OS16 - 6.G11.c Localization of FMD RNA and Viral Antigens in Different Tissues From Apparently Healthy Cattle and Buffalo Under Natural Condition in India: Pathology and Pathological Basis of Persistence - R. Ranjan

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

OS16 - 6.G11.c Localization of FMD RNA and Viral Antigens in Different Tissues From Apparently Healthy Cattle and Buffalo Under Natural Condition in India: Pathology and Pathological Basis of Persistence - R. Ranjan

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