Rajkumari Sanjukta

Pan-genome analysis of Aeromonas hydrophila, Aeromonas veronii and Aeromonas caviae indicates phylogenomic diversity and greater pathogenic potential for Aeromonas hydrophila

Aeromonas species are important pathogens of fishes and aquatic animals capable of infecting humans and other animals via food. Due to the paucity of pan-genomic studies on aeromonads, the present study was undertaken to analyse the pan-genome of three clinically important Aeromonas species (A. hydrophila, A. veronii, A. caviae). Results of pan-genome analysis revealed an open pan-genome for all three species with pan-genome sizes of 9181, 7214 and 6884 genes for A. hydrophila, A. veronii and A. caviae, respectively. Core-genome: pan-genome ratio (RCP) indicated greater genomic diversity for A. hydrophila and interestingly RCP emerged as an effective indicator to gauge genomic diversity which could possibly be extended to other organisms too. Phylogenomic network analysis highlighted the influence of homologous recombination and lateral gene transfer in the evolution of Aeromonas spp. Prediction of virulence factors indicated no significant difference among the three species though analysis of pathogenic potential and acquired antimicrobial resistance genes revealed greater hazards from A. hydrophila. In conclusion, the present study highlighted the usefulness of whole genome analyses to infer evolutionary cues for Aeromonas species which indicated considerable phylogenomic diversity for A. hydrophila and hitherto unknown genomic evidence for pathogenic potential of A. hydrophila compared to A. veronii and A. caviae.

Molecular Characterization and Computational Modelling of New Delhi Metallo-β-Lactamase-5 from an Escherichia coli Isolate (KOEC3) of Bovine Origin.

Emergence of antimicrobial resistance mediated through New Delhi metallo-β-lactamases (NDMs) is a serious therapeutic challenge. Till date, 16 different NDMs have been described. In this study, we report the molecular and structural characteristics of NDM-5 isolated from an Escherichia coli isolate (KOEC3) of bovine origin. Using PCR amplification, cloning and sequencing of full blaNDM gene, we identified the NDM type as NDM-5. Cloning of full gene in E. coli DH5α and subsequent assessment of antibiotic susceptibility of the transformed cells indicated possible role of native promoter in expression blaNDM-5. Translated amino acid sequence had two substitutions (Val88Leu and Met154Leu) compared to NDM-1. Theoretically deduced isoelectric pH of NDM-5 was 5.88 and instability index was 36.99, indicating a stable protein. From the amino acids sequence, a 3D model of the protein was computed. Analysis of the protein structure elucidated zinc coordination and also revealed a large binding cleft and flexible nature of the protein, which might be the reason for broad substrate range. Docking experiments revealed plausible binding poses for five carbapenem drugs in the vicinity of metal ions. In conclusion, results provided possible explanation for wide range of antibiotics catalyzed by NDM-5 and likely interaction modes with five carbapenem drugs.

Detection of Peste des petits ruminants virus and goatpox virus from an outbreak in goats with high mortality in Meghalaya state, India.

Aim: We describe a laboratory investigation carried out to confirm the etiology of the heavy mortality (37 animals died out of total 44, i.e. 84%) in goats in Ri-Bhoi district of Meghalaya, Northeast region of India in December 2015. The clinical signs observed were abortion, diarrhea, high fever (up to 104°F), pox lesion in the skin, and respiratory distress. Materials and Methods: The samples comprising whole blood, sera, and pox lesion were collected from the animals (n=7) from an outbreak for the screening of peste des petits ruminants (PPR) and poxviruses. The whole blood and sera were used for screening of PPR virus (PPRV) by sandwich enzyme-linked immunosorbent assay (ELISA) and antibody by competitive ELISA as well as detection of PPRV partial N gene by reverse transcription-polymerase chain reaction (PCR). The skin lesions were used for the detection of poxvirus by PCR. Results: The results showed the presence of PPR antigens (58-80%) in the samples by sandwich ELISA and antibody in all the sera samples ranging from 9% to 41% positivity in competitive ELISA. Four samples were positive for PPRV partial N gene. The skin lesion screened for poxvirus was also found to be positive for I3L gene of goatpox virus. Conclusion: We confirm the outbreak of disease in goats with high mortality is a case of mixed infection of PPR and goatpox detected for the first time in Northeast India.

Porcine Circovirus 2 in the North Eastern region of India: Disease prevalence and genetic variation among the isolates from areas of intensive pig rearing

Porcine Circovirus type-2 (PCV-2) is considered as a major threat to the piggery sector in India. To ascertain the epidemiological status and infection level of PCV2, a pilot study was undertaken to find out the prevalence of PCV2 in swine population by ELISA and PCR in the interior and border areas of Meghalaya which includes the area where accessibility and medical aid is a rare phenomenon. A total of 249 serum samples were collected from October 2014 to February 2016 from three divisions of Meghalaya: Khasi, Jaintia and Garo Hills Divisions. The mean positivity of PCV-2 antibodies in suspected sera was 83.93% whereas 62.25% of the suspected samples respectively were found to contain PCV2 as detected by PCR. Additional 190 tissue samples were collected during necropsy from both symptomatic and asymptomatic animals following reported outbreak in this region, which indicated a mean positivity of 18.94% (36/190); out of which 13 samples were subjected to sequencing to find out the genetic diversity of PCV2 amongst the field isolates. Molecular characterization and phylogenetic analysis of PCV2 isolates based on cap gene depicted genetic diversity among the strains in pig population of Meghalaya as the isolates belonged to PCV2a, PCV2b-1c and PCV2d genotypes; identification of the PCV2d genotype is probably the first report from Meghalaya. Four isolates forming an outlier group in the phylogenetic tree were arising out of natural inter-genotypic recombination between PCV2a and PCV2b. PCV2 being immunosuppressive in nature impairs the host immune response increasing the susceptibility to other co-infections leading to disease severity and high mortality in pig population. This baseline data gives a brief epidemiological status of PCV2 infection and circulating PCV2 genotype in this region which will be useful in the formulation of control and eradication programs in remotes areas of Meghalaya where accessibility is less and vaccination is a rare practice.

