Phenotypic and molecular characterization of strains of Ornithobacterium rhinotracheale isolated from poultry in Turkey

Ornithobacterium rhinotracheale has been identified as a newly emerging respiratory bacterial pathogen that has been causing significant economic losses to the poultry industry. In this study, a total of 30 isolates of O. rhinotracheale isolated (2 from layer pullets, 5 from broilers, 21 from turkeys and 2 standart strains) from chickens and turkeys were characterized by Polymerase Chain Reaction (PCR), RAPD-PCR and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis. The isolates of O. rhinotracheale identified by biochemical tests were also confirmed by PCR. Amplification product of 784 bp was obtained, which is corresponding to the expected size. All of them were characterized as O. rhinotracheale. The random amplified polymorphic DNA (RAPD) profiles using OPG11 primer and the protein profiles used in SDS-PAGE of the O. rhinotracheale strains show high variability in results of Neighbor Joining (NJ) and Unweighted Pair Group Method with Arithmetic Means (UPGMA). All field strains isolated from turkeys, broilers and layers show different RAPD and protein profiles.

Various pathogens (Turkey rhinotracheitis (TRT) virus, Newcastle Disease virus, Escherichia coli, Bordetella avium, etc.) have been identified as a cause to respiratory disease which are acting either as primary or secondary agent (Odor et al., 1997;van Empel and Hafez, 1999).O. rhinotracheale can be a primary or secondary cause which depends on strain virulence, adverse environmental factors (poor management, inadequate ventilation, high stocking density, poor litter condition, poor hygiene, high levels of respiratory ammonia), immunity state of flock, and presence of other infectious agents.The primary role of O. rhinotracheale in respiratory disease is open to debate (De Rosa et al., 1996;van Veen et al., 2000;Erganis et al., 2002;Hadimli et al., 2003;Canal et al., 2005).
O. rhinotracheale identified by Vandamma et al. (1994), after series of phenotypic and genotypic characterizations which includes protein profiles, DNA-DNA and DNA-rRNA hibridizations (Vandamme et al.,1994).Today, a total of 18 different serotypes which are marked from A to O have been reported (van Empel and Hafez, 1999).Of the chickens and turkeys, more than 95% of isolates are of serotype A (van Empel et al., 1997).The aims of the study was to biochemically and genotypically identify O. rhinotracheale strains isolated from layers, broilers and turkeys.In addition, genotypical differences was compared to among strains.

Bacteriological examinations
The number of 165 visceras (lung and liver, n=75) and swapes from trachea (n=90) were taken from 21 turkey flocks which were affected by respiratory disease (nasal discharge, gasping, ruffled feathers, suffered from occasional head oedema, severe choked breathing and weakness).Tissue samples were streaked on blood agar containing 7% sheep eritrocytes, supplemented with 5 mg/ml gentamycin and polymyxin B, and MacConkey Agar.The plates were incubated microaerobically in 37°C for 48-72 h.The biochemical identification was carried out as described previously (van Empel et al.,1997;Erganis et al., 2002).

Bacterial strain
A total 30 isolates of O. rhinotracheale were used in this study which consists of 2 from layer pullets, 5 from broiler (this isolates provided from Dr. TURKYILMAZ (Adnan Menderes University, Faculty of Veterinary Medicine, Department of Microbiology, Aydin, Turkey) and 21 from turkeys.The strains of O. rhinotracheale were isolated from turkeys and chickens in different flocks which are located in 3 different geographical regions of the Turkey.The 2 strains of O. rhinotracheale were also used as a standart strains.
SDS-PAGE analysis were used to determine polimorphisms among isolates of O. rhinotrachelae.The polimorphorfisms and similarity coefficient were calculated using Unweighted Pair Group Method with Aritmetic Means (UPGMA) and Neighbor Joining (NJ).

DNA extraction
A total of 30 O. rhinotracheale isolates were grown on blood agar for 48 h and washed in PBS at pH 7.2.The mixture was microfuged (10,000×g, 5 min, 4°C), and the cell pellets were resuspended in 385 µl of STE buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM etylelene daimine tetraacetic acid [EDTA]).The extraction of DNA of ORT isolates was made by using a commercial kit (Promega, Wizard® Genomic DNA Purification Kit (Cat.#A1120, USA).

PCR
Primers used in our study were of those reported by Van Empel and Hafez (1999), OR16S-F1 (GAG AAT TAA TTT ACG GAT TAA G) and the 20-mer oligonucleoitide OR16S-R1 (TTC GCT TGG TCT CCG AAG AT), which flanked a 784-bp DNA sequence within the 16S rDNA.The PCR mixture was consisting of 2 µl template DNA, 10 µl of PCR Buffer, 4 µl 10 mM dNTPs (in equal volumes of each Deoksinukleotide triphosphat), 3 µl of each primer (1.5 μM), 0.25 µl of Taq polimeraz (Promega) in a final volume of 50 µl.The amplification was performed under the following conditions in thermal cycler (Eppendorf, England): a denaturation step of 94°C for 5 min followed by 35 cycles of 94°C for 30 sn, denaturation at 58°C for 1 min, extension at 72°C for 1.5 min, and final extension at 72°C for 7 min.The PCR product was then analysed by agarose gel electrophoresis and visualised using ethidium bromide 0.5 μg/ml under ultraviolet light.A 100 bp DNA ladder (Promega, Maddison, USA) was used in all runs (Canal et al., 2005).

