A Preliminary Investigation on the Phenotypic Characteristics of Ornithobacterium rhinotracheale

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1 A Preliminary Investigation on the Phenotypic Characteristics of Ornithobacterium rhinotracheale ZAINI MOHD-ZAIN, TAN LIN JEE a and KAMARUZAMAN JUSOFF b Institute of Medical Molecular Biotechnology, Faculty of Medicine, Universiti Teknologi MARA, Shah Alam, Selangor. Malaysia a Veterinary Research Institute, Ipoh, Perak, Malaysia b Environment Science Centre. Yale University New Haven, CT 06511, U.S.A zainimz@salam.uitm.edu.my Abstract: - The phenotypic characteristics and pathogenicity of 18 isolates of Ornithobacterium rhinotracheale (ORT) obtained from chickens in Malaysia were investigated. Biochemical test results showed that these ORT isolates are made up of variants. All of the isolates were identified as serotype A. Two of these isolates were determined of their ability to cause embryo mortality and their clinical manifestations in specific-pathogen-free (SPF) chickens. Comparable to a reference strain, these two isolates were found to cause significantly (P<0.01) high number of embryo mortality. SPF chickens infected with these isolates did not produce any signs of respiratory syndrome but showed poor growth development. The results of this preliminary study have revealed some of the characteristics of the Malaysian ORT isolate which depict some potential consequences of infection in poultry industry. Key-words: - Ornithobacterium rhinotracheale, embryo mortality, growth retardation, chicken 1 Introduction Ornithobacterium rhinotracheale (ORT) can be considered as a newly identified bacterium in Malaysia since it was isolated from commercial broiler chickens for the first time in 2000 [1]. The bacterium which was previously known as Pasteurella-like organism before 1994 had been reported to be able to cause highly contagious disease in poultry [2]. It has been considered and proven to be a primary pathogen in broilers [3]. In turkeys and chickens, the infection of this organism has been associated with the reduction in egg production, poor growth performance and increased mortality rate [4]. Infected birds usually present with severe necrotizing fibrino-purulent septicaemia which may result in economic loss to poultry industries due to increased in condemnation [5]. In some cases, infected birds do not present with clinical signs [6]. Severity in chickens depends on the isolates and co-infection with other pathogens [7, 8]. The importance of ORT in poultry industries is usually overlooked because the clinical signs of ORT infection in chickens are relatively similar to infection by Avibacterium paragallinarum (APG) making diagnosis difficult [9]. This study attempts to provide some information on the characteristics of Malaysian ORT isolates to further understand the importance of this bacterium as a pathogen in chickens. 2 Materials and Methods 2.1 Bacterial Strains and Culture Media The 18 ORT strains used in this study were isolated between 2000 and 2007, from chickens that were submitted for disease investigation to Veterinary Research Institute (VRI), Ipoh, Malaysia. Isolation of the bacterium was carried out by culturing swabs of the nasal cavity, trachea and air sac onto 5% sheep blood agar [10]. Primary isolates were then subcultured onto ISSN: ISBN:

