Culture Based Isolation of Pathogenic Bacteria Associated with Respiratory Disease Complex in Broiler with Special Reference to Ornithobacterium rhinotracheale from India

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Respiratory diseases complex is the most serious disease affecting poultry and causes heavy economic losses in the poultry industry worldwide.In avian host, several bacteria, Pasteurella (P.multocida, P. gallinarum, P. haemolytica and P. anatipestifer), Klebsiella, Staphylococcus, Bordetella (B.avium) and Haemophilus (H.gallinarum) are involved in respiratory disease complex (Hafez, 2002;Hossain et al., 2013).
Escherichia coli are also associated with respiratory infection in chickens (El-Sukhon et al., 2002).
Ornithobacterium rhinotracheale (ORT) has recently been identified as causative agent for respiratory tract infections in poultry and other birds (Saif et al., 2003).Concurrent infection of young poultry with Klebsiella pneumoniae increased the severity of respiratory disease (Saif, 2003).ORT can be a primary or secondary etiological agent, depending on the strain, virulence, environmental factors, immune status of the host and the presence of other infectious agents (Van Empel and Hafez, 1999).ORT causes respiratory infections, such as weakness, pump-handled respiration, gasping,  Ornithobacterium rhinotracheale is a difficult bacterium to culture.It grows slowly and needs special growth conditions and so attempts at isolation are often negative and plates are overgrown by other bacteria (Hassanzadeh et al., 2010).In the USA, France, Belgium, Spain, Germany, Hungary, Israel, Korea, Japan and Taiwan, the outbreak of respiratory disease associated with ORT has been reported (Van Empel and Hafez, 1999).The aim of the present study was to isolate bacteria with special reference to ORT in broiler flocks associated with RDC in Gujarat and characterize the organisms using cultural characters, morphology and biochemical analyses.

Isolation of Organisms
A total of 60 samples comprising of trachea, larynx, lungs, exudates of infra orbital sinus and air sacs from broiler flocks were separately streaked on Brain-heart infusion medium (BHI) and MacConky Agar (MCA) plates for isolation of organisms.Plates were incubated aerobically at 37°C overnight.The plates were observed after 24 hours for the growth.The isolates were further inoculated on Blood Agar (BA) and Eosin Methylene Blue (EMB) Agar plates for their cultural characteristics.

Morphological and staining characters of isolates
The isolates were subjected to Gram staining for confirming the purity of cultures and morphological characters.

RESULTS AND DISCUSSION
A total of 60 samples from trachea, larynx, lungs, exudates of infra orbital sinus and air sacs in broiler flocks were cultured on BHI, MCA, EMB and BA plates for isolation of bacteria.Result show that fourteen samples produced growth on BHI agar.Among them, four isolates were found to be morphologically similar to Staphylococcus spp.confirmed by Grams staining revealed typical gram positive cocci in bunch (Fig. 1).
Further a total of 9 isolates showing lactose fermenting pink colonies on MacConkey agar (Fig. 2).However, non of the isolate with non lactose fermenting nature was obtained on MacConkey agar.Eight ioslates were confirmed as E.coli, which showed typical greenish metallic sheen on EMB agar as well as gram negative rods morphologically similar to E. coli when stained with Gram's Method of staining (Fig. 3).Similarly, colonies with large, mucoid, pink to purple colonies with no metallic green sheen on EMB agar medium and Gram negative coccobacilli in Gram's stained smear confirmed as Klebsiella spp.
The isolated bacterial colonies on blood agar plates were small, glistening, mucoid, haemolytic and dew drop like, and appeared as gram negative coccobacilli when stained with Gram's stain stain were identified as Pasteurella spp.Non haemolytic tiny colonies on blood agar and staining by Gram method revealed gram negative, highly pleomorphic, rod shaped bacteria identified as Ornithobacterium rhinotracheale (Fig. 4).
All the 17 isolates were characterized by biochemical tests viz.oxidase, catalase, urea and IMViC pattern (indole production, Methyl Red (MR) test, Voges-Proskauer (V.P.) test, citrate utilization on Simmon's citrate medium).The results have been summarized and presented in Table 1 and Fig The incidence of E.coli, Staphylococcus spp.O. rhinotracheale, Pasteurella spp.and Klebsiella spp.were 8 (47.06%), 4 (23.53%), 3 (17.65%), 1 (5.88%) and 1 (5.88%) in broiler flocks (Table 2).These results were in accordance to that reported by Mustafa  The results showed that the prevalence of RDC was 6.67% and 50.00% in apparently healthy and diseased broilers (Table 3).Similar findings have also been documented by Hassan et al., (2014) and Chaudhari, (2017).More number of bacteria were isolated from lung (58.33) followed by trachea (41.66), larynx (16.66), exudates of infra orbital sinus (16.66) and air sacs (8.33) in broiler birds (Table 3).The organism could also be isolated in pure culture in specimens from the trachea, lung or air sac exudates and these findings were in accordance with Murthy et al., (2008) and Pan et al., (2012).The frequency of isolation of ORT from various organs indicated that the isolates were most commonly recovered from the lungs, trachea and air sacs.In some birds, ORT was isolated from the lung, in association with Pasteurella spp.and E. coli.ORT was most frequently isolated from the respiratory tract of poultry, whereas Pasteurella spp.could be isolated from various organs, notably the liver (Murthy et al., 2008).

CONCLUSION
In conclusion, the emerging respiratory diseases complex in broiler caused by O. rhinotracheale, one of the emerged pathogen could be isolated either singly or concurrently with other bacteria, such as E.coli, Pasteurella spp.and Klebsiella spp., indicating its possible etiological role in the respiratory disease complex of broiler.Mixed or concomitant bacterial infections are significant and very severe but the synergistic role between O. rhinotracheale and other bacterial pathogens is yet to be ascertained.

Fig. 5 .Fig. 6 .
Fig. 5. O. rhinotracheale and Pasteurella isolate showing positive oxidase test . 5-7.ORT could be differentiated from other pathogenic bacteria by biochemical reactions.The present findings were coincided with the findings of Van Empel et al. (1997), Masdooq et al. (2008), Siddique et al. (2008), Hassanzadeh et al. 2010, Itoo et al. (2013) and Ashraf et al. (2015).Yilmaz et al. (2011) during their study of lung lesions of slaughtered broilers in slaughter house reported that E. coli ranked the first among the other bacteria isolated.Gowthman et al. (2012) found E. coli as major secondary invading pathogen in respiratory disease complex.

Table 1 .
Various biochemical characters of bacterial isolates

Table 2 .
Bacterial isolates from commercial broiler showing respiratory disease complex.

Table 3 .
Prevalence of RDC in apparently healthy and diseased broilers.