An updated comprehensive review on ornithobacteriosis: A worldwide emerging avian respiratory disease.

Ornithobacteriosis is an important emerging respiratory disease of domestic and wild birds caused by Ornithobacterium rhinotracheale (ORT) bacterium. The disease has been detected in some countries since 1980, which rapidly spread worldwide later on. Ornithobacteriosis can transmit either horizontally or even vertically. Infection with ORT is mainly characterized by respiratory distress, poor performance, acute death, and a drop in egg production. However, the most characteristic necropsy lesions of dead turkeys and chickens are yoghurt like airsacculitis and pneumonia, usually unilateral. Unfortunately, infection with ORT was misdiagnosed in most of the poultry flocks due to similarity with other respiratory pathogens and the lack of the ideal protocols for diagnosis. Recently, some molecular and serological techniques have been used to detect the infection. Treatment of ORT with antibiotics is very difficult and variable as a result of acquired resistance. Many vaccines have been developed to counteract such infection in broiler, layers, and breeder chicken and turkey flocks. Inactivated, live, and sub-unit vaccines have been used with satisfactory results. Thus, this review paper aimed to address ornithobacteriosis, emphasizing the distribution, transmission, clinical picture, diagnosis, and disease control.

Introduction

Respiratory infections of poultry are regarded as very important problems that cause high economic losses in the production system. One of these infections is ornithobacteriosis. It is a relatively novel emerging respiratory contagious disease among turkeys and chickens caused by Ornithobacterium rhinotracheale (ORT) bacterium. The bacterium is a highly polymorphic Gram-negative rod, non-motile or spore former, and belongs to genus nov., species nov. in the rRNA superfamily V and family Flavobacteriaceae (Vandamme ). Ornithobacteriosis induces severe adverse negative impact on the poultry industry worldwide. Poor growth rate, acute mortalities, increasing the medication costs, high condemnation rates at processing, and decreasing the quantity and quality of eggs and hatchability are the economic losses of infection (Chin ). The disease showed rapid evolution and spread all over the world with increase in the incidence rate. Ornithobacteriosis is mainly characterized by respiratory manifestations with the presence of yogurt-like fibrinous exudates in the airsacs and uni/or bilateral lung consolidation (Hafez, 1996; Banani ). However, the severity of the clinical picture is affected by the presence of other complicating infectious agents and non-infectious environmental conditions along with some virulence factors (van Empel and Hafez, 1999; Barbosa ). Accordingly, ornithobacteriosis may be regarded as a part of a complex of other respiratory viral and bacterial pathogens that synergize to induce the infection (Welchman ; Kursa ). Definitive diagnosis of ornithobacteriosis is based on isolation and identification of ORT bacterium using either conventional phenotypic methods (De la Rosa-Ramos ; Hassan ) and/or molecular techniques (Ellakany ; Veiga ; Hassan ; Karimi-Dehkordi ). Despite the fact that ORT infection can be successfully treated with antibiotics, the bacterium can rapidly develop antibiotic resistance (Devriese ). Therefore, some trials have been undertaken to produce inactivated, live, and sub-unit vaccines to counteract ORT infection (Lopes ; Schuijffel ; Ghasemipour ).

This review paper aimed to address ornithobacteriosis, emphasizing the distribution, transmission, clinical picture, diagnosis, and control of the disease.

The worldwide incidence and distribution

Ornithobacteriosis has been detected in domestic and wild birds with respiratory conditions in several countries worldwide. Early in 1987 in Hungary, Pasteurella-like organisms were isolated from ducks with respiratory signs. In addition, Riemerella anatipestifer like bacteria was found in turkeys showing respiratory affection in 1991 and 1992 in Germany. However, in 1991 in South Africa, highly pleomorphic Gram-negative rods were isolated from a 28-day-old broiler chicken flock suffering respiratory manifestations, mortalities, bad performance, foamy yoghurt like airsacculitis and pneumonia (van Beek ). Moreover, in the Netherlands and Germany in 1993, respiratory problems, poor growth rate, and acute mortalities have been observed in turkey and chicken broiler flocks (Hafez ; Hinz ; van Beek ). Later on, the disease was seen rapidly spreading across many countries like the USA (Charlton ), France (LeÂorat ), Israel (Bock ), Peninsula (Odor ; Salem ), Canada (Joubert ), and South Africa (Vandamme ; Buys, 1996; Travers, 1996; Goovaerts ).