2 chicken meat infusion (CMI) agar [11] and suspected ORT isolates were identified, characterized and serotyped according to established methods [9, 10]. PCR technique was used to confirm the isolates [12]. ORT isolates 9018/03 and 4393/04, were used in the embryo mortality study. These isolates were identified biochemically and confirmed to be ORT by PCR. An ORT strain B3263/91 of serotype A, which was originally isolated from diseased broiler chickens in South Africa was used as a reference strain (obtained from Paul van Empel, Intervet-International, The Netherlands). APG 6755/04 isolated from a layer chicken with infectious coryza was used in an experiment to compare the disease manifestations between APG and ORT infections. 2.2 Embryonated Eggs and Chickens Seven day-old embryonated eggs and four weekold White-Leghorn chickens used in this study were obtained from SPF flock maintained in a filtered air, positive pressure house at VRI. 2.3 Preparation of Inocula ORT and APG were grown on CMI agar for 48 hours in 8-10% CO 2 atmosphere. Each of the cultures was washed in phosphate buffered saline (PBS, ph7.2) and centrifuged at 12,000 g at 4 o C for 30 minutes. The pellets were collected and washed twice with PBS at 24,000 g for 15 minutes and were re-suspended in PBS to contain at least 1 x 10 8 CFU/ml of the organisms. They were diluted accordingly prior to egg inoculation and chicken infection. 2.4 Embryo Mortality A total of 160 embryonated SPF eggs were divided into three test groups and one control group. In each test group, 10 eggs each were inoculated via yolk sac with 100 µl of the inoculum containing 10 2, 10 3, 10 4, 10 5 and 10 6 CFU of each of the isolates, 9018/03, 4393/04 and B3263/91. Ten eggs in the control group were inoculated with the same volume of sterile PBS. All the eggs were candled daily for embryo mortality for 12 days. Embryos that survived until day 12 post-infection (p.i.) were sacrificed. All dead and live embryos were cultured to recover the inoculated organisms. 2.5 Pathogenicity Study in Chickens To distinguish the clinical manifestations produced by ORT and APG infections, a total of 40 chickens were weighed and were randomly divided into three groups (two test groups and one control group). Fifteen chickens were placed in each test group and 10 chickens in the control group. In each of the test groups, 10 chickens were inoculated intranasally with either 10 6 CFU of ORT 4393/04 or APG strain 6755/04. All the chickens in the control group were inoculated with 10 µl of sterile PBS via similar route. Five remaining chickens in each of the test groups were non-treated and were left as in-contact chickens. The chickens in all the groups were examined daily for signs of watery eyes, swollen head, coughing, sneezing, nasal discharge, rales and dullness. At day 7 p.i, all the chickens were weighed before they were euthanized. Post mortem examination was carried out to observe for macroscopic lesions. Their sinuses, tracheas and air sacs were swabbed for culture to recover the inoculated organisms. 3 Results 3.1 Bacterial Identification and Serotyping Based on biochemical test and enzymatic reactions, majority (10/18) of the isolates were observed to be phenotypically similar to the reference strain, B3263/91. Two other variants strains differed from the majority of the isolates in their fermentation of glucose and arginine dihydrolase (Table 1). All of the isolates including the variant strains produced an amplicon of 784 bp which confirmed them as ORT. All of the isolates were identified as serotype A. ISSN: ISBN:

3 3.2 Embryo Mortality Embryo mortality was first observed in all the test groups at day 3 p.i. which continued throughout the experiment (day 12 p.i). Inoculum dose as low as 10 2 CFU of isolates 9018/03 and 4393/04 was found to be able to kill chick embryos, comparable to the lethal effect of the reference strain, B3263/91 (Table 2). The highest number (26%) of mortality was recorded in the group inoculated with 4393/04 while the group inoculated with B3263/91 had the least number of mortalities. The control group did not have any embryo mortality. The number of embryo mortality between the test groups were not significantly different (P>0.01) but they were significantly higher (P<0.01) than the control group. The inoculated organisms were re-isolated from dead as well as live embryos from both test groups but none from the control groups. 3.3 Pathogenicity Study in Chickens It was observed that more chickens in the APGinfected group developed clinical signs of respiratory syndromes than those in the ORTinfected group (Table 3). Only one bird in the ORT-infected group had mild nasal discharge on day 2 p.i. which subsided by the next day. Six chickens, including three in-contacts in the APGinfected group presented with severe nasal discharge, watery eyes and swollen head which began on day 3 p.i. and persisted until day 7 p.i. None of the control bird developed any clinical signs. At necropsy (day 7 p.i), it was observed that the chicken in the ORT-infected group that had nasal discharge remained to have the mucous which was only limited in the nasal cavity, while the chickens (including in-contacts) in the APGinfected group had not only mucous in the nasal cavity but also cloudy air sacs. The tracheas of all the chickens in all the groups however, were normal. Upon culture of swabs from the various sites of the upper respiratory tract, there was higher recovery of inoculated organisms in chickens in the APG-infected group than the ORT-infected group. The inoculated organisms were also recovered from in-contact chickens. Between the test groups (excluding in-contacts), no significant difference (P>0.01) in the bodyweight gain was recorded, however, their bodyweight gain were significantly (P<0.01) lower than the control group. 4 Conclusion ORT infection in poultry has not been well recognized as a pathogen of importance until recently. This is because the clinical signs are similar to infectious coryza [9]. In this study, some phenotypic characteristics of the ORT isolated in Malaysia are revealed. Like in many other countries, serotype A seems to be the most prevalent serotype [13, 14, 15]. Although all the isolates were mainly isolated within the peninsular of Malaysia, variant strains that produce different biochemical reactions to typical strains of ORT would cause confusion during identification. The biochemical reactions displayed by these variants are similar to some of the variant strains isolated elsewhere [15, 16, 17]. The PCR technique was useful as a confirmatory test for ORT, which is in agreement to other reports [18, 19]. The ORT isolates examined in this study are shown to be able to cause mortality to chick embryos. Contrary to a report that ORT took nine days to kill a chick embryo [20], our isolates took three days [20]. The virulence of our isolates was comparable to that of the reference strain, B3263/91 and thus, indicating that our isolates are probably more virulent than the strains used in that report. The difference in the observations confirms the report that the pathogenicity of ORT isolates can be variable depending on the strains of the isolates [7]. The recovery of the inoculated ORT from chick embryos at the end of the experiment suggests that ORT can be transmitted via eggs. This observation is in agreement to van Veen et al. (2004) who observed respiratory tract lesions in SPF chicks that were placed in hatching ISSN: ISBN:

