Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Characterization of Pasteurella multocida and Riemerella anatipestifer of Ducks in Assam, India

Baristha Borah1, Ritam Hazarika2, Purabi Deka1, Deep Prakash Saikia2, Monuj Kumar Doley1, Snehangsu Sinha3,*, Rajeev Kumar Sharma1
1Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati-781 022, Assam, India.
2Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati-781 022, Assam, India.
3Department of Anatomy and Histology, College of Veterinary Science, Assam Agricultural University, Guwahati-781 022, Assam, India.
Background: Among the various bacterial pathogens P. multocida and R. anatipestifer cause varying degrees of mortality and morbidity leading to huge economic losses among the duck farming community. Considering the lack of information regarding the characterization of P. multocida and R. anatipestifer, a study was undertaken to establish the prevalence of these diseases in ducks from different parts of Assam, together with the capsular and virulence-associated gene characterization and antimicrobial resistance. 

Methods: A total of 235 samples comprising lungs, tracheal swabs, livers, heart blood and spleens collected from 47 diseased ducks, were screened for P. multocida and R. anatipestifer. The capsular and virulence-associated genes were amplified in isolated strains. The resistance pattern towards commonly used antimicrobial agents was studied by the disc diffusion method.

Result: The prevalence of duck septicemia and new duck disease was confirmed in ducks of Assam, India. Molecular tools were found to be highly specific besides phenotypic-based approaches in the detection, confirmation and differentiation of both bacteria due to phenotypic similarity. The antibiotic resistance patterns of both bacteria revealed a varying degree of sensitivity toward different antibiotics.
Duck is the second most important poultry species after chicken, reared for table egg production in India. Despite the natural resistance, ducks may be affected by many diseases prevalent in poultry. Among the various bacterial diseases, Pasteurella multocida associated duck pasteurellosis (fowl cholera) has been recognized as one of the prime causes of duck mortality (Amonsin et al. 2002), while Duck septicemia, caused by Riemerella anatipestifer is another important bacterial disease. In India, the disease has been reported in ducks from Meghalaya, Kerala and Assam (Priya et al., 2008, Hazarika et al., 2020). Due to their phenotypic similarity, species-specific molecular tests are recommended to differentiate P. multocida from R. anatipestifer (Ryll et al. 2001). Several host and pathogen-specific determinants are associated with the infections caused by P. multocida, among which, the capsular and virulence-associated genes are important (Katsuda et al. 2013). Despite the serious economic losses, the virulence factors associated with the pathogenesis of R. anatipestifer are yet to be explored.

Considering the increased popularity of duck farming in the North-Eastern part of the country, all-around supports are essential to raising disease-free duck farming by identifying the most common duck pathogens. Keeping the above facts in view, an investigation was attempted to characterize P. multocida and R. anatipestifer, prevalent in ducks from different parts of Assam.
The present investigation was initiated with 235 samples, comprising lungs, tracheal swabs, livers, heart blood and spleens collected from 47 diseased ducks. The clinical materials were obtained aseptically from the post-mortem section, College of Veterinary Science, Assam Agricultural University, as well as from the local markets and private duck farms in and around Guwahati, Assam during the period of January 2021-March 2022. The clinical samples of apparently healthy and diseased/dead ducks were subjected to bacteriological screening for P. multocida and R. anatipestifer.

The morphologically positive isolates were confirmed by simplex Polymerase Chain Reaction (s-PCR), targeting the species-specific (kmt1) gene (460 bp) and R. anatipestifer- specific Ribonuclease Z gene of 546 bp size. The genomic DNA (gDNA) was extracted from the respective isolates by the snap chill method. The P. multocida-specific PCR (PM PCR) was conducted as described by Deka et al., (2017). Similarly, the amplification of the R. anatipestifer-specific gene (RNAse Z gene) was done as reported by (Hazarika et al., 2020). The kmt1-positive isolates were subjected to molecular screening of capsular-specific gene(s) for capsular typing. The simplex capsular specific PCR (cap-PCR) was carried out, following the previously described protocol (Deka et al., 2017) with capsular specific primers for the hyaD-hyaC (type A), bcbD (type B) and dcbF (type D) gene. The reaction was performed with previously mentioned cycling conditions, followed by electrophoresis in a 1.5% agarose gel at 80 volts for 1 hr. The amplified products were visualized in a Gel documentation system (MiniLumi, DNR Bio-Imaging System, Israel). The identified capsular types of P. multocida were further subjected to molecular screening for distribution of certain virulence-associated gene(s), viz. the ompH, ompA87, fimA and nanB by standard s-PCR procedures (Tang et al. 2009) with the respective primers. An attempt was also made to characterize the R. anatipestifer isolate, for the ompA gene (1119 bp). The molecular screening was carried out with reported primer sequences and PCR conditions, as described by Yu et al. (2008).