In the different states of America, ORT has been isolated from turkeys and chicken flocks with respiratory signs and variable mortalities (DeRosa , 1997; Tahseen, 1997; Roepke ; Heeder ; Malik ; Walters, 2014; Hauck ). However, antibodies to ORT infection have been serologically detected in broiler and breeder’s sera in South America (Arns ), Netherlands (van Veen ), Southern Brazil (Canal ), Taiwan (Tsai and Huang, 2006), Thailand (Chansiripornchai ), and Pakistan (Siddique ).

Besides, the bacterium has been isolated and characterized from broiler chickens in the Rio Grande do Sul state of Brazil (Canal ) and Peru (Hung and Alvarado, 2001; Koga and Zavaleta, 2005), along with laying hens in Cuba (Espinosa ; Chávez ). Recently, Ha succeeded in isolating ORT from broiler chickens in New Zealand, while Szabó characterized ORT field isolates in Hungary. In Japan, Umali detected the possibility for isolation of ORT from the heart, liver, kidney, spleen, and ovaries of broiler chickens after systemic infection.

Regarding the Middle East countries, ORT has been characterized from Iranian (Rahimi and Banani, 2007; Asadpour ; Ghanbarpour and Salehi, 2009; Hassanzadeh ; Asadpour ; Mayahi ; Ghasemipour ), Turkish (Ozbey ; Türkyilmaz, 2005), and Jordanian (Roussan ) chickens and turkey flocks.

In Egypt, ornithobacteriosis was early detected in the 1990s, since ORT organism has been isolated alone or concomitantly with other bacteria from layers and broiler chicken and turkey flocks (Youssef and Ahmed, 1996; El-Gohary, 1998, El-Gohary and Awaad, 1998; El-Gohary ; El-Gohary and Sultan, 1999; Abd El-Ghany, 2000). Later, several studies have been carried out to isolate, characterize and treat ORT in different Egyptian governorates (Amal, 2002; Shihata and Ibraheem, 2004; Shahata ; Attia, 2008; Elbestawy, 2010; Hegazy ; Masoud ; El-Abasy ; Ellakany ; Hassan ). The incidence of ornithobacteriosis in Egyptian poultry flocks is summarized in Table 1.

Table 1.

The incidence of ornithobacteriosis in Egyptian poultry flocks.