4 incubators during hatch [21]. Our study has also shown that ORT infection can also be transmitted horizontally evident by the recovery of inoculated ORT from in-contacts chickens in close proximity with infected chickens. A study has demonstrated that ORT can cause respiratory tract lesions and growth retardation in both chickens and turkeys which is similar to infection by APG [22]. In the present study, respiratory symptoms shown by the chickens infected with ORT were less severe than those infected with APG, but both were equally able to retard growth in chickens. Although reports have shown that ORT alone causes severe respiratory symptoms in turkeys and chickens [2, 3, 4], the mild respiratory symptoms produced in our infected chickens signifies the low virulence of the ORT isolates. This is in accordance to other investigators that severity of clinical signs, duration of the disease and mortality in ORT infection are extremely variable depending on the strains [7, 23]. Besides strains, the breed of the chickens also has an influence on the severity of clinical symptoms [6] which possibly accounts for the mild respiratory symptoms that were observed in our infected chickens. It would be useful to investigate a large numbers of ORT isolates to further understand its pathogenicity pattern. It is concluded that ORT isolates examined in this study were lethal to chick embryos but causes mild respiratory symptoms in White Leghorn chickens. Growth retardation seems to be the most prominent clinical sign of ORT infection, as evident by the present study and other findings [2, 4, 22]. References: [1] Mahani, A.H.; Tan, L.J.; Maria, J.; Shukor, A.A.; Zuraidah, A.; Maizan, M.; Johara, M.Y. Isolation and identification of Ornithobacterium rhinotracheale from commercial broiler chickens with concurrent infectious bronchitis and colibacillosis. Proc. 2nd Intl. Congress CVA-Australasian Regional Symposium, Kuala Lumpur, 2001, pp [2] van Beek, P.N.; van Empel, P.C.; van den Bosch, G.; Storm, P.K.; Bongers, J.H.; du Preez, J.H. Respiratory problems, growth retardation and arthritis in turkeys and broilers caused by a Pasteurella-like organism: Ornithobacterium rhinotracheale or 'Taxon 28. Tijdschr Diergeneeskd. 119, 1994, [3] van Veen, L.; van Empel, C.P.; Fabria, T., Ornithobacterium rhinotracheale, a primary pathogen in broilers. Avian Dis. 2000, 44, [4] van Empel, P.; van den Bosch, H.; Goovaerts, D., Storm, P. Experimental infection in turkeys and chickens with Ornithobacterium rhinotracheale. Avian Dis. 40, 1996, [5] van Veen, L.; Gruys, E.; Frik, K.; van Empel, P. Increased condemnation of broilers associated with Ornithobacterium rhinotracheale. Vet. Rec. 147, 2000, [6] Sprenger, S.J.; Halvorson, D.A.; Nagaraja, K.V.; Spasojevic, R.; Dutton, R.S.; Shaw, D.P. Ornithobacterium rhinotracheale infection in commercial laying-type chickens. Avian Dis. 44, 2000, [7] Travers, A.F., Coetzee, L., Gummow, B., Pathogenicity differences between South African isolates of Ornithobacterium rhinotracheale. Onderstepoort J. Vet. Res. 63, [8] Travers, A.F. Concomitant Ornithobacterium rhinotracheale and Newcastle disease infection in broilers in South Africa. Avian Dis. 40, 1996, [9] Blackall, P.J. Infectious coryza: overview of the disease and new diagnostic options. Clin. Microbiol. Rev. 12, 1999, [10] van Empel, P.; van den Bosch, H.; Loeffen, P.; Storm, P., Identification and serotyping of Ornithobacterium rhinotracheale. J. Clin. Microbiol. 35, ISSN: ISBN:

