nach Testinfektion von Hühnern mit Haemophilus paragallinarum- Stämmen vom Serovar B Vier monovalente Versuchsvakzinen (VI, V, V und V7), die einen argentinischen Hämophilus paragallinarum-stamm (H) vom Serovar B enthielten, und verschiedene kommerzielle Vakzinen, entweder bivalent (V und V5), mit den Serovaren A und C, oder trivalent (V6), mit den Serovaren A, B und C, wurden geprüft. Dazu wurden die Vakzinen 6 bis Wochen alten Hühnern einmalig oder zweimalig (im Abstand von Wochen) subkutan oder intramuskulär verabreicht. Drei bis 7 Wochen nach der letzten Impfung wurden geimpfte und ungeimpfte Hühner durch intrasinale Inokulation mit argentinischen Hämophilus paragallinarum-stäramen vom Serovar B testinfiziert. Als die vakzinierten Tiere einer starken Testinfektion mit dem Vakzinestamm H ausgesetzt wurden, erwiesen sich einige Versuchsvakzinen, aber keine der kommerziellen Vakzinen als schützend. Die in Bouillon hergestellten Vakzinen (V, V und V7) schützten wirksamer als die in Hühnerembryonen produzierte Vakzine (V1). Die Unwirksamkeit der kommerziellen trivalenten Vakzine V6 könnte mit der Herstellungsmethode zusammenhängen. Die Vakzine V7 schützte sowohl gegen die Testinfektion mit dem Vakzinestamm (H) als auch gegen Testinfektionen mit argentinischen Stämmen vom Serovar B (H6, Hll und H1). Diese Ergebnisse bestätigen die Notwendigkeit, regionale Stämme vom Serovar B in die Zubereitung lokaler Vakzinen einzubeziehen. RESUMEN Evaluación de vacunas inactivadas de coriza infecciosa en gallinas expuestas a cepas de la serovar B de Haemophilus paragallinarum Se administró a gallinas de 6 y semanas de edad por vía subcutánea o por vía intramuscular una sola dosis o dos dosis (a intervalos de semanas) de cuatro vacunas monovalentes experimentales (V1, V, V, V7) que contenían una cepa (H) argentina de la serovar B de Haemophilus paragallinarum así como tres vacunas comerciales, bien del tipo bivalente (V y V5) que contenían las serovars A y C o una trivalente (V6) que contenía las serovars A, B y C. Entre las tres y siete semanas tras la última vacunación las gallinas vacunadas y no vacunadas fueron inoculadas por vía intrasinusal con cepas de H. paragallinarum de la serovar B argentina. Cuando las gallinas vacunadas fueron intensamente expuestas a la cepa vacunal H algunas de las vacunas experimentales protegieron mientras que ninguna de las vacunas comerciales fueron capaces de proteger. Las vacunas experimentales manufacturadas en caldo de cultivo (V, V, V7) protegieron más eficazmente que la vacuna producida en embrión de pollo (V1). El fallo de la vacuna comercial trivalente V6 para proteger pudiera estar relacionado con el método de producción. La vacuna V7 protegió frente a la infección con la cepa vacunal (H) o frente a tres cepas argentinas de la serovar B (H6, H11, H1). Estos resultados confirman la necesidad de incluir cepas regionales de la serovar B en la formulación de vacunas locales.