Findings	References
The incidence rate of ORT was demonstrated as 8.6% in layer chicken flocks showing depressed egg production.	Youssef and Ahmed, 1996
The vertical transmission of ORT is possible as the bacterium has been associated with hatching problems in chicken and turkey eggs.	El-Gohary, 1998
Concomitant ORT and E. coli infections has been recorded in chicken broilers with an incidence rate of 8.5% for ORT.	El-Gohary and Awaad, 1998
Both ORT and Pasteurella haemolytica have been isolated from commercial larger chickens in percentage of 4.3%.	El-Gohary et al., 1998
ORT has been isolated from meat-type breeder chickens in incidence of 3.2%.	El-Gohary and Sultan, 1999
The incidence of ORT infection in 75 broiler chicken farms representing different Egyptian governorates was carried out. The bacterium has been biochemically and molecularly characterized from air sacs (3.5%), lungs (2.2%), trachea (0.44%), pericardium (0.22%) and liver (0.22%). All isolates were belonging to serotype A, and were sensitive to amoxicillin and chloramphenicol. The results of pathogenicity test revealed that the isolated ORT strains were pathogenic for 2 weeks old chickens, while other infection with E. coli and infectious laryngotracheitis increased the severity of the clinical picture.	Abd El-Ghany, 2000
The incidence of ORT infection in chicken flocks in Upper Egypt (Assuit governorates) has been demonstrated in percentage of 5.8%.	Amal, 2002
In El-Sharkia governorate, ORT has been isolated and characterized rabbits.	Shihata and Ibraheem, 2004
ORT was isolated from chicken embryos and layers and this indicated possibility of vertical transmission of the bacterium. In addition, the in-vitro antibiotic sensitivity test results showed that amoxicillin, enrofloxacin and tetracycline were the most effective antibiotics against ORT.	Shahata et al., 2006
In Kafrelshikh governorates, ORT infection has been detected and investigated in rabbit’s flocks.	Attia, 2008
Both ORT and M. gallisepticum have boon discovered in chicken flocks in El-Behera and Kafr El-Sheikh governorates. The rate of ORT isolation was 7.27%.	Elbestawy, 2010
The positive correlation between the presence of antibodies against ORT and decreased body weight in broilers has been proven. Besides, ORT isolates resistant to gentamycine, norofloxacin, ciprofloxacin, cefotaxim, sulphamethoxazole trimethoprim and colistin sulphate, but were sensitive to amoxicillin, ampicillin and doxycycline.	Hegazy et al., 2015
Five broiler’s ORT strains have been detected and showed 94%–98% similarity to some American and Asian ones after sequencing of 16S rRNA.	Masoud et al., 2015
Interestingly, ORT has been isolated and characterized from 21 out of 300 (7%) diseased rabbits showed respiratory manifestations, decreased feed intake with poor performance and expectoration of blood stained mucus just prior to death In Kafrelshikh governorate. All ORT isolates were serologically belonging to serotype A. All of ORT strains were sensitivity to sulphamethoxozole/trimethoprine, spiramycin, neomycin, ampicillin, amoxicillin, ciprofloxacin and tetracycline, while non-sensitive to penicillin, streptomycin, clindamycin, lincomycin, gentamycin, vancomycin and colistin sulphate. Induction of experimental ORT infection in 3-months-old rabbits were successfully carried out and the animals showed respiratory disease picture. Moreover, treatment of experimentally infected animals with sulpha-trimethoprine and coconut oil relief the severity of the lesions.	El-Abasy et al., 2016
Conventional isolation of ORT revealed presence of the bacterium in an incidence rate of 11.66%. Broilers and layers isolates of ORT were identified molecularly using PCR. These ORT isolates were closely related to Asian, European, and American strains (98%–100%). The role of live infectious bronchitis vaccines on the severity of ORT infection was investigated after experimental infection of broiler chickens. The results revealed that live infectious bronchitis vaccines that are usually used in the Egyptian poultry field may concomitantly increase the pathogenicity of ORT infection. This combination can led to decreasing in body weight, weight gain, and increasing in feed conversion ratio.	Ellakany et al., 2019
In this study, the incidence of ORT infection in broiler chickens in Assiut governorate was 17.77% (32/180) through isolation and phenotypic identification methods, but it was 3.33% (6/180) using molecular techniques. Serologically, all the tested ORT isolates were serotype A. Moreover, 100% of ORT isolates were sensitive to colistin and doxycycline, 50% of isolates were sensitive to ampicillin and streptomycin, and 16.67% of isolates were sensitive to neomycin and trimethoprim. Meanwhile, all of the isolates were resistant to gentamycin, amoxycillin and cephradine with 100% incidence. The results of the pathogenicity test in broiler chickens declared that living ND attenuated vaccine as La Sota exaggerated the clinical signs and lesions of ORT experimental infection.	Hassan et al., 2020

The causative agent and the virulence factors

As a result of the difficulties in isolation and characterization of ORT bacterium, it was early named as Pasteurella like (van Empel and Hafez, 1999), Kingella like (van Beek ), polymorphic Gram-negative rode bacterium (Charlton ), and TAXON 28 (van Empel and Hafez, 1999). However, ORT was genotypically and taxonomically classified in the early 1990’s as a new genus and species (Hafez ; Vandamme ). The species rhinotracheale belongs to genus Ornithobacterium. The bacterium was classified as a Gram-negative and highly pleomorphic rod of the rRNA superfamily V, in the taxonomic neighborhood of the genera Cytophaga, Riemerella, and Flavobacterium (Vandamme ; Canal ). The genus Ornithobacterium belongs to the family Flavobacteriaceae (Hafez ), which also includes the genus Riemerella with R. anatipestifer and the genus Coenonia with C. anatina. Besides a new species named Candidatus Ornithobacterium hominis sp. nov., ORT is the only species described within the genus Ornithobacterium.

Before the first taxonomic identification of ORT bacteria, misdiagnosis of infection was common, and the causative agent was attributed to some other bacteria such as Pasteurella, Riemerella, Bordetella, or Haemophilus (Hafez ; Bragg ) as well as other viruses as Pneumovirus (Marien ).

The Pathogenicity and persistence of ORT organisms in the host are influenced by environmental conditions, biofilm formation, and coinfection with other organisms (Marien ; De la Rosa-Ramos , 2018). Serotypes A, B, C, D, and E of ORT showed different virulence factors with variable adherence profiles (Chansiripornchai ; De Haro-Cruz ). The tissue’s adherence and colonization with ORT are associated with the presence of some virulence factors such as hemagglutinin, neuraminidase, and other glycoprotein (Kastelic ; De la Rosa-Ramos ). Hemolytic isolates of ORT have been described and differentiated from R. anatipestifer isolates (Walters, 2014). Figure 1 shows the factors that influence the severity of ornithobacteriosis infections.

