Molecular Studies of Rabies in Trade Dogs and Detection of some RNA Viruses in Bats in Plateau State, Nigeria.
ABSTRACT
Rabies, the cause of an almost invariably fatal encephalomyelitis, remains a public health threat in Nigeria where the dog is the main reservoir. Ribonucleic acid (RNA) viruses are most commonly implicated in emerging and reemerging diseases.
Bats are established natural reservoir hosts for many emerging viral zoonoses of enormous human and animal health impacts.
Bat and dog meats are consumed by some ethnic groups in Nigeria.This study determinedthe characteristics of rabies virus in slaughtered dogs, detected some RNA viruses in bats and serologically detected some lyssaviruses in bats from Plateau State.
Brain tissues were obtained from 532 slaughtered dogs and the direct fluorescent antibody test (DFAT) was used to screen for rabies antigen.
TheDFATpositive brain tissues were subjected to the reverse transcription polymerase chain reaction (RT-PCR). Bats (356) consisting of Eidolon helvum(244),Epomophorusfranqueti(12), Epomophorusgambianus(5), Chaerophonpumila(33), La via frons (3), Nycterismacrotis(18), Rhinolophuslanderi(37), and Rhinopomamicrophylum (4) were collected from 8 locations during 2010- 2011.
Bat brain samples (356) were tested for Lyssavirus antigen by DFAT. Modified rapid fluorescent focus inhibition test (RFFIT) was used to test serum samples from 76 bats for antibodies to Lagos bat (LBV), Mokola (MOKV), Duvenhage (DUVV), West Caucasian bat (WCBV), Shimoni bat (SHBV) and the classical rabies virus strain (CVS-11).
Using pan-viral RT-PCR with consensus degenerate primers, rectal swabs from bats (n=95) were screened for some RNA viruses. Neighbour joining (NJ) was used to construct phylogenetic trees for sequences obtained with 1000 bootstrap values. DFAT detected rabies antigen in 92 (17.29%) dog brain tissues, of which 4(4.35%) were positive by RT-PCR.
The N gene of all 4 were amplified and sequenced and all had a 99% homology to other previously documented rabies viruses identified from Nigeria. Among these 4 samples, one was further isolated and its entire genome was characterized and phylogenetic analysis showed it belongs to the Africa 2 lineage.
No Lyssaviruswas detected in the 356 bat brains. Of 76 bat sera tested, 28.94% (22/76) had neutralizing antibodies to LBV, MOKV, and SHBV. Twenty one (27.6%) neutralized LBV, 6(8%) neutralized MOKV and 18(24%) neutralized Shimoni bat virus.
All the positive sera originated from the straw-coloured fruit bat (Eidolon helvum) with the exception of one Chaerophonpumila serum which neutralized both LBV and SHBV. Four samples neutralized both MOKV and LBV. No serum neutralized CVS-11, WCBV and DUVV. Among the 95 bat faecal swabs screened, eight (8.42%) were positive for Coronavirus(CoV).
Seven of the identified CoVs had an average of 95% homology to the Ghana bat CoV and one had 97% identity to the Kenya bat CoV (BtKy56). All eight sequences clustered with the BetaCoV group of the SARS-CoV.
One bat faecal sample was positive for Paramyxovirus with 70% homology to the J-virus from Kenya and one specimen was also positive for Reovirus with sequence relating closely (93%) to the mammalian Orthoreovirus.
Sixteen specimens (16.84%) were positive for Rotavirus (RV) RNA of which phylogenetically, 13 sequences were related to RV A, 1 to RV B and 2 to RV C. None of the faecal swabs was positive for Arenavirus, Influenza virus, and Rhabdoviruses.
The continued detection of rabies virus in trade dogs and other lyssaviruses in bats show their importance in the epidemiology of lyssaviruses in Nigeria.
The detection of Coronavirus, Paramyxovirus, Reovirus and Rotavirus in feacal samples of the bats is an indication of the role that bats may play in the emergence of viral diseases.
