EPIDEMIOLOGICAL AND MOLECULAR STUDIES ON RIEMERELLA ANATIPESTIFER INFECTION IN DUCKS

Assiut University web-site: www.aun.edu.eg

EPIDEMIOLOGICAL AND MOLECULAR STUDIES ON RIEMERELLA ANATIPESTIFER INFECTION IN DUCKS

DOHA ABD ALRAHMAN AHMED; MOSTAFA SAIF ELDIN;

RAGAB SAYED IBRAHIM AND OMAR AMEN

*Department of Avian and Rabbit Diseases, Faculty of Vet. Medicine, Assiut University, Egypt.

Received: 23 December 2020;     Accepted: 31 December 2020

INTRODUCTION

Amongthe global leading problems confronting duck industry is Riemerella anatipestifer infection that implicates in acute and chronic conditions and can develop into epizootic infectious polyserositis in domestic ducks, mainly young, with a mortality of up to 90% (Sandhu, 2008; Wang et al., 2014; Majhi    et al., 2020). The R. anatipestifer is a short to filamentous rod-shaped, Gram-negative, singly  or  in  pairs,  non-motile,  non-spore-

Corresponding author: Doha Abd Alrahman Ahmed

E-mail address: dohabdalrahman780@gmail.com

Present address: Department of Avian and Rabbit Diseases, Faculty of Vet. Medicine, Assiut University, Egypt.

forming bacterium that is capsulated with Indian ink (Hess et al., 2013; Shancy et al., 2018). Clinically, the infected birds show lethargy, nasal discharge, swollen sinuses, dyspnea, diarrhea and neurologic disturbances (Sandhu, 2003; Wu et al., 2020). Ducklings under 5-weeks old, die within 1 to 2 days after appearance of clinical signs, but the older may survive longer (Deif et al., 2015; Shancy et al., 2018). Fibrinous pericarditis, perihepatitis air-sacculitis, and meningitis with severely congested liver and spleen are the main gross lesions (Sandhu, 2008; Chikuba et al., 2016; Shancy et al., 2018). Conventional microbiological examination is a helping tool in R. anatipestifer detection but being time consuming and laborious, recent R. anatipestifer specific PCR method is developed revealing great success in fast, accurate and reliable identification of the bacteria (Wang et al., 2012; Soman et al., 2014). Antimicrobial agents and improved biosecurity are currently applied to prevent and control R. anatipestifer infection in waterfowl farming; however, the increasing resistance to common antibiotics in R. anatipestifer seriously challenges the treatment (Chen et al., 2012). Tracing the literature back, only few attempts in Assiut were done by Shahata and Sokkar, (1977); Ibrahim and Shahata, (1991); and Ibrahim and Abd Al-Azeem, (2005) to study this problem in ducks. So this study aimed at investigating incidence of R. anatipestifer infection in diseased ducks, and determining the antibacterial susceptibility pattern and pathogenicity of the prevalent R. anatipestifer isolates in Assiut Province.

MATERIALS AND METHODS

Sampling:

Altogether, 120 specimens (60 livers, 20 lungs and 40 naso-tracheal swabs) from diseased or freshly dead ducks aging 1-18-weeks old were collected under complete aseptic condition, over the period from January 2020 to September 2020, from the different diagnostic laboratories in Assiut, Egypt, and transported to the laboratory of Faculty of Veterinary Medicine-Assiut University.

Bacteriological and biochemical examination:

A methylene blue stained film was examined from each specimen. Swabs from each organ were inoculated separately into trypticase soy broth and incubated at 37^oC for 24 hours. Then, a loopful from the incubated broth was streaked onto 10%-sheep blood agar (BA) and incubated at 37^oC under anaerobic conditions for 24 hours. The suspected colonies were sub-cultured into MacConkey agar at 37 ^oC for 24 hrs. and were stained with gram’ stain to be examined microscopically. The purified suspected R. anatipestifer colonies were biochemically identified (Indole production, Urease, Catalase, Oxidase, and Litmus milk tests). Also, sugars (sucrose, glucose, lactose, fructose, maltose, dulcitol, salicin, D-mannitol, galactose) fermentation were tested according to Quinn et al. (2002).    