Isolation of O. rhinotracheale
The 21 O. rhinotracheale strains were isolated from 21 turkey flocks that were suffering respiratory diseases.While 19 isolates were isolated from lungs and trachea of turkeys in 2 different companies in same providence, 2 strains were recovered from another providence.Also, 7 isolates (previously 5 isolated from broilers and 2 layers) were added from our culture collection.In addition, 2 strains were used as references strains.

Biochemical reactions
All isolates reacted positively in the p-nitrophenyl-b-Dgalactopyranoside (PNPG), urease and oxidase tests, but, were negative to catalase, nitrate reduction, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, indole, TSI, and growth on MacConkey agar.Acid produced from glucose, mannose, lactose, maltose, and fructose were variable.For all isolates, no acid was produced from sucrose, sorbitol, and dulcitol.Not all strains were having hemolysis.

SDS-PAGE analysis
The dendogrames of protein profiles by SDS-PAGE of all ORT strains are shown in Figure 1.The protein profiles of O. rhinotracheale strains were shown high variability in results of NJ and UPGMA methods.All of field strains isolated from turkeys, broilers and layers were shown different protein profiles.However, it has shown that the isolates of broilers, layers or turkeys among themselves were given similar bands.In addition, although isolates of turkey, 2 strains (10 and 11) isolated from different region and flocks were formed to be different bands than others.

PCR
The isolates of ORT identified by biochemical tests were also confirmed by PCR.Amplification product of 784 bp was obtained which was corresponding to the expected size (Figure 2).All of them were determined as O. rhinotracheale.The dendogrames of RAPD-PCR profiles of all ORT strains are shown in Figure 4. Using OPG-11 primer, the RAPD profiles of the ORT strains were shown high variability in results of NJ and UPGMA methods.All of field strains isolated from turkeys, broilers and layers were shown different RAPD profiles (Figure 3).The isolates of O. rhinotracheale isolated from layers and broilers were characterized as to be different than isolates of turkeys.Also, the isolates of turkey were observed as to be similar  to each other.

