Microbiological

Communication

Biosci. Biotech. Res. Comm. 9(3): 457-462 (2016)

Biotyping and antibiogram of

Riemerella anatipestifer

from ducks in Kerala

Surya, P. S.

, Priya, P. M.

* and Mini, M.

MVSc Scholar, Department of Veterinary Microbiology, College of Veterinary and Animal Sciences,

Mannuthy, Thrissur, Kerala-680651.

Assistant Professor, e of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala-680651.

Professor and Head Department of Veterinary Microbiology, College of Veterinary and Animal Sciences,

Mannuthy, Thrissur, Kerala-680651.

ABSTRACT

New duck disease caused by Riemerella anatipestifer is a new disease emerged in Kerala from 2008 onwards. Six R.

anatipestifer isolates responsible for the disease were isolated from suspected ducks from different outbreak areas of

the state and were identi ed. Since the ecological, morphological and cultural characteristics of R. anatipestifer are

more or less similar to Pasteurella multocida, the disease is often confused with duck pasteurellosis and misdiagnosed.

R. anatipestifer infection is also characterized by the presence of bipolar organisms in blood smear and impression

smears of organs as in the case of P. multocida, but the size is little larger. The detection and identi cation of the

causative bacterium, from ducks with signs and lesions consistent with the acute or chronic form of the disease, is one

of the most important aspects of disease diagnosis. Hence, a study was conducted to isolate the agent of new duck

disease and stating its differential biotyping characters from that of P. multocida. They were differentiated using tests

like indole production, gelatin liquefaction, ornithine decarboxylases utilization and fermentation of glucose. The

antibiogram pattern was determined to advocate the choice of drug for the purpose of treatment. All the R. anatipes-

tifer isolates were sensitive to chloramphenicol, cipro oxacin, enro oxacin, nor oxacin, gentamicin, clindamycin,

doxycycline and cefuroxime.

KEY WORDS: ANTIBIOGRAM, BIOTYPING, KERALA, NEW DUCK DISEASE,

PASTEURELLA MULTOCIDA, RIEMERELLA ANATIPESTIFER

457

ARTICLE INFORMATION:

*Corresponding Author: priya@kvasu.ac.in

Received 18

July, 2016

Accepted after revision 7

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

458 CHARACTERISATION OF

RIEMERELLA ANATIPESTIFER

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Surya, Priya And Mini

INTRODUCTION

God’s gift of beautiful water bodies at various localities

of Kerala are acting as ideal environment for duck rear-

ing. Regular vaccination against duck plague and duck

pasteurellosis carried out in the state greatly reduced

their incidence. When we succeed in controlling the

existing disease, due to known and unknown global

environmental changes, several new diseases are emerg-

ing. One such disease is the new duck disease in Kerala,

reported since 2008 (Priya et al., 2008). It is an enzootic,

contagious, often primary septicemic disease of domes-

ticated ducklings (Fulton and Rimler, 2010).

In addition to ducks, it also infects geese, turkey,

chicken, wild birds and domestic pigs (Segers et al.,

1993). In young ducklings, it results in a mortality rate

as high as 75 per cent and in adult birds, it ranges from

20 to 40 per cent. The causative agent is Riemerella

anatipestifer, a Gram- negative rod shaped, non-motile,

non-sporulating bacterium.

In India, the disease has been reported in ducks

from Assam and Kerala (Shome et al., 2004 and Priya

et al.,2008). Both R. anatipestifer and Pasteurella mul-

tocida reveal bipolarity in blood smear on Lieshman’s

/ Giemsa staining.Both the organisms share common

ecological and morphological characters . Hence, the

 eld veterinarians are often unable to distinguish these

two organisms due to their phenotypic similarity. Here

comes the need of isolation and identi cation of the

agent. The present study discussed in detail on direct

microscopic examination, right clinical samples to be

collected, selection of cultural media and its incubation

condition and the differential biochemical characters of

R. anatipestifer from that of P. multocida. These param-

eters are highly useful at  eld level to con rm the dis-

ease, (Sun et al., 2012, Pala and Radhakrishnan 2014.,

Soman et al., 2014).

MATERIAL AND METHODS

Live and dead ducks (128) from the disease suspected

outbreak areas were brought to the Department of Vet-

erinary Microbiology was used for sample collection.

Detailed post mortem examination was conducted to

observe various gross lesions. Heart blood smears and

impression smears of liver and spleen were stained by

Leishman’s stain for the presence of bipolar organisms.

Samples of heart blood, liver, spleen, lungs and brain

were collected aseptically and streaked on ten per cent

bovine blood agar. They were incubated microaerophili-

cally in a candle jar at 37°C for 48 hours. The bacte-

rial isolates were identi ed based on morphological and

staining reactions, cultural and biochemical characters.