Molecular identification by using Polymerase chain reaction (PCR):

Purified R. anatipestifer genomic DNA was obtained by boiling according to Soman      et al. (2014). Briefly, pure colony was suspended in 5 ml of phosphate buffered saline (PBS) and centrifuged at 3000×g for 10 min at 4°C (repeated thrice until obtaining pellet). The pellet was washed twice in PBS, re-suspended in 100 μl of nuclease free water, boiled for 10 min, chilled in ice for 30 min. and centrifuged at 3000×g for 5 min at 4°C. Finally, the supernatant was collected and used as template DNA. R. anatipestifer species specificprimer set (Forward (F): (5´-TTACCGACTGATTGCCTTCTA-3´ and Reverse (R): (5´-AGAGGAAGACCGAGG ACATC-3´) was used for amplification.  

The PCR reaction mixture (25μl total volume) contained 12.5μl Master mix (One  master mix (Gene Direx) Code No. MB203-0100), 9.5μl PCR grade water, 1μl Forward primer (10pM/µL), 1μl Reverse primer (10pM/µL), 1μl Template DNA (25μl total reaction). The reaction was conducted in Veriti thermocycler (Applied biosystems, Germany) following the cycling conditions described by Shancy et al. (2018). Accurately, an initial hot start at 94°C for 5 minutes, followed by 35 cycles, each consisting of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute; and a final extension step at 72°C for 4minutes. The amplified products (5µl) were detected on ethidium bromide-stained 1.5% agarose gel by visualizing them with UV light in comparison to molecular size of 100-1.500bp DNA ladder (RTU, Cat.No.DM001. R500, 11bands).

Determining the Antibacterial Susceptibility Pattern of R. anatipestifer using Standard Disk Diffusion method:

In-vitro susceptibility of R. anatipestifer isolates to 14 antibacterial agents (Ampicillin (10μg); Amoxycillin (10μg); Gentamicin, (10μg); Streptomycin, (10μg); Spectinomycin (100μg); Cephradine (30μg); Erythromycin (15μg); Chloramphenicol (30μg); Doxycycline (30ug) Oxytetracycline (30μg); sulfa+trimethoprim (25μg) and Flumequine (30μg)) was investigated according to Bauer et al. (1966) following Clinical Laboratory Standards Institute (NCCLS, 1999). CLSI, 2010; and CLSI, 2018).

Detection of minimum inhibitory concentration (MIC):

The anti-Riemerella effect of Spectinomycin,Streptomycin, Erythromycin, Florphenicol, Sulphaquinoxaline, Doxycycline, Cephradine, Gentamycin, Amoxicillin and Lincomycin was checked in microtiter plate 96 wells using double fold micro-dilution method against all obtained R. anatipestifer in a density of 10⁵ CFU (CLSI, 2018). The concentration of each antimicrobial was 10 μg/mL, 2.56 μl of each antimicrobial was added in 2 wells of the first row of plate then 50 μl tryptone soya broth with bacteria was added to all wells. Another 50 μl tryptone soya broth with bacteria was added to first row of plate (wells of antimicrobials) then two-fold serial dilution technique was made and discard the last 50 μl. The bacterial inoculum broth was taken as a positive control and another broth without bacterial inoculum was considered as a negative control. The microtiter plates were incubated at 37ºC for 24 hours and examined for the lowest concentration showing no detectable growth (MIC).

Pathogenicity testing:

A total number of 40 one-day-old molar ducks were purchased from (El-Shams Company, Assiut) and reared in clean well ventilated pens. Birds were divided in to two groups (20 per each) and provided with antibiotic-free commercial ration and water ad-libitum. Daily till 14 days old, tracheal swabs were obtained from each group and inoculated in trypticase soy broth for 24 hr and plated on trypticase soy agar to exclude previous Riemerella infection. At day 15, birds in the first group were inoculated intramuscularly with 0.5ml broth culture containing CFU/ml of R.anatipestifer isolate .Birds in the second group were treated with sterile broth and kept as negative control.

