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).
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).
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).
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).
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.
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).