5 [11] Zain, Z.B., Iritani, Y. Serotyping of Haemophilus paragallinarum isolated in Malaysia. J. Vet. Med. Sci. 54, [12] Hafez, H.M. Diagnosis of Ornithobacterium rhinotracheale. Int. J. Poultry Sci. 11, 2002, [13] El-Sukhon, S.N.; Musa, A.; Al-Attar, M.; Studies on the bacterial etiology of airsacculitis of broilers in Northern and Middle Jordan with special reference to Escherichia coli, Ornithobacterium rhinotracheale and Bordetella avium. Avian Dis. 46, 2002, [14] Turan, N.; Ak, S. Investigation of the presence of Ornithobacterium rhinotracheale in chickens in Turkey and determination of the seroprevalance of the infection using the enzyme-linked immunosorbent assay. Avian Dis. 46, 2002, [15] Canal, C.W.; Leao, J.A.; Rocha, S.L.; Macagnan, M.; Lima-Rosa, C.A.; Oliveira, S.D.; Back, A. Isolation and characterization of Ornithobacterium rhinotracheale from chickens in Brazil. Res. Vet. Sci. 78, 2005, rhinotracheale strains and examination of their transmission via eggs. Acta Vet. Hung. 49, 2001, [21] van Veen, L.; Vrijenhoek, M.; van Empel, P. Studies of the transmission routes of Ornithobacterium rhinotracheale and immunoprophylaxis to prevent infection in young meat turkeys. Avian Dis. 48, 2004, [22] Bragg, R.R.; Greyling, J.M.; Verschoor, J.A. Isolation and identification of NADindependent bacteria from chickens with symptoms of infectious coryza. Avian Pathol. 26, 1997, [23] van Empel, C.P.; Hafez, H.M. Ornithobacterium rhinotracheale: a review. Avian Pathol. 28, 1999, [16] Hinz, K.H.; Blome, C.; Ryll, M. Acute exudative pneumonia and airsacculitis associated with Ornithobacterium rhinotracheale in turkeys. Vet. Rec. 135, 1994, [17] Vandamme, P.; Segers, P.; Vancanneyt, M.; van Hove, K.; Mutters, R.; Hommez, J.; Dewhirst, F.; Paster, B.; Kersters, K.; Falsen, E. Ornithobacterium rhinotracheale gen. nov., sp. nov., isolated from the avian respiratory tract. Int. J. Syst. Bacteriol. 44, 1994, [18] Hung, A.L.; Alvarado, A. Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from Peru. Avian Dis , [19] Koga, Y.; Zavaleta, A.I. Intraspecies genetic variability of Ornithobacterium rhinotracheale in commercial birds in Peru. Avian Dis. 2005, 49, [20] Varga, J.; Fodor, L.; Makrai, L. Characterisation of some Ornithobacterium ISSN: ISBN:

6 Table 1. Biochemical and enzymatic characteristics of O. rhinotracheale isolated in Malaysia. Test Reference B3263/91 O. rhinotracheale isolates Variant 1 Variant 2 Variant 3 Nitrate reduction Catalase Urease Indole Growth on MacConkey Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Fermentation of fructose lactose maltose galactose glucose No. of isolates Table 2. Number of embryo mortality in 7 day-old chicken embryonated eggs artificially infected with O. rhinotracheale. Inoculated Strain No. of embryo mortality No. of Dose of inoculum (CFU) eggs inoculated Total No. of ORT re-isolated (%) Dead Survived P-value to control group Nil (control) (0) 100 (0) - B3263/ (18) 41 (82) P< / (22) 39 (78) P< / (26) 37 (74) P<0.01 ISSN: ISBN:

7 Table 3. Experiment to determine the clinical manifestations, gross lesions and recovery of organisms from four week-old SPF chickens experimentally infected with O. rhinotracheale. Inoculated organism Clinical Signs a Gross lesion b Recovery of organisms c Average bodyweight gain (g) O. rhinotracheale Inoculated 1/10 1/10 3/ In-contact 0/5 0/5 3/ A. Inoculated 3/10 10/10 10/ paragallinarum In-contact 3/5 5/10 5/ Nil (Control) 0/10 0/10 0/ Numerator denotes the number of positive chickens and denominator denotes the total number of chickens used. a Clinical signs include eye tearing, swollen head, coughing, sneezing, nasal discharge, rales and dullness. b Gross lesions seen at post mortem examination which include presence of mucous in nasal cavity and trachea, consolidated lung and cloudy air sac. c Inoculated organisms re-isolated from nasal cavity, trachea, lung and air sac. ISSN: ISBN: