Isolation and Molecular Characterization of Riemerella anatipestifer from Domesticated Ducks of Assam, India

Abstract

Background: Riemerella anatipestifer (R. anatipestifer) is a gram negative, microaerophilic, non-motile, bipolar bacteria. High genetic diversity and molecular differentiation were reported among field isolates. Although the bacterium causes one of the most economically important duck diseases in the north-eastern region of India, little work has been done on isolation, identification and molecular characterization of the bacteria. Hence, the present investigation was undertaken with a view to characterize the R. anatipestifer isolates from ducks of Assam.Methods: Phenotypic and molecular identification of R. anatipestifer isolates from domesticated ducks of Assam, India were carried out during the period from February, 2019 to January 2020. A total of 624 samples (Ocular swab, throat swab, liver, spleen, kidney, brain, heart, lung) from ducks comprising of apparently healthy, ailing and dead ducks were collected from five districts of Assam, India were processed to isolate and identify the bacteria. The tentative identification of the bacteria was done based on phenotypic characteristics viz., colony morphology, growth characteristics and biochemical reactions. All the phenotypically positive isolates were further subjected to molecular identification based on PCR assay targeting 16S rRNA gene and ERIC sequence.Result: The bacteria could be isolated from different field samples. The highest percentage of the samples that yielded the bacteria are from brain (76%) followed by spleen (74%) of dead ducks and less number of ocular swab (33%) from apparently healthy ducks were found positive. Sequencing of the amplified product of the selected R. anatipestifer isolates targeting 16S rRNA gene revealed homology percentage of 96.5-100%. Further, sequences representing five geographical locations were submitted to NCBI gene bank. Phylogenetic studies of the isolates indicated that there is prevalence of at least two genetically different strains of R. anatipestifer in the study area. The study suggested that the R. anatipestifer infection is endemic in Assam causing varying rate of morbidity (39%) and mortality (53%) and molecular based confirmation is necessary besides phenotypic identification.