TABLE OF CONTENTS
Title Page ……………… i
Declaration ……. iii
Certification .. iv
Acknowledgements ……… v
Dedication ….. viii
Table of Contents ….. ix
List of Tables ….. xii
List of Figures ……….. xiii
List of Plates …. xv
List of Appendices … xvi
List of Abbreviations and Symbols…. xvii
Abstract …………. xviii
CHAPTER 1:INTRODUCTION
1.1 Background to the Study ……… 1
1.2 Statement of Research Problem . 2
1.3 Justification ………. 4
1.4 Aim………. 6
1.5Objectives ………… 6
1.6Research Questions ….. 6
CHAPTER 2:LITERATURE REVIEW
2.1The Rabies Virus ………. 8
2.2 Rabies in Nigeria ……… 8
2.2.1 Rabies in dogs and other animals …….. 9
2.2.2Sub-clinical rabies ………. 12
2.2.3Dogmeat consumption in Nigeria ….. 12
2.3 Lyssaviruses and Bats …. 13
2.3.1 Bat lyssaviruses in Africa ……… 14
2.3.2 Insectivorous bat-associated rabies …… 17
2.3.3Bat rabies and host switching ………. 19
2.4 Coronaviruses in Bats ……. 20
2.5Paramyxoviruses in Bats …. 22
2.6Reoviruses in Bats … 24
2.7 Rotaviruses in bats ……. 25
CHAPTER 3:MOLECULAR STUDIES OF RABIES IN TRADE DOGS IN PLATEAU STATE NIGERIA
3.1 Introduction ……. 27
3.2 Materials and Methods ….28
3.2.1 Studyarea …. 28
3.2.2 Selection of dog markets … 28
3.2.3 Dawaki and Amper markets. ……. 29
3.2.4 Direct flourescent antibody test (DFAT) .. 32
3.2.5 Reverse transcription polymerase chain reaction (RT-PCR) ….. 33
3.3 Results ….. 39
3.4 Discussion …. 48
3.5 Conclusion … 50
CHAPTER 4:DETECTION OF ANTIGENS AND ANTIBODIES TO LYSSAVIRUSES IN BATS IN PLATEAU STATE
4.1Introduction …… 52
4.2 Bat Samples ..54
4.2.1Study area …. 54
4.2.2 Bat survey and sample collection … 54
4.2.3Bat identification ……….. 58
4.3 Lyssavirus Diagnosis …….. 58
4.3.1Lyssavirus antigen detection by DFA test on bat brain tissues. …….. 58
4.3.2 Modified rapid fluorescent focus inhibition test (RFFIT) detection of virus neutralizing antibodies in batsera 59
4.3.3 Reading and interpretation of results … 61
4.4 Results ……………….. 63
4.4.1Bats sampled …..63
4.4.2 Lyssavirus neutralising antibody and antigen detection ……… 63
4.5Discussion . 68
CHAPTER 5: SEARCH FOR SOME RNA VIRUSES IN BATS CAPTURED FROM PLATEAU STATE, NIGERIA
5.1 Introduction … 71
5.2 Materials and Methods 73
5.2.1 Study area ……….. 73
5.2.2 Bat survey, sampling and identification … 73
5.2.3 Molecular techniques used for detection of selected RNA viruses at the CDC, GA. …… 73
5.2.4 TOPO® TA cloning with Invitrogen pCR 4.0 …….. 83
5.3Results …… 91
5.4. Discussion …….. 108
CHAPTER 6: GENERAL DISCUSSION AND CONCLUSION
6.1General Discussion …….. 113
6.2 Conclusion ……….. 115
6.3Recommendations …………. 115
6.4Limitations of the Study ….. 116
REFERENCES ……….. 117
APPENDICES…….135
INTRODUCTION
1.1 Background to the Study
Rabies virus (RABV), genus Lyssavirus and family Rhabdoviridae, is enzootic throughout most of the world. The domestic dog (Canisfamiliaris) acts as the principal vector in Africa and Asia (Knobel et al., 2005; 2007).
However, a range of mammalian carnivores also can act as hosts (Nel et al.,1993; Swanepoel et al., 1993; Rupprecht et al.,1995; Real et al., 2005; Velasco-Villa et al., 2005; Hass and Dragoo, 2006; Childs et al., 2007; Davis et al., 2007).
Bats are established as the natural reservoir hosts for many emerging viral zoonoses and more than 60 viral species have been detected in various regions of the world with enormous human and animal health impacts.
Some of these viruses include: Lyssaviruses (Calisher et al., 2006), paramyxoviruses (Murray et al., 1995; Chua et al., 2000; Drexler et al., 2012), filoviruses (Leroy et al., 2005; Calisher et al., 2006), coronaviruses (Li et al., 2005; Tong et al., 2009; Quan et al., 2010), influenza virus (Tong et al., 2012)and reoviruses (Esona et al., 2010; Wong et al., 2012).
Viruses from bats could be transmitted to humans via several means such as: bites, exposure to infected saliva, faecal aerosols and contact with infected tissue materials or infected host (Wong et al., 2012).
REFERENCES
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