Riemerella anatipestifer infection in ducks has become an emerging problem in many countries nowadays. In Bangladesh, huge number of ducks are died every year due to diagnostic error with other organisms like Pasteurella spp., Salmonella spp., Escherichia coli, Duck Plague, Duck viral hepatitis and Avian Influenza due to their phenotypic similarities which was also observed by other studies (Alexander et al. 2003; Kardos et al. 2007; Pala et al. 2013).
In our study we conducted a short survey on susceptible age group, morbidity, mortality, clinical features, vaccination and treatment used commonly by veterinary surgeon (VS) in the respected Upazila. The ducklings at 2–8 weeks of age were highly prone to this disease and maximum mortality were found at 4–8 weeks of age in mid-May to July according to the statement of farmers and VS. The maximum morbidity and mortality was observed during June to July at the 8–10 weeks of age group by (Sarker et al. 2017) whereas the outbreak was also reported during summer (Haque 1987). The mortality rate in ducklings were higher than adult ducks which in agreement with (Doley et al. 2003). The survey revealed both the morbidity and mortality were 75–80% and 40–45% on the basis of farmer’s history. In a previous study, researchers showed the mortality rate 35–65% in Bangladesh (Sarker et al. 2017). The morbidity and mortality vary depending on the age (below 8 weeks), co-infection (E. coli, Salmonella spp., P. multocida and so on) and other stress factors (environment, climate conditions, nutrition) which is up to 75% (Subramaniam et al. 2000; Crasta et al. 2002; Yu et al. 2008). However, all the affected ducks were found showing the clinical signs-tremors of head and neck, paddling their legs, incoordination, and circular movement, mild coughing, sneezing, diarrhoea and moreover, the owners of the affected flocks claimed the same observations which support the recent study (Hazarika et al. 2020). The post mortem changes were observed haemorrhage and congestion on the liver, perihepatitis having normal size of liver and pericarditis with white lichee like covering on heart, and haemorrhage on the trachea and lung and (Sarker et al. 2017) stated widespread haemorrhage and congestion in the body cavity, grey colored necrotic foci on liver, enlarge kidney and haemorrhages in the sub-epicardial region of conical heart. One study (Chikuba et al. 2016) found whitish, gelatinous and fibrinous exudates covering on the heart and liver surface.
In this study, we collected ocular and oropharyngeal swab from affected live birds and liver, heart and lung from dead ducks for chronological study and used LB broth, nutrient agar, TS agar, EMB agar, SS agar, Mac Conkey agar, Bovine blood agar, duck blood agar for isolation of R. anatipestifer. The occurrence of R. anatipestifer in ocular swab was low only 10% where oropharyngeal swab was 30% in affected live birds. In previous study it was recorded that R. anatipestifer is common flora for pharyngeal and laryngeal swab (Ryll et al. 2001; Chang et al. 2019). In addition, in case of dead ducks the occurrence was 35% and 25% for heart and liver respectively.
The bacterium in bovine blood agar produced small, grey and non-haemolytic appearance and pearl like appearance on duck blood agar with 24 hours incubation at 37\(℃\) both for microaerophilic and aerophilic incubator. Similarly, the same colonies were found on blood agar with 5% CO2 at 37\(℃\) for 24 hours. Bovine blood agar at 10% level has been reported to be useful for the primary isolation of the organism at 37\(℃\) in an atmosphere enriched with 5–10 per cent CO2 for 24 h was used for the growth (Crasta et al. 2002; Priya et al. 2008). The organisms were also grown on 10% sheep blood agar plates in an atmosphere enriched with 5% CO2 described by (Priya et al. 2008). Recently, (Majhi et al. 2020) concluded the cultural characteristics on various medium like small, non-haemolytic colonies on blood agar; smooth, grey, glistening and dewdrop like colonies on Nutrient agar, the bacteria grew but did not produce metallic sheen on EMB agar. In contrast, we found no growth was on EMB agar, Mc Conkey agar which agreed with (Pala et al. 2013; Surya et al. 2016; Shancy et al. 2018), and SS agar. Moreover, the bacteria showed smooth, circular, and greyish on TSA.
