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Serological investigation of bat rabies in Southern China

Serological investigation of bat rabies in Southern China. Changchun Tu. Diagnostic Laboratory on Rabies and Wildlife Associated Zoonoses, Ministry of Agriculture of China. Rabies Transmission Cycle. rabid domestic animal. rabid wild animal. Exposed human. Intermediate host.

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Serological investigation of bat rabies in Southern China

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  1. Serological investigation of bat rabies in Southern China Changchun Tu Diagnostic Laboratory on Rabies and Wildlife Associated Zoonoses, Ministry of Agriculture of China

  2. Rabies Transmission Cycle rabid domestic animal rabid wild animal Exposed human Intermediate host Natural reservoir Ten of 11 LVs have chiroptera as natural reservoirs. Infected human Disease and death Dead end host

  3. Bat LV in Southeast Asia • The neutralizing antibodies against ABLV and EBLV were found in insectivous and • frugivorous bats recently in: • The Phillipines • Cambodia (only country reporting natural infection of RABV in bats ) • Thailand • Bangladesh • China?

  4. RABIES IN CHINA • Human rabies deaths/cases in China between 2000 to 2006 (MOH,China)

  5. RABIES IN CHINA

  6. A.Yunan: 1 site B.Guangxi: 3 sites C.Guangdong: 5 sites D.Hainan: 2 sites Where we sampled ?

  7. 大黄蝠 How many sampled and analyzed ? Sampled: 1287insectivorous and frugivovousbats for blood specimen, belonging to 13 species. Assayed: 685 blood specimens in 8 species (7 insect and 1 fruit bats) had sufficient volume for iFAT and mFAVN.

  8. Methods A iFAT: Bat serum (4× & 8× dilution) is added to fixed monolayer infected with RABV then probed by FITC-conjugated protein A/G. Needs 20 ul serum . B FAVN: needs 200 ul serum C mFAVN: needs 50 ul serum • The most part of bat sera were screened by iFAT, then subjected to conventional FAVN for confirmation if having sufficient volume. (at IVS,Changchun,China) • Small part of bat sera were tested directly by mFAVN. (at VLA, Weybridge, UK).

  9. A BC Negative cntrl Results iFAT--control Positive cntrl: dog (A) and mouse hyperimmune serum (B), human anti-rabies serum (C) added respectively onto RABV infected monolayer, then probed by FITC-protein A/G. Negative cntrl: mock-infected monolayer.

  10. A B Negative cntrl Results iFAT--control Positive cntrl: rabbit anti NiV virus serum (A) and mouse anti NiV N serum (B), added respectively onto monolayer infected by recombinant baculovirus expressing NiV N protein, then probed by FITC-protein A/G. Negative cntrl：mock-infected monolayer

  11. Bat no. LX0093 (100×) Bat no. HN0047 (200×) Results iFAT– bat serum sample detection In fruit bats: 12/514 sera were seropositive. In insect bats:1/122 bat sera were seropositive;

  12. Results Viral neutralization • FAVN: Only 15/514 fruit bat serum samples, including 3 iFAT positive, had sufficient volume for FAVN. The titers of these 3 sera were 0.33 IU/ml, 0.15 IU/ml and 0.08 IU/ml. Two methods were 100% coincident • mFAVN: 2/171 insect bat sera were positive, both showing titer of 0.38 IU/ml. • Low VN titers also reflected low antibody level in natural infection.

  13. Results and Discussion • Total positive rate: 15/685 (2.2%). • Antibody response was very low as indicated by: iFAT: specific, but limited and faint cytoplasmic fluorescence FAVN: fruit bat: 0.33 IU/ml, 0.15 IU/ml, 0.08 IU/ml mFAVN: insect bat: 0.38 IU/ml, 0.38 IU/ml. • Two methods are 100% coincident • Positive species: Rousettus leschenaulti ——frugivorous, 12 samples Rhinolophus ferrumequinum ——insectivorous, 1 sample Rhinoophus blythi ——insectivorous, 2 samples • Positive fruit bat distributed in four sampling sites of three Provs, while positive insect bat in only one sites of Guangdong Prov.

  14. Results Ratio of rabies seropositive bat Sample collection sites:6in Guangdong; 1 in Yunnan; 2 in Guangx; 1 in Hainan.

  15. Discussion • There is no labeled second antibody specifically prepared to detect bat immunoglobulin • FAT is a golden standard method recommended by WHO and OIE for rabies diagnosis, in which the result can be read readily and accurately under microscopy by eyes • To further evaluate the effect of our iFAT the FITC-Protein A/G mixture was also used to detect serum antibodies of human, mouse rabbit and pig against other viral antigens on fixed cell sheets and specific cytoplasmic green fluorescence were seen as well (data not shown), indicating the versatility of FITC-Protein A/G for binding immunoglobulin of different mammals. • Low percent of bat brain tissues showed positive RT-PCR but negative in FAT and MIT, the result was unable to be confirmed by additional methods, therefore not incorporated in this presentation.

  16. Conclusion • China has the natural infection of RABV in different bat species. • FITC-protein A/G is a powerful tool to detect bat immunoglobulin in serological study. • Our research is a primary work and more extensive studies need to be done before systematic description of bat in China for natural infection state of lyssavirus.

  17. 谢谢! Thank You!

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