DISCUSSION
The diagnosis of naturally occured O. rhinotracheale infection may commonly be difficult, according to findings of clinical and histopathological examinations (van Beek et al., 1994;Hafez, 1996).Because, similar clinical symptomes in respiratory diseases caused by many microorganisms were often formed (Travers et al., 1996;Szalay et al., 2002).Therefore, the diagnosis of O.
rhinotrachelae infection based on isolation and identification of bacteria (van Empel et al., 1997;Banani et al., 2001).The levels of antibodies after infection can be determined in early 5 days, otherwise barely determined in later stages.For this reason, O. rhinotracheale infections in infected poultry houses and/or the respiratory system can be under-diagnosed or misdiagnosed (van Leen et al., 2005).Furthermore, the slow-growing O. rhinotracheale strains can be overgrown by other more rapidly reproducing bacteria and may also be overseen due to their highly variable biochemical properties or their bypassing of clinical practitioners.
A B Therefore, in the event of O. rhinotracheale infections, there is a need for the identification and confirmation of the causative agent (Tsai and Huang, 2006;Hassanzadeh et al., 2010).In this study, all the O. rhinotracheale isolates have been isolated from chickens and turkeys suffering respiratory diseases.However, a relationship between the number of samples from flocks with respiratory diseases and the rate of isolation was not intended.
PCR is fast, sensitive, and specific method to identify and characterize bacterial strains and is an alternative method to determine the etiologic agents of respiratory diseases in poultry (Amonsin et al., 1997;Hung and Alvarado, 2001;Özbey et al., 2004;Hassanzadeh et al., 2010;Tachil et al., 2010).However, the cost and availability of equipments, as well as incorrect manipulation of the reagents with the amplification of contaminants, may lead to the misinterpretation of the results (Koga and Zavaleta, 2005).Many researchers (Amonsin et al., 1997;Hung and Alvarado, 2001;Özbey et al., 2004;Hassanzadeh et al., 2010;Tachil et al., 2010) have used the PCR method with success for confirmation of isolated and identified strains and the detection of O. rhinotracheale DNAs in tissues.Kiliç et al. (2009), reported that O. rhinotracheale strain inoculated alone (4.8×10 8 cfu) into 14 day old broiler chickens using the aerosol route.After inoculation, microbiological (culture) and molecular (PCR) analyses were performed on different parts of respiratory system (sinuses, larynx, trachea, lungs and air sacs) and on visceral organs.Finally, O. rhinotracheale strain was successfully reisolated in culture and PCR from samples of trachea and lungs.All isolates positive by O. rhinotracheale culture were also positive by PCR and culture negative samples were also PCR negative.In a study conducted by Hassanzadeh et al. (2010), in slaughtered broiler chickens, it was reported that the number of ORT-positive tracheal swapes and tissue samples increased from 4 to 7 with the use of the PCR.Due to the overgrowth of the causing agent by other bacteria, it is indicated that the isolation yields of this particular bacterium is rather low (Hassanzadeh et al., 2010).In an another study, O. rhinotracheale DNA was detected in 75 samples (19.93%) of broiler turkeys in Iran (Doosti et al., 2011).In the present study, all the O. rhinotracheale isolates obtained from layer hens, broiler chickens and turkeys shown the same bands (784 bp) using PCR.This result suggests that the PCR can be used for confirmation of isolates as part of common diagnosis in field (Hassanzadeh et al., 2010;Doosti et al., 2011).
SDS-PAGE can be used for confirmation of O. rhinotracheale isolates and demonstration of similarities or differences between isolates.Several researchers (van Empel, 1998;van Damme et al., 1994;Amonsin et al., 1997;Lopes et al., 2000;Hung and Alvarado, 2000) have been reported a high rate of similarity for the outer membrane and/or proteins of the outer membrane between O. rhinotracheale strains of different origin and/or different serotypes.Banani et al. (2001), reported that 59 O. rhinotracheale isolates they isolated from broiler chickens and commercial layer hens with clinical signs of respiratory disease belonged to serotype A. Based on SDS-PAGE results; they also pointed out to the presence of close similarities between the protein profiles of these isolates.These researchers (van Empel, 1998;van Damme et al., 1994;Amonsin et al., 1997;Lopes et al., 2000;Hung and Alvarado, 2000) reported minor differences between isolates and predominating similarities among some strains.Furthermore, Özbey et al. (2004), reported that the 6 O. rhinotracheale strains isolated from the trachea (n=5) and lungs and trachea (n=1) of slaughtered broiler chickens yielded similar protein profiles when SDS-PAGE was performed.
In the present study, although the isolates obtained from broiler chickens, layer hens and turkeys displayed close similarities, they yielded rather different bands.It was noted that strains of O. rhinotracheale isolated from layers, broilers and turkeys formed distinct clusters.While the layer and broiler isolates has existed in same cluster, turkey isolates took part in different group.Tsai and Huang (2006), reported that 40 O. rhinotracheale strains were isolated from 28 chickens and 12 in Taiwan.All isolates were identified using culturing and confirmed using PCR.The 16S ribosomal RNA (rRNA) sequences of 23 Taiwanese O. rhinotracheale isolates showed high identity (98-100%) to sequences.
It is reported that RAPD-PCR method was relatively simple and easy to use for the characterizations of O. rhinotracheale and gave a level of discrimination.Most genotypes could be identified by this method using one or two primers only: OPG 11 (D:0.786)or OPG 11 (D:0.850)(Leroy-Setrin et al., 1998).Both Leroy-Setrin et al. (1998) and Van Empel (1998), showed that no relationship between serotypes and either ribotypes or RAPD types could be established Özbey et al. (2005), reported that RAPD assay showed almost similar DNA profiles in 6 ORT isolates of serotypes A, B, D and E. In this study, eight random-amplified-polymorphic DNA (RAPD) types by UPGMA method were found among the O. rhinotrachelae strains.In addition, O. rhinotracheale isolates of layers, broiler and turkey by SDS-PAGE analysis were demonstrated 19 different clusters.Tachil et al. (2007), reported that fingerprinting methods may be more discerning tool for characterization of O. rhinotrachelae isolates than the serological tests using the agar gel precipitation test.Also, fingerprinting method can potentially be a valuable tool in identifying an isolate from a clinical outbreak of O. rhinotracheale infection for development of autogenous vaccine.

Conclusion
As the study reveal, O. rhinotracheale strains isolated from different geographical regions, sources and animal species (turkeys, broiler chickens and layer hens) were Erganis et al. 87 confirmed using PCR.The protein profiles of these strains were determined using SDS-PAGE and their similarities were evaluated using RAPD-PCR.It is considered that further studies are required to confirm the O. rhinotracheale strains via different molecular methods such as using pulse-field gel electrophoresis, the random-amplification-fragment-length-polymorphism (AFLP) method, and entire length of the 16S rRNA gene or the sequence divergence.

Figure 1 .
Figure 1.The dendogrames of SDS-PAGE analysis by NJ (A) and UPGMA (B) methods in strains of O. rhinotracheale.

Figure 4 .
Figure 4.The dendogrames of RAPD-PCR profiles by NJ (A) and UPGMA (B) methods in strains of O. rhinotrachele.