Since P. multocida is the most confusing organism with

R. anatipestifer, duck isolate of P. multocida serotype A

(maintaining in the department) was used for compari-

son as a negative control. Antibiotic sensitivity pattern

of the isolates was determined by standard disc diffusion

method (Bauer et al., 1966).

RESULTS AND DISCUSSION

Examination of heart blood smears and liver impression

smears revealed bipolar organisms which are relatively

larger in size than P. multocida, indicating the importance

of examination of heart blood and impression smears

from liver and spleen. Pillai et al. (1993) also noted the

size difference of bipolarity between these two organisms.

Since the clinical signs and gross lesions of new duck

disease are similar to diseases like duck pasteurellosis and

E. coli infection, the gold standard method of diagno-

sis is the isolation of the bacteria from clinical materials

in suitable media. So the isolation was tried from heart

blood, lung, liver, spleen, ovary and brain. In acute stage

of the disease, the organism could be readily isolated from

heart blood, liver, spleen, lungs and brain (Pathanasophon

et al., 1994). Bisgaard (1995) suggested that R. anatipesti-

fer often resulted in chronic salpingitis in surviving duck

and geese. So isolation was also tried from ovary of the

infected bird. According to Gooderham (1996), the best

source of isolation of the organism was brain.

In this study, though isolates were obtained mainly

from heart blood and liver, chance of contamination

was less in brain. The primary isolation was carried out

in  ve to ten per cent bovine blood agar. According to

Rimler et al. (1998) no selective and/or indicative media

had been used for the isolation of R. anatipestifer and

the isolation of the organism from clinical materials

was sometimes dif cult due to the overgrowth of other

organisms (Higgins et al., 2000). Chocolate agar (Leavitt

and Ayroud, 1997), ovine blood agar (Crasta et al., 2002)

and ten per cent bovine blood agar (Priya et al. 2008

and Pala et al. 2014) have reported to be useful for the

primary isolation of R. anatipestifer.

The incubation carried out in a candle jar with mild

tension at 37°C for 48 h was found to be optimum

for the culture of R. anatipestifer from clinical materials.

Smith et al. (1987) suggested that the organism preferred

microaerophilic environment for initial isolation. Segers

et al. (1993) reported that the organism grew best at

temperature 35 to 37°C after a primary isolation in a CO

enriched atmosphere. The  ndings of the present study

are in agreement with the observations made by earlier

workers.

Following incubation of clinical samples in bovine

blood agar, convex, entire, transparent and butyrous

colonies suggestive of R. anatipestifer obtained from six

birds were designated as RA1 to RA6. These observa-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CHARACTERISATION OF

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Surya, Priya And Mini

FIGURE 2. Variable morphology of R. anati-

pestifer

tions are in accordance with the  ndings of Smith et al.

(1987) and Songer and Post (2005). All the isolates were

non-haemolytic on blood agar, except one (RA2), which

produced a clear zone of haemolysis after 48 h of incu-

bation (Fig. 1), indicating it may be a different strain

or serotype, since more than 20 serotypes of R. anati-

pestifer have been reported worldwide (Sandhu. 2008).

Hinz et al. (1998b) recorded that among 123  eld strains

of R. anatipestifer, 25 strains displayed  haemolysis

on blood agar after 24 h to 48 h of incubation. In the

present study, out of the 128 birds screened, samples

from six birds were showing colonies suggestive of R.

anatipestifer.

Smears from culture, stained by Gram’s staining

revealed Gram negative organism with a variable mor-

phology varying from short rods to  lamentous forms

(Fig. 2). Similar  ndings were reported by Baba et al.

(1987) and Leavitt and Ayroud, (1997). The biochemical

characteristics of the isolates and its comparison with

DP1 are given in Table 1. The isolates did not grow on

MacConkey agar. They were catalase and oxidase posi-

tive and unreactive to O-F test, as reported by Carter

and Wise (2004) and Pala et al. (2013). According to

Hinz et al. (1998a) and Ryll et al. (2001), R. anatipestifer

is characterized more by the absence than the presence

of speci c phenotypic properties. The second stage bio-

chemical reactions used for characterization of R. anati-

pestifer (Segers et al., 1993) were almost identical for

all the six isolates. Variations were observed only in the

presence of urease and fermentation of sugars. Similar

 ndings have been reported by Pillai et al. (1993), Van-

canneyt et al. (1999), Bernardet et al. (2002) and Shome

et al. (2004). According to OIE (2008), P. multocida and

R. anatipestifer could be differentiated using tests like

indole production, ornithine decarboxylase utilization

and gelatin liquefaction. On the basis of morphological,

cultural and biochemical characteristics, all the isolates

were identi ed as R. anatipestifer and were differenti-

ated from P. multocida.