Fig. 1.

Factors influence the severity of ornithobacteriosis infections.

Host susceptibility and transmission of infection

Ornithobacteriosis is incriminated in infection of all commercial avian species and wild birds in many countries of the world. There is a wide range of birds that could be infected with ORT or carry the bacterium in their respiratory tracts. The bacterium is present in the apparent healthy captive and free-ranging non-galliform species. Turkey, chicken, duck, goose, guinea fowl, gull, ostrich, partridge, pheasant, pigeon, quail, and rook showed ornithobacteriosis (Charlton ; Anonymous, 1995; Devriese ; Buys, 1996; Hafez, 2002). Infection with ORT was first described in turkeys, the main susceptible host (Hafez, 1996; Karimi-Dehkordi , Kursa ), as well as in chickens (Roussan ; Hassan ). It has been reported that the incidence rate of ORT in turkeys was higher (41%) than that in chickens (6.9%) (Hauck ). Besides, the disease has been reported in the Egyptian Muscovy and Balady ducks (El-Abasy, 2008), and the bacterium has been molecularly detected in pigeons and birds of prey (Tsai and Huang, 2006; Thieme ). Interestingly, ORT has been demonstrated in rabbit farms with respiratory problems in some Egyptian governorates (Shihata and Ibraheem, 2004; Attia, 2008; El-Abasy

Ornithobacteriosis spreads horizontally through inhalation and direct contact or indirectly through the drinking water (Chin and Charlton, 2008). Provide strong evidence of vertical transmission (from the hen to the egg through the ovary), the entrance of the bacterium via eggshell is different. It is also probable since ORT was isolated from reproductive organs, infertile and hatching eggs, and from dead embryos (Tanyi /1996; El-Gohary, 1998; Shahata ). The bacterium was also found on the eggshells and in the yolk sacs of day-old- chicks but at very low incidence (1%) (van Empel, 1997). This type of transmission can occur either trans-ovarian or by cloacal contamination (van Empel, 1997). Experimental studies showed that ornithobacteriosis infected turkey breeder hens showed survival of the bacterium in the ovary and oviduct without signs (Back , 1998a; Nagaraja ).

Wild birds may also be considered as an important source of infection to the commercial poultry flocks. Ornithobacteriosis is regarded as a threatening but not a zoonotic disease (Cobb and Smith, 2015; World Organization for Animal Health, 2018).

Clinical signs and lesions

Birds infected with ORT showed reduced food intake, decreased weight gains, sneezing, nasal discharge, wet eyes with lacrimation, sinusitis, facial edema followed by coughing, dyspnea, prostration, and death (Canal ; Rahimi and Banani, 2007; Asadpour ). Sudden death with or without respiratory signs has been found in chickens with nervous manifestations (Chin ). Certain reported cases showed that ornithobacteriosis might induce sudden death due to meningitis (van Empel and Hafez, 1999). Experimental infection of chickens and turkeys with ORT revealed decrease in body weight and growth retardation (van Empel ; Ellakany ). The clinical picture of ORT could be vanished within a week or become more complicated in the presence of other pathogens or even non-recognized as an ORT infection anymore (van den Bosch, 2001).

The necropsy findings of ornithobacteriosis are associated with sinusitis, tracheitis, pericarditis, airsacculitis, peritonitis, and exudative pneumonia (Amonsin ). However, the most characteristic post-mortem lesions are the presence of foamy white, “yoghurt-like” exudate in the airsacs, predominantly in the abdominal airsacs and fibrino-purulent pneumonia (Hinz ; Banani ). Moreover, in South Africa, subcutaneous oedema over the cranium and severe osteitis without respiratory affections have been detected in 28-day-old broiler chickens (Goovaerts ).