RESULTS

Out of the 120 examined ducks, 20 were R. anatipestifer infected with a prevalence rate of 16.6%. The most common encountered signs observed in the examined birds were sinusitis, decrease body weight, locomotor disturbances, nervous signs and arthritis. Pericarditis, peri-hepatitis, and airsacculitis were the main postmortem lesions (fig. 1A, B).

Isolation, Bacteriological and biochemical identification:

Methylene blue stain for tissue smears and blood films of the suspected samples showed typical bipolar cocco-bacillary organisms (fig. 2A, 2B). The produced colonies showed morphological characteristics typical to R. anatipestifer on the used culture media (smooth, convex, transparent, glistening, dew drop like, mucoid on the trypticase soy agar and non-hemolytic in blood agar) (fig. 3A, 3B). No bacterial growth was detected on MacConkey agar.

Gram’s stained films showed gram-negative coccobacilli that were bipolar in recent cultures. The isolated suspected bacteria showed no evidence of motility on the semisolid agar media. Isolates were urease, catalase and oxidase positive, slow alkaline change of litmus milk. Indole production was negative and could not ferment sugars (glucose, fructose, maltose, sucrose, lactose, Salicin, Dulcitol, and Galactose).

Molecular characteristics of the obtained isolates:

Out of the 20 biochemically positive R. anatipestifer isolates, 10 isolates produced the typical band of the R. anatipestifer specific gene (546 bp) during the molecular examination with a prevalence rate of 8.33% (fig. 4)

Antimicrobial Susceptibility of the isolated R. anatipestifer:

The isolated R. anatipestifer showed in-vitro sensitivity mostly against amoxicillin, doxycycline, and Flumequine and all isolates were absolutely resistant to streptomycin, chloramphenicol, ampicillin, erythromycin, Spectinomycin, and Cephradine(Table 1, Fig. 5).

Minimum inhibitory concentration (MIC):

On the basis of MIC test for 10 different antimicrobials it was found that all
isolates were resistant to 4 antimicrobials (100%) (Lincomycin, erythromycin, sulphaquinoxaline, Spectinomycin. 9 isolates were resistant to 3antimicrobials (90%) (Streptomycin, Cephradine, florphenicol). 9 isolates were sensitive to amoxicillin (100%). 9 isolates were sensitive to doxycycline (90%). 3 isolates were sensitive to gentamicin (30%). As shown in table (2), table (3) and fig. (6).

Pathogenicity test:

The ducks aged 14 days were inoculated intramuscularly with 0.5ml of broth culture containing CFU/ml of R.anatipestifer isolate NO (1). Clinical picture observed after inoculation was nasal and ocular discharge, mild sinusitis, and congestion of beak, depression, ataxia, ruffled feathers, nervous signs, arthritis and greenish white diarrhea. Two birds died per acutely after 24 hours post inoculation by (I/M route) with septicemic picture (congested lung and enlarged congested liver and spleen). Post 7 days infection, necropsy findings observed as perihepatitis, air sacculitis and pericarditis as shown (fig. 7A&B, 8A&B, 9A&B, 10, 11A&B&C). Control groupshowed No (sings, lesions and deaths). Re isolation of the inoculated organism from experimentally infected ducks was successful.

Fig. 1: (A) Sinusitis in naturally R. anatipestifer infected ducks.(B) pericarditis, perihepatitis, air sacculitis due to natural infection.

Fig. 2: (A) Methylene blue stained blood smear showing bipolar coccobacilli from R. anatipestifer infected duckling. (B) Gram’s stained film from a recent culture showing gram negative bipolar cocco-bacilli.                         

Fig. 3: (A) Dew drop like, mucoid colonies on trypticase soy agar. (B) Dew drop like non-hemolytic colonies on blood agar                                       

Fig. 4: Agarose gel electrophoresis 1.5% stained with ethidium bromide showing PCR products of R. anatipestifer specific gene illuminate (546pb) detected in biochemically positive colonies. Lane M: 100 bp ladder as molecular size DNA marker. Lane (+): control positive (pure R. anatipestifer strain). Lane (-): control negative.  Lanes 1 to 10: Positive samples.

Table 1: Antimicrobial sensitivity profile of R. anatipestifer isolates.

Fig. (5): Antimicrobial sensitivity profile R. anatipestifer isolates.