Once the organism reaches the brain, chemotherapy

is of limited value. Hence, a variety of chemotherapeutic

agents have been used in the early stage of the disease

itself to treat the infection. As there is often a wide vari-

ation in the responsiveness of R. anatipestifer to these

agents, in vitro drug sensitivity testing is essential for

the selection of an appropriate drug in a given situation.

All the isolates used in the present study were subjected

to antibiotic sensitivity testing. Among the 26 antibiot-

ics used, cipro oxacin, enro oxacin, nor oxacin, doxy-

cycline, gentamicin, clindamycin, cefuroxime and chlo-

ramphenicol appeared to be the most effective drugs as

all the isolates tested were found to be sensitive to these

agents. Sensitivity to enro oxacin against R. anatipes-

tifer was reported by Turbahn et al. (1997). With regard

to the sensitivity of cipro oxacin, gentamicin, chlo-

ramphenicol and doxyxycline, the results of the present

study are in agreement with those of Shome et al. (2004)

and Priya et al. (2008).

All the isolates showed resistance to methicillin, met-

ronidazole, oxacillin, penicillin G, polymyxin B, eryth-

romycin and sulphadiazine. Other antibiotics tested

showed variable sensitivity pattern (Fig. 3). Several

workers reported the high sensitivity of R. anatipestifer

to penicillin G, erythromycin and polymyxin B (Baba et

al.,

1987; Pathanasophon et al., 1991 and Pathanasophon

et al., 1994). Chang et al.,(2003) conducted in vitro and

in vivo antibiogram using ceftiofur and 16 commonly

used antibiotics against 50 isolates of R. anatipestifer .

Their results revealed that penicillin, cephalothrin, cefti-

ofur, chloramphenicol,  umequine and kanamycin are

the effective antibiotics.

In contrast to that, the study conducted by Zhong

et al., (2009) showed that the isolates were resistant

to penicillin, ampicillin, tetracycline and sensitive to

enro oxacin, chloramphenicol and neomycin.

FIGURE 1. Haemolysis produced by

RA2 on blood agar

460 CHARACTERISATION OF

RIEMERELLA ANATIPESTIFER

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Table 1: Biochemical characteristics of Riemerella anatipestifer

TESTS RA1 RA2 RA3 RA4 RA5 RA6 DP1

Gram’s reaction - - - - - - -

Motility - - - - - - -

Growth microareobically - - - - - - -

Growth aerobically - - - - - - -

Growth on MacConkey agar - - - - - - -

Haemolysis on blood agar - + - - - - -

Catalase + + + + + + +

Oxidase + + + + + + +

O-F test - - - - - - F

Indole production - - - - - - +

Methyl-red test - - - - - - -

Voges-Proskauer test - - - - - - -

Urease + - - + + + -

H2S production - - - - - - -

Nitrate reduction - - - - - - +

Citrate utilization - - - - - - -

Gelatin liquefaction + + + + + + -

Ornithine decarboxylase - - - - - - +

Sugar fermentation

Dextrose - - - + + - +

Galactose - - - + - - +

Lactose - - - - + - -

Fructose - - - - + + +

Sucrose - + - + - + +

Xylose - - - - + - +

Mannose - - - - - - -

Maltose - - + - + + -

Mannitol - - - - - - +

Sorbitol - - - - - - +

Dulcitol - - - - - - -

Adonitol - - - - - - -

Inositol - - - - - - -

Salicin - - - - - - -

Inulin - - - - - - -

Arabinose - - - - - - +

Trehalose - - - + - + -

Melibiose - - - - - - -

Cellobiose - - - - - - -

Rhamnose - - - - - - -

Raf nose - + - - - - -

Zhang et al. (2014) stated the usefulness of levami-

zole as immunostimulant in the administration of adju-

vanated vaccine. Zhong et al. (2009) suggested that R.

anatipestifer drug resistance pro les changed over time.

So to reduce the irresponsible use of antibiotics, disc dif-

fusion analysis should be done for effective antibacterial

treatment.

Earlier, Sun et al. (2012) reported the prevalence of

multi- drug resistant R. anatipestifer isolates from China.

Manju et al., (2014) noticed that cipro oxacin, enro-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CHARACTERISATION OF

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461

Surya, Priya And Mini

 oxacin and gentamicin gave a wider zone of inhibition

where as the R. anatipestifer isolates tested were resist-

ant to amoxicillin, chloramphenicol and co-trimoxazole.

The variations in the antibiogram of the isolates in the

present study could be attributed to the indiscriminate

use of antibiotics either to treat the disease condition or

their increased use as feed additives, which might have

resulted in acquired drug resistance.

ACKNOWLEDGEMENTS

Authors are thankful to The Dean, College of Veterinary

and Animal Sciences, Mannuthy, Thrissur for providing

necessary facilities to carry out the work.

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