Actually, the lesions become more severe if other complicating infectious pathogens are associated with ORT infection and often lead to death (Abd El-Ghany, 2000; Chin ). Accordingly, the severity of the clinical picture of ornithobaxteriosis, disease duration, and mortality rates are extremely variable and influenced by the virulence of the bacterial strain, the immune status of the host, the environmental conditions like bad management, poor ventilation, overcrowding, poor litter quality, bad hygiene, and high ammonia levels along with the presence of concurrent or secondary infections (Travers, 1996; Bisgaard ). For instance, administration of living Newcastle (ND) La Sota vaccine at 5 days before ORT challenge induced a more serious increase in airsacculitis and pneumonia scores compared to both ORT challenge and ND La Sota vaccine administration alone (van Empel ). Furthermore, Abd El-Ghany (2000) revealed that ORT strains were pathogenic for 2-week-old chickens, while co-infections with Escherichia coli (E. coli) and infectious laryngeotracheitis increased the severity of the clinical picture. Pan demonstrated that the experimental infection of broiler chickens with ORT could induce a mortality rate of around 50%. In comparison, mixed co-infection of ORT with H9N2 avian influenza virus (AIV) led to a higher mortality rate of 70% and 90%, respectively, if ORT inoculation was simultaneously made with H9N2 or if H9N2 AIV was inoculated after 3 days. In the same context, ORT infection alone could induce a disease condition with mortalities, but co-infection with Streptococcus zooepidemicus was more lethal (Pan ). Recently, Ellakany confirmed that concomitant experimental ORT infection and Mycoplasma gallisepticum increased the severity of clinical respiratory signs and lesions and hurts the performance and growth parameters of broiler chickens.

The histopathological lesions of ORT were represented as granulomatous pneumonia, tracheitis, and fibrinous airsacculitis (van Empel and Hafez, 1999; Abd El-Ghany, 2000; Chin ; Ellakany ). Kilic described the microscopic lesions after experimental ORT infection in chickens as focal epithelial hyperplasia along with necrosis and inflammatory lesions of the lamina propria in the upper respiratory tract, air sacs, as well as around bronchioles and some lung areas.

Laboratory diagnosis

Signs and lesions associated with ornithobacteriosis are of little value and not sufficiently specific to diagnose the disease since a similar clinical picture could be seen in other infections (Hafez and Sting, 1996). Hence, diagnosis of ornithobacteriosis mainly relies on phenotypic and molecular detection of the bacterium or immunogenic detection of antibodies (Ellakany ).

Despite conventional ORT isolation method could be difficult owing to the overgrowth by other opportunistic bacteria (Churria , 2012), it is still necessary for serotyping, determination of in vitro antimicrobial sensitivity test as well as production of autogenous vaccines (Vandamme ; Hafez and Sting, 1996; Hegazy ). Techniques of isolation and identification may differentiate ORT bacterium from other similar respiratory bacteria as Pasteurella multocida, E. coli, or Avibacterium paragallinarum.

For successful isolation of ORT, samples should be from the airsacs, lungs, and trachea after natural and experimental infections (Joubert ; Abd El-Ghany, 2000; Welchman ; Hauck ; Gavrilović ). Moreover, ORT bacterium was isolated from the nasal mucosa and orbital sinuses swabs of infected turkeys (Karimi-Dehkordi ). Isolation of the bacterium from the heart, liver, kidney, spleen, ovaries, and brain is suggestive after systemic infection (van Beek ; Nagaraja ; Umali ). Samples should be collected from suspected flocks as early as possible. Tissues and swabs could be maintained at 4°C for 2 days or at −20°C for 5 days without adverse effect on the variability of ORT organism or growth of other bacteria (Numee ).

Samples should be inoculated on 5%–10% sheep blood agar media and incubated under microaerophilic (5%–10% CO2) or anaerobic conditions for 24–48 hours. As ORT is a growing fastidious organism, it needs special media’s supplement and special environmental conditions (Travers, 1996). Most ORT isolates showed resistance to gentamicin or polymyxin. So, adding these antibiotics (5–10 µg/ml) to the media can suppress other contaminating overgrowing bacteria such as Pseudomonas, Portus, and E. coli species (Back ; Hassan ). Positive cultures of ORT appear as non-hemolytic, pinpoint to pinheaded, gray to grayish white, circular, convex, and reddish glow colonies with a distinct butyric acid odor (Shahata ). Some isolates of ORT showed incomplete β hemolysis, especially after 96 hours of incubation. In addition, the bacterium can grow in brain heart infusion broth and on trypticase soya agar but not on MacConkey agar (Roepke ; Post ; Asadpour ; Mayahi ; Hassan ).

Microscopic identification of stained smears from suspected ORT colonies showed Gram-negative, highly pleomorphic, non-motile, or spore-forming rods (van Empel and Hafez, 1999; Chin and Charlton, 2008; Espinosa ; Chin ).