Table 2: MIC of 10 R. anatipestifer isolates.

Table 3:MIC breakpoint of R. anatipestifer isolates.

Fig. (6): MIC of 10 R. anatipestifer isolates.

Fig. (7): Experimentally infected ducks show diarrhea, incoordination, ruffled feathers, and anorexia.

Fig. (8): Experimentally infected ducks show (A) arthritis, and (B)nervous signs

Fig. (9): (A)Experimentally infected ducks show ocular and nasal discharge and congested beak. (B): duck on the right was control and the other on the left was infected duck was infected and show loss of weight

Fig. (10): congested septicemic liver of experimentally infected duck 24hours post-inoculation.

Fig. (11): Illustrates (A)perihepatitis, (B) air sacculitis and (C) pericarditis after 7-days post-inoculation of the isolated organism.

DISCUSSION

R. anatipestifer is one of the problems retarding duck industry worldwide causing infectious polyserositis of ducklings, with a mortality up to 90 % (Sandhu, 2008; Wang et al., 2014; Majhi et al., 2020). Our study was designed to determine the prevalence of R. anatipestifer infection in ducks and evaluate the antibacterial susceptibility and pathogenicity of the obtained R. anatipestifer isolates. In the current work, 20 suspected isolates of the organism were recovered from different duck farms in Assiut Province.

The most common encountered signs observed in the examined birds were sinusitis, decrease body weight, locomotor disturbances, arthritis and nervous signs. These findings were recorded by several authors as Leibovitz, (1972); Leavitt and Ayroud, (1997); Sandhu, (2003); Aparna and Renjith, (2012); Hess et al. (2013) and Wu et al. (2020). Postmortem lesions included lungs congestion, enlarged pinkish liver and enlarged purple spleen, enteritis, congested beak, pericarditis, perihepatitis, airsacculitis, meningitis, osteomyelitis, caseous salpingitis, chronic arthritis and cellulitis. These lesions aligned with those recorded by Aparna and Renjith, (2012).

The produced colonies showed morphological characteristics typical to R. anatipestifer on the used culture media.Thecolonies were  smooth, convex, transparent, glistening, dew drop like, mucoid on the trypticase soy agar  and non-hemolytic in blood agar, no bacterial growth was detected on macConkey agar.These findings matched with Leavitt and Ayroud, (1997); Shancy et al. (2018) and Majhi et al. (2020).On the other hand some isolates were haemolytic, particularly when plates incubated longer than 48 hrs (Brogden, 1989; Shancy et al., 2018). Among 123 field strains of R. anatipestifer, Hinz et al. (1998) recorded that 25 strains showed β-haemolysis on blood agar after 24h to 48h incubation.

Concerning cellular morphology of our R. anatipestifer isolates, they appear as bipolar cocco-bacillary organisms by methylene blue stain and gram-negative coccobacilli that were bipolar in recent cultures by gram’s stain. These results are in agreement with these   described by Pillai et al. (1993); Hess et al. (2013); Pala et al. (2013) and Shancy et al. (2018).

In the present study, biochemical tests revealed that R. anatipestifer isolates were slow alkaline change of litmus milk; indole production was negative while oxidase, catalase and urease were positive. These findings agree with (Brogden et al., 1982; Soman et al., 2014; Deif et al., 2015 and Surya et al., 2016). The results of sugar fermentation of our isolate revealed that most isolates could not ferment sugars including (glucose, fructose, maltose, sucrose, lactose, salicin, dulcitol, and galactose). These findings were also reported by (Brogden et al., 1982 and Bernardet et al., 2002). On the other hand, Priya et al. (2008) and Deif et al. (2015) reported that this bacteria ferments mostly lactose, maltose, dextrose and sucrose.