Gram staining revealed gram negative, coccobacilli, short rod shaped organism and in addition, (Pillai et al. 1993; Heba et al. 2015) agreed with the same findings and larger in size than P. multocida (Surya et al. 2016). For more accurate diagnosis conventionally biochemical tests including Indole production, Methyl- red test (MR), Voges-Proskauer (VP) test, H2S production, and Oxidase and Catalase test, sugar fermentation (glucose, lactose, maltose, mannitol, dextrose, sucrose) test have been used and produced the similar results mentioned in (Sarker et al. 2017); fermentation of dextrose, lactose, maltose, mannitol, glucose, sucrose, MR test positive, VP and Indole negative, and Catalase positive. Similarly, oxidase test positive and H2S negative mentioned by (Surya et al. 2016; Shancy et al. 2018).
The PCR is considered as gold standard, regarding specificity, sensitivity, and reliability for detecting microbial causal agents of diseases (Soman et al. 2014). In this study, PCR was performed targeting the Riemerella species specific gene designed by (Kardos et al. 2007) and gyrB gene primarily. A band of 546 bp size was observed by species specific primers at annealing temperature 54\(℃\) according to (Rubbenstroth et al. 2013). Researchers (Kardos et al. 2007; Soman et al. 2014; Hazarika et al. 2020) stated the same amplicon size at their studies. Interestingly, the gyrB gene was more accurate, sensitive and reported as a specific biological marker for detection of R. anatipestifer at molecular level (Udayan et al. 2019). In addition, (Yamamoto and Harayama 1995) explained gyrB is present in all bacterial strains universally. In our study all bacterial isolates showed 162 bp amplicon size using the same conditions found in (Udayan et al. 2019). We furthermore applied groEL gene for more specific confirmation of R. anatipestifer isolates in this study and found specific and consistent amplicon at 271 bp. GroEL is the member of molecular chaperon family GroE system, together with groES (Babu et al. 2014). The groEL gene based PCR assays have already been established by various researchers in the last decades (Kim et al. 2010; Hossain et al. 2012; Siddique et al. 2017; Siddique et al. 2019), proven as a powerful phylogenetic marker (Junick and Blaut 2012).
The positive isolates were then tested for antibiotic resistance profile with different groups of antibiotics. Among them large groups of antibiotics such as Beta-lactams (Penicillin G, Cephradin), Aminoglycosides (Streptomycin, Neomycin, and Gentamycin), Penems (Meropenem), and Macrolids (Erythromycin) elicited 100% resistant, while Cefuroxime presented 80.95% resistant among R. anatipestifer isolates and Ceftriazone was found susceptible to R. anatipestifer. Subsequently, Sulphonamides (Cotrimoxazol), Phenicols (Florfenicol) and Quinolones (Levofloxacin) showed 100% susceptible to all isolates. The high percentage of intermediate resistance pattern were found in Colistin which was 33.33% and only 4.76% in Ciprofloxacin. A large number of antimicrobial agents had been used for controlling the infection of R. anatipestifer and reducing the significant economic losses at a field level by various study over the time period. In vivo susceptibility testing revealed sensitive to Enrofloxacin, Ciprofloxaxin, Ofloxacin and Neomycin by (Hazarika et al. 2020) ,and furthermore, several studies stated that Ciprofloxacin, Gentamycin, Polymyxin-B, Chloramphenicol, Norfloxacin, Doxycycline, Gentamicin, Clindamycin and Cefuroxime were sensitive to R. anatipestier (Zhong et al. 2009; Surya et al. 2016; Majhi et al. 2020). In contrast, Methicillin, Sulfadiazine, Penicillin-G, Metronidazole, Erythromycin, oxacillin, polymyxin B, sulphadiazine, Cefuroxime and Ampicillin were found resistant to R. anatipestifer by (Surya et al. 2016; Majhi et al. 2020). Moreover, gentamicin as well as cefazolin (Hazarika et al. 2020), and penicillin, ampicillin, tetracycline were also showed resistant (Zhong et al. 2009). Only three antibiotics like Streptomycin, Lincomycin and Doxycycline were found intermediate sensitive to R. anatipestier (Hazarika et al. 2020; Majhi et al. 2020). So, it is concluded that R. anatipestifer drug resistance profiles changed over time which is also agreed by (Zhong et al. 2009).