Regarding the biochemical identification, ORT isolates are positive oxidase and negative catalase (van Empel ; Chin and Droual, 1997; Hafez, 1998; van Empel, 1998; Ryll ). Commercial kits (API-20 NE and API-ZYM) are used for the biochemical characterization of ORT isolates. The bacterium is positive for arginine dihydrolase, β- galactosidase, gelatin liquefaction, and Voges-Praskauer tests, while negative for L-lysine decarboxylase, ornithine decarboxylase, and H2S production tests (Chin and Charlton, 2008; Hassan ). Regarding sugar fermentation tests, ORT reveals positive sucrose, arabinose, lactose, fructose, galactose, and maltose, but negative glucose, mannitol, inositol, and sorbitol (Rahimi and Banani, 2007; Mayahi ).

There is a great possibility to reduce the detection rate of ORT after culturing due to the presence of tiny colonies, slow growth of the bacterium, and the need for enriched media and microphonic conditions (Zahra ). Therefore, molecular detection of ORT DNA using polymerase chain reaction (PCR) and gene sequencing of 16S rRNA and rpoB genes are now used for the routine diagnosis (Hafez and Beyer, 1997; Veiga ). These recent techniques are regarded as very important and fruitful tools for the definitive detection of infection (Ozbey ; Banani ; Ellakany ). Moreover, they are fast, sensitive, and specific for the characterization of bacterial strains (Hung and Alvarado, 2001; Li and Diao, 2009; Montes De Oca-Jimenez ; Veiga ).

Many types of modified PCR techniques are used for the characterization of ORT. Enterobacterial repetitive intergenic consensus-PCR, repetitive element palindromic-PCR, random amplified polymorphic DNA-PCR, and multilocus sequence typing have been developed (Szabó ). Great variations among ORT isolates have been found using phylogenetic analyses of the 16S rRNA gene (Banani ; Montes de Oca-Jimenez ). Lately, Veiga suggested using ORT rpoB gene for partial sequencing of isolates from different avian species.

The phylogenetic relationship indicated the existence of a greater genetic variability (Montes De Oca-Jimenez ; Veiga ), particularly between ORT strains from different hosts (Thieme ).

Serotyping of ORT isolates has been carried out using agar gel precipitation test and enzyme-linked immunosorbent sssay (ELISA) with specific antisera against 18 (A–R) serotypes (van Empel ; van Empel, 1998; Türkyilmaz, 2005; Wu ; Hassan ). Within ORT species, several serotypes and strains with different virulence are present (Ryll ). The different ORT serotypes have no direct relationship with virulence. Rapid slide agglutination test was also used to type ORT isolates (Back ). Reports indicated that all the tested ORT isolates belong to serotype A, which is the most prevalent among strains of chickens (94%) and turkeys (57%) (Siddique ). Serotypes A, B, D, and E are most common in turkeys, while serotypes F, K, and M are sometimes isolated from chickens and turkeys (van Empel and Hafez, 1999). A cross-reaction between serotypes B and A and between serotypes I and L were observed after using rapid serum plate agglutination test in layers. Besides, cross-reactions have been detected for serotypes A, E, and I, but not with serotype C (Türkyilmaz, 2005). Serological identification of ORT is hampered by limitations, such as cross-reactivity between strains (Szabó ). Due to the difficulties in serotyping methods, the presence of un-determined new serotypes of ORT has been suggested (Numee ; De la Rosa-Ramos ).

To overcome these disadvantages, a wide range of techniques have been implemented over the last years. The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become more efficient than biochemical tests for routine laboratory diagnosis of microorganisms as it is a rapid, reliable, and direct technique for the identification (Alispahic ). In the recent study of Alispahic , the molecular characterization of 47 ORT field strains derived from Austrian turkey farms was carried out using MALDI-TOF MS and whole genome sequencing techniques. The results of MALDI-TOF MS revealed that most ORT strains were grouped within one cluster although they comprised of different serotypes, except serotypes F, K, and M that formed a different cluster. The whole genome sequencing results confirmed that the previous data indicated that serotypes F, K, and M were clearly different from the other ORT strains and may belong to different Ornithobacter species.