Randomly selected isolates representing the respective capsular types of P. multocida along with R. anatipestifer recovered from duck were screened for their outer membrane protein (OMP) profile. The OMP fraction was extracted from the selected isolates, following the standard protocol (Wheeler et al. 2009). The sample mixtures were subjected to electrophoresis in stacking (5.0%) and resolving (12.0 %) gels to determine the protein profile, as per the protocol described by Laemmli et al. (1970). A pre-stained molecular weight marker was also run along with the samples.

In vitro drug resistance of P. multocida and R. anatipestifer isolates were tested against a panel of antimicrobial agents, viz., penicillin G (10 unit), sulfamethoxazole/trimethoprim (23.75/1.25 μg), doxycycline (30 μg), sulfadiazine (300 μg), amoxicillin (10 μg), cloxacillin (10 μg), ampicillin (10 μg), erythromycin (10 μg), tetracycline (30 μg), ofloxacin (5 μg) and cefotaxime (30 μg) (Hi-Media, Mumbai) by using the disc diffusion method as per the performance standards M31-A3 of the Clinical and Laboratory Standards Institute.
Isolation and molecular characterization of P. multocida and R. anatipestifer isolates 
The screening of 235 clinical samples comprising of lungs, tracheal swabs, liver, heart blood and spleen of diseased/dead ducks with involvement of respiratory and nervous system revealed P. multocida in 32(13.62%) samples. Recovery of R. anatipestifer was possible only in one tracheal swab (0.43%) of a duck showing severe nervous symptoms (Table 1).

Table 1: Isolation of P. multocida and R. anatipestifer from clinically affected duck.

Among the P. multocida isolates, the highest recovery was observed in tracheal swabs (10), followed by lung and heart blood (9 each) and liver (4), while the affected spleens could not reveal P. multocida. Demonstration of species-specific KMT1 (460bp) gene could further confirm all the 32 isolates to be P. multocida (Fig 1A).

Fig 1: Amplified gene products in capsular type A and D Pasteurella multocida. A=Species specific KMT1 gene (460bp), B= Type A specific hyaD-hyaC gene (1044 bp), C= Type D specific dcbF gene (657 bp), D= ompH gene (1000 bp), E= ompA87 gene (838 bp), F= fimA gene (866 bp) in the field isolates.

Similarly, the lone R. anatipestifer isolate exhibited the species-specific RNAse Z (546 bp) gene (Fig 2A). Further screening of the confirmed isolates of P. multocida of clinically affected ducks revealed 18 (56.25%) isolates as capsular type A (Table 1), bearing capsular type-specific hyaD-hyaC gene of 1044bp size (Fig 1B), while 5(15.63%) were found to exhibit the type D specific dcbF gene of 657bp amplicon size (Fig 1C). The remaining 9 isolates were recognized as untypable (UT). Exploring for certain virulence-associated genes in the P. multocida isolates could indicate the distribution of the ompH gene (1000bp) along with the ompA87 gene (838bp) in all the capsular type A and D isolates (Fig 1D and 1E). Demonstration of the fimA gene (866 bp) was possible in all the type D isolates, while 14 of the type A isolates could exhibit the fimA gene (Fig 1F). None of the capsular types A and D could reveal the nanB gene. All the untypable isolates were also found to bear the ompH gene, while five of the untypable isolates could exhibit the ompA87 gene (Table 2). The untypable isolates were found to be lacking in the fimA and nanB genes. The only isolate of R. anatipestifer recovered from a diseased duck could exhibit the OmpA gene of 1119bp size (Fig 2B).

Fig 2: Amplified gene products in Riemerella anatipestifer isolate; A= species-specific RNAse Z gene (546 bp), B= ompA gene (1119 bp).

Table 2: Distribution of virulence-associated gene(s) in Pasteurella multocida isolates.