Detection of classical swine fever virus E2 gene in cattle serum samples from cattle herds of Meghalaya

The present study focused on the detection and genetic characterisation of 5′ untranslated region (5′UTR) and E2 gene of classical swine fever virus (CSFV, family Flaviviridae, genus Pestivirus) from bovine population of the northeastern region of India. A total of 134 cattle serum samples were collected from organised cattle farms and were screened for CSFV antigen with a commercial antigen capture enzyme linked immunosorbent assay (Ag-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). A total of 10 samples were positive for CSFV antigen by ELISA, while all of them were positive in PCR for 5′UTR region. Full length E2 region of CSFV were successfully amplified from two positive samples and used for subsequent phylogenetic analysis and determination of protein 3D structure which showed similarity with reported CSFV isolate from Assam of sub-genogroup 2.1, with minor variations in protein structure.

Characterization of Marek’s disease virus and phylogenetic analyses of meq gene from an outbreak in poultry in Meghalaya of Northeast India

AbstractThe aim of the present study was to characterize the virus from the lesions and histopathology of organs associated with mortality in Kuroiler (dual purpose variety of poultry developed and marketed by Keggfarms Pvt. Ltd, India) birds suspected of Marek’s disease. Among 1047 birds from two farms of different location with 5.5 and 34% mortality, two types of lesion were observed in post mortem examination; tumors in vital organs—liver, spleen, kidney, lung and ovaries and generalized small nodular tumour in the abdominal cavity. Molecular characterization based on detection of ICP4 gene showed the presence of Marek’s disease virus (MDV) from tissues and cell culture adapted isolates in Madin Darby Canine Kidney cell lines. Histopathological examination revealed multinucleated immature lymphoid cells infiltration in the organs. Phylogenetic analysis of the isolates based on meq gene showed the isolates belongs to cluster I genotype of MDV. This is for the first time the MDV virus is characterized from an outbreak in the poultry flock in farmer’s field affecting production in Meghalaya state of North east India.

Evidence of BVDV in Pigs from North Eastern Part of India- Genetic Profiling and Characterisation

Introduction: The work has been attempted to detect and genetically characterise the nature of Bovine Viral Diarrhea Virus (BVDV) isolates from the porcine population of the north east. Methods and Material: The samples have been collected over a two year period and are from areas where there is a mixed and integrated rearing of livestock in close proximity. The isolates were identified, cloned and sequenced using BVD specific genomic primers for two important domains viz., E-2 and 5’ UTR. Results: Porcine BVD Sequences were analysed phylogenetically. Divergence in 3 sequences is noted in the 5’ UTR region that are forming a clear outlier group while E-2 sequences are coming close to BVDV group but forming a separate cluster.

Comparison of agarose gel electrophoresis and RNA-PAGE for rapid detection of rotavirus from faecal samples

Rapid and correct diagnosis of rotavirus infection is very important to offer immediate treatment. In the present study, ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and agarose gel electrophoresis (AGE) methods were standardised and compared for the detection of rotaviruses. Double-stranded RNA (dsRNA) of rotavirus was extracted from five positive faecal samples by QIAamp Viral RNA Mini Kit and subjected to RNA-PAGE where the concentration of separating gel was varying, i.e. 7.0%, 8.5% and 12.5%. The bands of the RNA segments were clearly separated on 7.0% and 8.5% separating gel. Extracted dsRNA was also subjected to AGE where the concentration of gel was varying at 0.8%, 1.2%, 1.6% and 2.0% containing ethidium bromide. All the 11 bands of 11 segments of dsRNA were clearly visualised in all the concentrations of gel while 1.2% gel showed clear and separated bands of ladder as well as samples. Different concentrations of rotavirus, i.e. 100–1000 ng, were also loaded on standardised RNA-PAGE and AGE, separately. However, both the methods showed same detection level up to 100 ng particles of dsRNA. Further, the time required for extraction of dsRNA from five samples, their PAGE running and staining by silver stain took around 6–7 h which was comparatively more than the AGE separation, took only 2–3 h for complete procedure. Extracted dsRNA of rotavirus from 100 stool samples of children and 42 faecal samples of piglets were subjected to standardised RNA-PAGE and AGE separately for detection of rotavirus. Interestingly, 38 (38%) samples from children and 3 (7.14%) from piglet were found to be positive for rotavirus by AGE. However, RNA-PAGE could detect only 34 (34%) samples from children and 3 (7.14%) from piglets. Out of 38 positive samples from children, 32 showed long electrophoretic patterns and remaining 6 showed short electrophoretic pattern while that for 3 piglet samples, all showed long electrophoretic pattern. Thus, from our results, it can be concluded that the AGE is highly sensitive (91.17%), marginally less specific (90.90%), reproducible, superior, efficient, less laborious, cost-effective and time-saving than the RNA-PAGE for rapid diagnosis of rotavirus from faecal samples of humans as well as animals.