Bacteriological examination revealed a recovery of 20 R. anatipestifer isolates with a prevalence rate 16, 6%. These results agreed with previous studies reported by Ibrahim and Abd Al-Azeem, (2005). The prevalence rate of R. anatipestifer infection ranged from 11% - 84.4%% in ducks worldwide as observed by Huang, (2008); Priya et al. (2008); Wang et al. (2012); Chen et al. (2015) and Majhi et al. (2020). Seventy-six R. anatipestifer isolates were detected, and the prevalence in the ducks and geese were 12.3% (46/375) and 8.0% (30/375), respectively in central Taiwan recorded by Chang et al. (2019). In contrast, Deif et al. (2015) reported that recovery of 20 R. anatipestifer isolates with a prevalence rate 16.7%. Out of 69 samples collected from diseased ducks (more than 7 weeks old) revealed isolation of 14 R. anatipestifer isolates (11.7%). While out of 51 samples collected from diseased ducklings (1-7 weeks old), revealed isolation of 6 isolates (5%). Comparing the results obtained from ducks and ducklings showed a higher prevalence rate of ducks (11.7%) than ducklings (5%).

According to molecular characterization 10 out of 20 R. anatipestifer isolates with prevalence rate (8, 33%) were positive in PCR assay which is considered to be a useful laboratory tool for the definitive identification of suspected R. anatipestifer isolates due to absence of selective and/or indicative media for isolation (Rubbenstroth et al, 2009), it was some time difficult to isolate the organism from clinical samples due to overgrowth of other organism. These was described by Higgins et al. (2000) and Cultural and biochemical characteristics based identification of R. anatipestifer is time consuming, laborious, and require several days to complete (Soman et al., 2014). Characterization of R. anatipestifer by traditional methods is often not sufficient because of phenotypic diversity (Deif et al., 2015). Our results are in disagreement with those described by Wang et al. (2012) who revealed that Using gyrB-PCR to livers of diseased ducks, 46% riemerellosis incidence rate was recorded in China.

In our recent study, the results of antimicrobial susceptibility test of the isolates showed that the most effective antibiotics were amoxicillin, doxycycline, Flumequine. While all isolates showed absolutely resistant to streptomycin, chloramphenicol, ampicillin, erythromycin, Spectinomycin and Cephradine. ‎Sandhu, (2001) stated that Sulfadimethoxine-trimethoprim was effective in reducing mortality. According to Chang et al. (2003); Zhong et al. (2009) and Deif et al. (2015), all isolates were of high sensitivity to Ciprofloxacin, Norfloxacin, Gentamycin, Chloramphenicol, Polymyxin-B and with moderate sensitivity to Doxycycline, and resistance to penicillin G, Metronidazole, Sulfadiazine, Methicillin, Ampicillin, Cefuroxime, Erythromycin. On the basis of MIC test for 10 different antimicrobials it was found that all isolates were resistant to 4antimicrobials (100%) (Lincomycin, erythromycin, sulphaquinxaline, Spectinomycin. 9 isolates were resistant to 3antimicrobials (90%) (Streptomycin, Cephradine, florphenicol). All isolates were sensitive to amoxicillin (100%). Nine isolates were sensitive to doxycycline (90%). 3 isolates were sensitive to gentamicin (30%). Ibrahim and Hussein, (2000) and Ibrahim Abd Al-Azeem, (2005) studied susceptibility of R. anatipestifer to different antimicrobials using MIC, they recorded complete susceptibility to penicillin, amoxicillin, enrofloxacin, lincospectin, Oxytetracycline and cephalosporin. Complete resistance to aminoglycosides (streptomycin, gentamycin) and sulfadimethoxin was demonstrated.

The recorded gross lesions in birds that exposed to the experimental infection with R. anatipestifer were pericarditis, perihepatitis, air sacculitis and septicemic lesions especially in liver, spleen and myocardium. Our observation on gross lesions are similar to findings of Tripathy et al. (1980) and Ibrahim and Shahata, (1991) who reported that pericarditis and variable degree of air sacculitis were common lesions, Bayoumi, (1988) who recorded hemorrhage and septicemic picture and Hatfield and Morris, (1988) who observed clinical signs of diarrhea and incoordination in ducklings , severe pericarditis , thickening of air sacs and fibrinous pericarditis in  intramuscularly inoculated group of ducklings, Per-acute death with septicemic picture had been seen by  Ibrahim and Abd Al-Azeem, (2005). The current results are in disagreement with those described by Asplin, (1956) who said that no clinical signs were observed in any experimental group of duckling.

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