Contrary to the low percentage of recovery in our study, Mbuthia et al., (2008) reported the isolation of P. multocida from 25.9% of apparently healthy ducks. Based on the observation of the kmt1 as a marker gene, Deka et al., (2017) opined that PM-PCR is a rapid, robust and highly specific confirmatory method for P. multocida, irrespective of serotypes. The capsular type A, followed by D and F have been established as the most prevalent capsular type of P. multocida in Indian duck. Like the Indian reports, the predominance of capsular type A, followed by type D was also previously recorded in ducks (Eldin et al., 2016). Recovery of untypable strains of P. multocida was found to be common in avian hosts (Arumugam et al., 2011). The loss of capsules following sub-cultivation might be a probable explanation for rendering circulated P. multocida in animal environments untypable (Dziva et al., 2008). Among the available literature from India, a low recovery rate of R. anatipestifer from suspected cases of duck septicemia was reported by Surya et al. (2016). A previous investigation, considered to be the first information from Assam on the prevalence of duck septicemia-like disease revealed recovery of R. anatipestifer from ocular and pharyngeal swabs of clinically affected ducks (Hazarika et al., 2020). Their study could demonstrate both the R. anatipestifer species-specific RNAse Z (564 bp) and gyrB (162 bp) genes as a suitable marker in the identification process. Contrary to the present observation, a recent study from Assam, India could record a higher recovery rate of R. anatipestifer from the brain (76%) and spleen (74%) of suspected duck septicemia cases. Phylogenetic studies of the isolates revealed at least two genetically different strains in the study areas and suggested the R. anatipestifer infection as endemic in Assam with variable morbidity and mortality (Doley et al., 2021).
Outer membrane protein profiling of the P. multocida and R. anatipestifer isolates
The OMP fractions of representative isolates of the P. multocida type A and D revealed an almost similar protein profile. Both the isolates exhibited a total of 14 polypeptide bands with approximate molecular weight (MW), within the range of 14 to 63 kDa and above (Fig 3A).

Fig 3: Outer Membrane protein profile of (A) Pasteurella multocida (Type A, D and untypable) and (B) Riemerella anatipestifer.

Among those visible protein bands, 14, 35, 37, 42 and 48 kDa proteins, shared by both types appeared as major proteins. A total of 10 polypeptide bands could be visible in the OMP fraction of a randomly selected UT strain of P. multocida, of which proteins of approximately 14, 32 and 42 kDa MW exhibited distinct bands. Among the five visible polypeptides in the OMP fraction of the lone R. anatipestifer isolate, a band of an approximate size of 40kDa was recognized as the predominant protein (Fig 3B). The available data on the prevalence of virulence-associated gene(s) in P. multocida of avian origin reflected the consistent distribution of ompH and ompA in capsular types A and D of P. multocida. However, there may be extensive molecular mass heterogeneity in the OmpA and OmpH proteins (Deka et al., 2017). Among the adhesion-associated genes, the fimA was identified in association with the pathogenic strains of P. multocida (Ewers et al. 2006). Contrary to the present observation, the distribution of the nanB gene was recorded previously in both type A and D isolates of P. multocida, even in the un-typable strains. Among the scanty information exploring virulence-associated gene(s) of R. anatipestifer, a study by Ahmad et al. (2017) could establish the utilization of ompA as a useful component in the field of diagnosis and control strategies for new duck disease. The ompA gene was found highly conserved among the R. anatipestifer isolates.

During a proteomic study on Indian isolates (type B) and the vaccine strain of P. multocida, the 32, 37, 72 and 89 kDa proteins appeared as the immunogenic OMPs (Somshekhar et al. 2014). The predominance of the 31, 33 and 37 kDa proteins was also previously reported from India in the OMP fraction of P. multocida serotype B: 2 (Tomer et al., 2002). However, the nucleotide sequence analysis of the ompA gene in the duck septicemia-associated R. anatipestifer isolate, the encoded protein of 387 amino acids with a molecular mass of 42 kDa as the major predominant, species-specific antigen in R. anatipestifer (Subramaniam et al. 2000).
Antimicrobial resistance pattern of the isolates
 All the P. multocida isolated from suspected cases of duck pasteurellosis revealed penicillin G, sulfamethoxazole/trimethoprim, sulfadiazine, cloxacillin, erythromycin and tetracycline resistant (Table 3), while resistance towards ampicillin and doxycycline was recorded in 76.0 and 68.0% of the isolates, respectively.

