OS16 - Open Session 2016 Cascais, Portugal 26 - 28 /10/2016 EuFMD Sessions\Open Session\Archive-2018\Open 2016 Cascais- Portugal\PPT presentations\

1. ICAR- DIRECTORATE OF FOOT AND
MOUTH DISEASE
Institute of FMD
FAO Regional Leading Diagnostic
Laboratory for SAARC
INDIAN COUNCIL OF AGRICULTURAL RESEARCHAn ISO-9001: 2008 Certified laboratory
Localization of Foot-and-Mouth Disease viral RNA and viral
antigens in different tissues from apparently healthy cattle and
buffalo under natural condition in India
R. Ranjan*,1, J. K. Biswal 1, S. Saravanan1, B.B. Dash1, S. Nandkumar2, L. Rodriguez3, J. Arzt3, B. Pattnaik1
1ICAR-Directorate of FMD, Mukteshwar, Nainital, Uttarakhand, India
2Chief Disease Investigation Office Palode, Kerala, India
3Foreign Animal Disease Research Unit, USDA/ARS PIADC, NY, USA
*drrajraj@gmail.com

2. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
New Delhi

3. ICAR-Directorate of FMD
Mukteshwar-263138, India
DFMD, Mukteshwar
IC-FMD, Bhubaneswar

4. ICAR-Directorate of FMD
Mukteshwar-263138, India
Organizational set up Directorate of FMD
CENTRAL
LABORATORY,
Mukteshwar
International Center
for FMD
Bhubaneswar
• NRC on Yak, Dirang
• NRC on Mithun, Nagaland

5. ICAR-Directorate of FMD
Mukteshwar-263138, India
• Foot and mouth disease (FMD) is considered as a serious threat to
the livestock industry and marginal farmer worldwide and it is a
major barrier to worldwide trade in animals and animal products.
• Following recovery from the acute form of FMD virus (FMDV)
infection, a variable, yet substantial proportion of ruminants become
persistently infected i.e. carriers in both vaccinated and non
vaccinated cattle and buffalo.
• There is paucity of literature dealing with FMDV persistence/viral
RNA localization in apparently healthy cattle and bovine under
natural condition in FMD endemic region.
• The current study was envisaged to determine tissue-specific
localization of FMDV in different tissues obtained from apparently
healthy cattle and buffaloes under natural condition to provide the
basis for effective control strategies.

6. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
Sample collection and its processing
SerumOesophageal pharyngeal fluid (OPF) Tissues (11)
middle tongue (MTNG), ventral soft
palate (VSP), dorsal soft palate 1 (DSP1,
cranial part), DSP 2 (caudal part),
palatine tonsil (PTON), dorsal
nasopharynx 1 (DNP 1, cranial part),
DNP 2 (caudal part) middle
retropharyngeal lymphnode (MRPLN),
submandibular lymphnode (SMLN), right
popiliteal lymphnode (RPOP) and heart
(Ht)
Anti-NSP antibody status
(Mohapatra et al., 2011)
Ist in 50% (v/v)
buffered glycerin for
virus isolation (VI) and
FMD viral genome
detection by mPCR.
2nd in OCT
embedding medium
for pathological
studies (Arzt et al.,
2009)
• After collection of OPF, it was kept
at -80oC freezer until further use.
• Virus isolation (VI) and its genome
detection was carried out.
• VI from the OPF and tissues was also
carried out by chemical transfection
method (Bisht et al., 2014).
Cattle (n=22)
Buffaloes (n=70)

7. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India

8. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
r3AB3
ELISA
r3AB3
ELISA
Total
Positive Negative
FMDV
RNA
(OPF)
Positive 4 10 14
FMDV
RNA
(OPF)
Negative 0 56 56
Total 4 66 70
FMDV RNA (tissue) FMDV RNA (tissue) Total
Positive Negative
FMDV RNA
(OPF)
Positive 14 0 14
FMDV RNA
(OPF)
Negative 4 52 56
Total 18 52 70
r3AB3 ELISA r3AB3 ELISA Total
Positive Negative
FMDV
RNA
(tissue)
Positive 4 14 18
FMDV
RNA
(tissue)
Negative 0 52 52
Total 4 66 70

9. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
a b c d
a. Ventral soft palate (thin black arrow) and palatine tonsil (thick black arrow),
b. dorsal soft palate (DSP) demarcated into DSP1 (thick black arrow) and DSP2 (thin black
arrow),
c. dorsal nasopharynx (DNP) divided into two part DNP1 (thin black arrow) and DNP2
(thick black arrow),
d. middle retropharyngeal lymph node.

10. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
21 tissues were found positive for FMDV RNA while 25 tissues were found positive
for FMDV antigen from 18 buffaloes by immunomicroscopy.
Types of tissue FMD viral Ag
positive %
Types of tissue FMD viral Ag
positive %
Types of tissue FMD viral Ag
positive %
1. Tongue 00 4. Dorsal soft palate 2 44 7. Palatine tonsil 16
2.Ventral soft palate 6 5. Dorsal nasopharynx 1 17 8. Middle retropharyngeal lymph node 0
3.Dorsal soft palate 1 28 6. Dorsal nasopharynx 2 28 9. Sub mandibular lymph node 0

11. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
b. Dorsal nasopharynx
SG
E CE
GC
GC
E
E
GC
SG
11
Figure: Light microscopy of dorsal nasopharynx (DNP) collected from different buffaloes,
sections showing variation in number of crytps and thickening of epithelium, salivary glands
(SG) and germinal centres (GC) within the connective tissue of the lamina propria below the
respiratory epithelium (E). H&E- 40x.

12. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
DSP1DSP2
Nucleus FMDV Ag MergePanctoykeratin
Colocalization of FMDV antigen in sub-epithelial regions of DSP

13. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
Nucleus FMDV Ag MergePanctoykeratin
DNP1DNP2

14. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
VSP-IFA
Nucleus FMDV Ag MergePanctoykeratin
VSP-IHC

15. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
Figure. Immunofluorescence of different tissues collected from apparently healthy buffalo
showing negative reaction, (a) DSP, (b) DNP, and (c) PTON, where nuclei- blue, FMDV capsid
protein- red, pan cytokeratin-green. Primary antibody- FMDV rabbit polyclonal antibody, pan
cytokeratin mouse monoclonal antibody. Secondary antibody- goat anti rabbit IgG (H+L)
Alexa fluor-594 and goat anti mouse IgG (H+L) Alexa fluor-488. Bar= 50μm.
a b c

16. OS16
ICAR-Directorate of FMD
Mukteshwar-263138, India
• Infectious FMDV was not isolated from any tissues samples.
• Overall, 25 of the 1012 tissue- contain FMDV genomic RNA; 21 of these
tissues were from buffalo, whilst 4 were from cattle.
• No considerable changes were observed in any tissues at microscopic level
by light microscope. However, respiratory epithelium thickening and
number of crypts was found more in buffaloes DSP than cattle.
• Interestingly, all the samples found positive by immunomicroscopy are
of buffalo origin, thereby suggesting a higher incidence and/or longer
duration of persistence of FMDV in buffaloes than cattle.
• Among these tissues, samples with highest prevalence of detection of
FMDV antigen by immunomicroscopy were DSP2 followed by DSP1,
DNP2, then DNP1 and PTON.
Conclusions

17. ICAR-Directorate of FMD
Mukteshwar-263138, India
Acknowledgements
• Scientific and technical staff of ICAR- PDFMD, Mukteshwar
• Indian Council of Agricultural Research, New Delhi
• Mr. Basant, Uttam Nath Goswami, Shyam Lal Tamta, and Mr.
B. Das.
• Drs. Rodriguez and Arzt were supported by ARS-CRIS
Project 1940-32000-057-00D. Additional funding was
provided by the U.S. Department of State, Biosecurity
Engagement Program through the USDA, ARS Office of
International Research Programs.
• Staffs of Slaughter house.

18. ICAR-Directorate of FMD
Mukteshwar-263138, India
Himalayan view from PDFMD, Mukteswar 18
Thanks for
your kind
attention