1. 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………

2. 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

3. 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.

4. Gaurikund Outbreak-Kedarnath (2009)

5. Severe nasal mucopurulent discharge-pony in Gaurikund

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

7. 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

8. 16S rRNA DNA sequence >1500 bp
Full length gene

9. NCBI

10. EzBioCloud-EzTaxon

11. 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

12. 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

13. 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

14. Actinobacillus equuli
Strain Eq163F magnification 4x 5% SBA

15. Actinobacillus equuli Eq28B

16. Phylogenetic analysis of
Actinobacillus equilli

17. Bordetella bronchiseptica

18. Bordetella bronchiseptica
Eq24E & Eq128

19. Phylogenetic analysis of
Bordetella bronchiseptica

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

22. Streptomyces albidoflavus Eq106 semen sample
NRCE RAC 2012
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23. Corynebacterium spp. Horse-Ladakh
23 November 2017
© NRCE - Hisar

24. 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

25. 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

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

27. Nocardiopsis alba Eq135
NRCE RAC 2012
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28. 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.

29. 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.

30. THANKS
Streptococcus spp, cocci arranged in long chains, human boil pus
NRCE RAC 2012
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