High seroprevalence of ORT was demonstrated among broiler and breeders chicken and turkey flocks in several regions worldwide using ELISA (van Empel ; Canal ; Hegazy ). For instance, Hafez and Sting (1996) detected antibodies to ORT in 79% of broiler breeder chicken flocks, and 26% and 55% of broiler chickens and meat turkey flocks, respectively. Furthermore, Ryll demonstrated the presence of antibodies in 96.6% of the sera of broiler turkeys. In Iran, antibodies of ORT were found in 205 (44.2%) out of 463 broiler chickens and in 340 (72%) out of 472 breeder chicken serum samples (Allymehr, 2006). Another study showed the presence of antibodies in the sera of 289/460 (83%) broiler breeder flocks (Asadpour ). Moreover, of the 420 serum samples, 134 (31.9%) were positive for ORT antibodies (Ghanbarpour and Salehi, 2009). High (100%) seroprevalence to ORT was detected in broiler breeder chicken flocks in Brazil (Canal ) and layer chicken flocks in the United States (Heeder ).

It is important to note that antibodies of ORT and other pathogens were detected using ELISA. For example, antibodies of ORT and turkey rhinotracheitis virus (Hafez, 1997a, 1997b) and Chlamydia psittaci were demonstrated (Hafez ).

Prevention and control

The prevention of ornithobacteriosis in the poultry production system should be considered since the disease has become endemic worldwide. Adoption of strict biosecurity measures and husbandry practices is critical. All in-all out policy should be applied. Thorough cleaning and disinfection of poultry houses are essential to avoid the possibility of ORT re-infection or spreading, especially in endemic areas. In vitro study of Hafez and Schulze (2003) declared that a concentration of 0.05% aldehydes and organic acids (formic and glyoxyl) disinfectant preparations effectively inactivated ORT bacteria within 15 minutes. The in vitro antibiotic susceptibility pattern of ORT strains is greatly inconsistent. It depends on the locality, the source of strain, the inherent genetic differences between bird breeds, and the routinely used antibiotics in the area (Odor ; Malik ; Türkyilmaz, 2005; Mayahi ). In addition, the mutation of ORT plasmids plays an important role in developing antibiotic resistance (Back ). For instance, an increase in the minimal inhibitory concentration of enrofloxacin from 0.03 to 0.25 mg/ml for ORT treatment in turkeys was due to mutations in the gyrA gene (Marien ). Different classes of antibiotics, even the recently used ones, became inefficient against ORT strains, maybe due to transfer of the resistance among them (Devriese ) or increase in the resistance for different drugs (Cauwerts ). As a result of frequently acquired resistance, the treatment of ornithobacteriosis is difficult and cannot be effectively achieved through antibiotics (Devriese ). An early study by van Beek declared that the oral treatment of ORT infected turkeys using enrofloxacin and trimethoprim/sulphonamide was not effective. However, twice injections of tetracyclines and penicillin gave good results. Treatment of ORT-infected birds with 250 ppm amoxicillin and 500 ppm chlortetracycline in the drinking water for 3–7 days was effective in relieving infection (Hafez, 1997b). In Germany, Hafez (1996) observed that 90%–100% of the ORT strains were sensitive to tetracycline, chloramphenicol, and amoxicillin, while they were resistant to enrofloxacin, gentamycin, neomycin, and trimethoprim/sulphonamide. Although 90% of the abovementioned isolates were resistant to enrofloxacin in Germany, they were sensitive to the same antibiotic in Belgium and France (Devriese ; Dudouyt ; Roger and LeÂorat, 1997). Moreover, Chin and Droual (1997) demonstrated that water treatment with amoxicillin, tetracycline, and chloramphenicol was satisfactory. Isolates of ORT in France showed resistance to gentamicin and colistin but sensitivity to amoxicillin, spectinomycin, and tylosin (Roger and LeÂorat, 1997). In USA, 100% of ORT revealed susceptibility to ampicillin, penicillin, spectinomycin, erythromycin, and tylosin, 79.4% were susceptible to neomycin, tetracycline, and sarafloxacin, and the rest of the isolates were susceptible to streptomycin, gentamicin, and trimethoprim (Nagaraja ). Strains of ORT strains in the Netherlands showed susceptibility to amoxicillin, tetracycline, enrofloxacin, and trimethoprim/sulphonamid (van Veen ). However, later on, the sensitivity of these ORT strains to amoxycillin and tetracycline decreased from 62% to 14%. Even four out of the strains were non-susceptible to enrofloxacin the combination of trimethoprim-sulphonamide. In Mexico, Soriano declared that ORT strains were sensitive to amoxicillin, enrofloxacin, and oxytetracycline, while resistant to gentamicin and fosfomycin. In addition, Mohd-Zain demonstrated that 100% of ORT strains were resistant to ampicillin, enrofloxacin, and sulfanomide/trimethoprim, while they were sensitive to chloramphenicol. In the study by Asadpour , the authors found that all ORT strains were non-sensitive to enrofloxacin, ciprofloxacin, erythromycin, tetracycline, while all of them were sensitive to ceftriaxone. Moreover, two strains (66.70%) showed moderate susceptibility to amoxicillin and florfenicol. Churria reported that all isolates of ORT were resistant to gentamicin. Most of them were resistant to enrofloxacin, erythromycin trimethoprim-sulfamethoxazole, doxycycline, and fosfomycin, while all of them were sensitive to ampicillin and florfenicol. The recent Egyptian study of Hassan revealed that 100% of circulating ORT strains were non-susceptible to gentamycin, amoxycillin, and cephradine, while 100% were susceptible to colistin and doxycycline, 50% to ampicillin and streptomycin, and 16.67% to neomycin and trimethoprim.