Table 3: Antimicrobial Resistance profile of Pasteurella multocida and Riemerella anatipestifer.

Amoxicillin and ofloxacin were found to be effective antimicrobial agents for all the P. multocida isolates. Contrary to the P. multocida isolates, the single isolate of R. anatipestifer was found to be sensitive to most of the antimicrobial agents under test. However, the isolate was found to be resistant to penicillin G, sulfadiazine, ofloxacin and cefotaxime. An absolute resistance against sulfadiazine was previously demonstrated among 123 Indian isolates of P. multocida from different avian species; while chloramphenicol might be a more effective antibiotic for the treatment of P. multocida infection. The study could also reveal the emergence of multidrug-resistant strains of P. multocida among Indian poultry (Shivachandra et al. 2004). Development of resistance towards enrofloxacin, ciprofloxacin, ofloxacin, gentamicin, amikacin, ampicillin and penicillin were seen in a majority of avian P. multocida isolates and recognized as a major hurdle for the Indian poultry industry (Balakrishnan et al. 2012).

The available literature on resistance patterns in the Indian isolates of R. anatipestifer could reveal most of the isolates are resistant to penicillin G, trimethoprim, ampicillin, amoxicillin and tetracycline (Hazarika et al., 2020). Due to the regular use in the treatment or as feed additives, the gradual development of sulfadiazine resistance among the R. anatipestifer isolates was also recorded. Ofloxacin and cefotaxime were also previously recorded as ineffective for R. anatipestifer isolates (Surya et al. 2016).
Based on the results obtained during the present study on the characterization of P. multocida and R. anatipestifer of duck origin, the prevalence of duck septicemia and new duck disease was confirmed in Assam, India. The present study also revealed the presence of concurrent infection of both R. anatipestifer in P. multocida in the same host. Molecular tools were found to be highly specific besides phenotypic-based approaches in the detection, confirmation and differentiation of both bacteria due to phenotypic similarity. The antibiotic resistance patterns of both bacteria revealed a varying degree of sensitivity towards different antibiotics. Hence, one must be careful in choosing a specific antibiotic regime in the face of a disease outbreak. However, as the sample size in the present study was limited, there is a further need for an in-depth systemic study on epidemiology for the prevention and control of duck diseases

  1. Ahmad, S.M., Priya, P.M., Mini, M. and Kumar, V.K. (2017). Outer membrane protein A (OmpA) based polymerase chain reaction (PCR) for the detection of Riemerella anatipestifer infection in Ducks of Kerala, India. International Journal of Science and Environmental Technology. 6(4): 2694-2699.

  2. Amonsin, A., Wellehan, J.F.X., Li, L.L., Laber, J., Kapur, V. (2002). DNA finger printing of Pasteurella multocida recovered from avian sources. Journal of Clinical Microbiology. 40(8): 3025-3031. 3025-3031.2002.

  3. Arumugam, N.D., Ajam, N., Blackall, P.J., Asiah, N.M., Ramlan, M., Maria, J., Yuslan, S., Thong, K.L. (2011). Capsular serotyping of Pasteurella multocida from various animal hosts-A comparison of phenotypic and genotypic methods.  Tropical Biomedicine. 28(1): 55-63. PubMed: 21602769.

  4. Balakrishnan, G. and Roy, P. (2012). Isolation, identification and antibiogram of Pasteurella multocida isolates of avian origin. Tamil Nadu Journal of Veterinary and Animal Science. 8(4): 199-202. 

  5. Deka, P., Bhattacharyya, D.K., Sharma, R.K., Saikia, G.K., Pathak, N., Barkakati, B., Neher, S., Gogoi, A. (2017). Capsular typing and virulence gene profiling of Pasteurella multocida isolated from chickens. International Journal of Chemical Studies. 5(5): 1928-1933.

  6. Doley, M.K., Das, S., Sharma, R.K., Borah, P., Sarma, D.K., Buragohain,  L., Hazarika, R., Baruah, N. (2021). Isolation and molecular  characterization of Riemerella anatipestifer from domesticated ducks of Assam, India. Indian Journal of Animal Research.

  7. Dziva, F., Muhairwa, A.P., Bisgaard, M., Christensen, H. (2008). Diagnostic and typing options for investigating diseases associated with Pasteurella multocida. Veterinary Microbiology. 128(1-2): 1-22.

