Role of Veterinary Type Culture Collection in capturing Equine Microbial Biodiversity R.K. Vaid, Taruna Anand, T. Riyesh, Mamta Tigga, K. Shanmugasundaram, N. Virmani, Jitendar Singh , B.C. Bera, Shashank Bardwaj, S. Barua , Yash Pal, B.K. Singh, B.R. Gulati, B.N. Tripathi, R.K. Singh Principal Scientist, National Centre for Veterinary Type Cultures, National Research Centre on Equines, Sirsa Road, Hisar (Haryana)   I am going to highlight the role………

Outbreak Attendance and Sample Collection From 2007, The equine disease outbreaks of industry (Stud farms), defense (ITBP, Police, RVC), and farmers were attended and outbreak history and metadata was collected. Majority of samples were collected from Uttarakhand, Rajasthan, Himachal Pradesh, J&K, Delhi and Haryana. Samples included Nasal swabs, ocular swab, tonsillar swab, preputial swab, stomach contents of aborted fetus, vaginal swab, PM samples of body tissues (lung, liver, kidney, vegetative endocarditis), soil, mare milk, stallion semen, pyometra, foal diarrhoea, cervical swab, mule/horse/donkey dung, whole blood etc. Coming to M&M from 2007 we attended to disease outbreaks in equines- horse, mule, donkeys

Outbreak Attendance and Sample Collection The pathological samples collected were brought to laboratory on ice and processed for microbial isolation by standard methods. Bacterial Isolates were purified and identified by phenotypic and biochemical testing. Selected isolates were identified upto species level by 16S rRNA gene cloning, sequencing, phylogenetic analysis and homology searches using 3 ribosomal DNA databases.

Gaurikund Outbreak-Kedarnath (2009)

Severe nasal mucopurulent discharge-pony in Gaurikund

Isolates from mare abortions Pure Escherichia coli colonies Streptococcus spp. hemolysis 23 November 2017 © NRCE - Hisar

16S rRNA gene PCR amplification for Identification Aeromonas spp. Bordetella spp. Streptococcus spp. Corynebacterium spp. Pseudomonas spp. Staphylococcus spp. Rhodococcus equi (3 strains) 1.5 kb M = 1 kb DNA ladder L1- L6 = PCR products

16S rRNA DNA sequence >1500 bp Full length gene

NCBI

EzBioCloud-EzTaxon

RESULTS 392 bacterial isolates were obtained from 170 samples. The identification of microbial isolates obtained from disease outbreaks in equine has lead to discovery of a diverse set of bacterial isolates. Out of the 31 genera, 18 were Gram- negative

Genera identified (31) Actinobacillus, Bordetella, Aeromonas, Escherichia, Klebsiella, Pseudomonas, Enterobacter, Serratia, Pantoea, Kluyvera, Providencia, Citrobacter, Shigella, Comamonas, Acinetobacter, Flavobacterium,, Achromobacter, Ignatzchineria. 13 were Gram-positive viz., Rhodococcus, Streptococcus, Enterococcus, Staphylococcus, Corynebacterium, Streptomyces, Nocardia, Nocardiopsis, Microbacterium, Bacillus, Brevibacillus, Lysinibacillus, and Barrientosiimonas

Out of these many strains were hitherto unreported from equines in India Actinobacillus, Bordetella, Kluyvera, Providencia, Comamonas, Ignatzchineria. Streptococcus equi ssp. ruminatorum, Nocardia otitidiscaviarum, Nocardiopsis alba, Microbacterium spp., Barrientosiimonas humi

Actinobacillus equuli Strain Eq163F magnification 4x 5% SBA

Actinobacillus equuli Eq28B

Phylogenetic analysis of Actinobacillus equilli

Bordetella bronchiseptica

Bordetella bronchiseptica Eq24E & Eq128

Phylogenetic analysis of Bordetella bronchiseptica

Nocardia asteroides from granulomatous pneumonia-Horse identified as Nocardia otitidiscaviarum Acid fast Nocardia spp. showing branching filaments (mod. AF stain 1000x)

Streptomyces albidoflavus Eq106 semen sample NRCE RAC 2012 11/23/201711/23/201711/23/2017

Corynebacterium spp. Horse-Ladakh 23 November 2017 © NRCE - Hisar

Brevibacillus spp. from equine dung Reclassified recently from Bacillus spp, Used as Biocontrol agent, Isolated from AIDS patients and clinical cases of peritonitis in humans. Produces Gramicidin a non-ribosomal antimicrobial peptide. 23 November 2017 © NRCE - Hisar

Enterococcus asini from horse dung Previously isolated from ceacal contents of donkey (Vaux et al 1998); Cells are Gram-positive cocci, ovoid and occur mostly, in pairs or short chains. Non-pigmented and non-motile. 23 November 2017 © NRCE - Hisar

Moraxella spp. Ladakh equine 23 November 2017 © NRCE - Hisar

Nocardiopsis alba Eq135 NRCE RAC 2012 11/23/201711/23/201711/23/2017

Preservation The Cultures are Cryopreserved in Glycerol at -80º C. The Cultures are preserved by Lyophilization. The significant isolates are Accessioned. The cultures are distributed for research work.

CONCLUSIONS We have detected and identified previously unreported pathogenic bacteria from equines such as Actinobacillus equlli, Bordetella bronchiseptica, Streptococcus equi ssp.ruminatorum, Nocardia otitidiscaviarum etc. Detection and identification of these microbiota indicates that a subset of known cultural bacterial biodiversity of animal remains unexplored or underreported in India. The ex-situ conservation for the characterization of new emerging animal microbes in clinical and epidemiological context is critical for their ecological understanding in health and disease.

THANKS Streptococcus spp, cocci arranged in long chains, human boil pus NRCE RAC 2012 11/23/201711/23/201711/23/2017