From the above-mentioned studies, it could be concluded that most ORT strains became resistant to the majority of the used antibiotics in the field (Watteyn ; Umali ). Therefore, vaccination may be a promising and effective strategy to counteract ornithobacteriosis. Inactivated, live, and recombinant sub-unit vaccines of ORT have been developed with variable results (Gornatti Churria ). An early trial has been done to vaccinate day-old-broiler chickens and turkeys with autogenous inactivated oil adjuvant bacterin (Bock ). Moreover, this type of bacterin significantly reduced ORT lesion scores after vaccination of broilers at 26-days-old (van Empel and van den Bosch, 1998). However, it has been found that vaccination of birds at 8-week-old was more effective than vaccination at 4-week-old to avoid interfering with maternal immunity (Gopala Krishna Murthy ). Vaccination of breeders using inactivated bacterin was found to be effective and protective against the development of pathologic changes of ORT infections in the progeny (Bisshop, 2005). No cross-protection between serotypes was induced after vaccination with bacterins in oil adjuvant (Bock , 1997). Many types of inactivating substances and adjuvants were added to 18 ORT vaccines to choose the best one (Gopala Krishna Murthy ). It has shown that a vaccine containing mineral oil adjuvant induced the highest immune response and the lowest respiratory lesions in vaccinated birds. Cauwerts also observed decreasing mortalities and increasing production of the offspring from the vaccinated breeders. Vaccination of breeder broiler chicken flocks with inactivated ORT bacterin containing serotype A induced 39% increase in the production rate and 22.3% decrease in progeny loss (De Herdt ). Autogenous inactivated oil adjuvant ORT bacterins showed a successful reduction of ornithobacteriosis outbreaks in Turkey (Erganis ), Israel (Chin ), and Iran (Ghasemipour ). Mention if there is cross protection between different serotypes of ORT. It has been shown that inactivated or subunits vaccines of ORT mostly give low or only partial cross-protection and not always for all serotypes. However, live types vaccines can provide this cross protection (Schuijffel , 2006).

van Empel and van den Bosch (1998) found that the vaccination of breeders against ornithobacteriosis using live vaccine provided satisfactory protection against pneumonia and airsacculitis in their progeny until 28 days of age. The protective efficacy of a live, temperature-sensitive mutant ORT vaccine against a bacterial challenge has been evaluated in day-old turkey poults (Lopes ). The vaccine strain colonized the upper respiratory tract and recovered 13 days post vaccination with protective humoral immune response.

Nevertheless, the presence of different ORT serotypes within the bacterium species represent a major challenge in vaccine production. So, the production of recombinant or sub-unit vaccines becomes urgent to induce homologous and heterologous protection along with the rapid immune response (van Empel and Hafez, 1999; Schuijffel ). Schuijffel showed that recombinant subunit vaccine containing eight encoded cross-reactive antigens induced homologous and heterologous protection against ORT challenge as well as production of protective antibodies.

Conclusion

Despite the continuous progress in ORT characterization in almost all countries around the world, there is a gap of knowledge and a lack of information in some aspects. Thus, more research is needed. For example, the mechanism and pathogenesis of ORT infection in the host, the development of more recent diagnostic tools, and the design of treatment and vaccination protocols are still in need. Besides, it is necessary to include some national monitoring programs for emerging respiratory affections like ornithobacteriosis to avoid the adverse economic losses caused by such infection.