  8. Eldin, W.F.S., Reda, L.M. (2016). Epidemiological prevalence of Pasteurella multocida in ducks. Japanese Journal of Veterinary Research. 64(Suppl. 2): S251-S255. 

  9. Ewers, C., Lübke-Becker, A., Wieler, L.H. (2006). Pasteurella multocida insight into the virulence determinants of a heterogeneous bacterium. Berliner und Munchener Tierarztliche Wochenschrift. 9(10): 367-386.

  10. Hazarika, R., Doley, M.K., Sarmah, H., Deka, P., Sharma, R.K. (2020). Isolation and molecular identification of Riemerella anatipestifer from ducks in Assam. International Journal of Current Microbiology and Applied Sciences. 9(3): 319- 327.

  11. Katsuda, K., Hoshinoo, K., Ueno, Y., Kohmoto, M., Mikami, O. (2013). Virulence genes and antimicrobial susceptibility in Pasteurella  multocida isolates from calves. Veterinary Microbiology. 167(3-4): 737-741.

  12. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259): 680-685.

  13. Mbuthia, P.G., Njagi, L.W., Nyaga, P.N., Bebora, L.C., Minga, U., Kamundia, J., Olsen, J.E. (2008). Pasteurella multocida in scavenging family chickens and ducks: Carrier status, age susceptibility and transmission between species. Avian Pathology. 37(1): 51-57. 03079450701784891.

  14. Priya, P.M., Pillai, D.S., Balusamy, C., Rameshkumar, P., Senthamils,  P. (2008). Studies on outbreak of “New duck disease” in Kerala, India. International Journal of Poultry Science. 7(2): 189-190.

  15. Ryll, M., Christensen, H., Bisgaard, M., Christensen, J.P., Hinz, K.H., Köhler, B. (2001). Studies on the prevalence of Riemerella anatipestifer in the upper respiratory tract of clinically healthy ducklings and characterization of untypable  strains. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health. 48(7): 537-546.

  16. Shivachandra, S.B., Kumar, A.A., Biswas, A., Ramakrishnan, M.A., Singh, V.P., et al. (2004). Antibiotic sensitivity patterns among Indian strains of avian Pasteurella multocida. Tropical Animal Health Production. 36(8): 743-750. 

  17. Somshekhar, S.H., Veeregowda, B.M., Suryanaray, V.V.S., Leena, G., Dhama, K. and Chakraborty, S. (2014). Outer Membrane  Protein (OMP) profiles of Pasteurella multocida isolates associated with haemorrhagic septicaemia by SDS-PAGE and Western blot analysis. Asian Journal of Animal and Veterinary Advances. 9(8): 513-518. ajava.2014.513.518.

  18. Subramaniam, S., Huang, B., Loh, H., Kwang, J., Tan, H. M., Chua, K.L. and Frey, J. (2000). Characterization of a predominant  immunogenic outer membrane protein of Riemerella anatipestifer. Clinical and Diagnostic Laboratory Immunology. 7(2): 168-174. 174.2000.

  19. Surya, P.S., Priya, P.M., Mini, M. (2016). Biotyping and antibiogram of Riemerella anatipestifer from ducks in Kerala. Bioscience  Biotechnology Research Communications. 9(3): 457-462.

  20. Tang, X., Zhao, Z., Hu, J., Wu, B., Cai, X., He, Q., Chen, H. (2009). Isolation, antimicrobial resistance and virulence genes of Pasteurella multocida strains from swine in China. Journal of Clinical Microbiology. 47(4): 951-958. 10.1128/JCM.02029-08.

  21. Tomer, P., Chaturvedi, G.C., Minakshi, M.P., Malik, P. and Monga, D.P. (2002). Comparative analysis of the outer membrane protein profiles of isolates of the Pasteurella multocida (B:2) associated with haemorrhagic septicaemia. Veterinary Research Communications. 26(7): 513-522. https://

  22. Wheeler, R. (2009). Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants. Bioscience Horizons. 2(1): 1-12. https://

  23. Yu, C.Y., Liu, Y.W., Chou, S.J., Chao, M.R., Weng, B.C., Tsay, J.G.,  Chiu, C.H., Ching, W.C., Long, L.T., Chang, C.C., Chu, C. (2008). Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms. Avian Pathology. 37(3): 273-279.https://

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