VECTOR-BORNE AND ZOONOTIC DISEASESVolume 3, Number 3, 2003© Mary Ann Liebert, Inc.

Research Paper

Rabies in Wildlife in Latvia

SANITA VANAGA,1 REINA VAN DER HEIDE,2 RAFAELS JOFFE,1

and WIM H.M. VAN DER POEL2

ABSTRACT

In the Baltic States, lyssaviruses are often detected in wildlife and presumed to constitute an important publichealth hazard. In order to decrease rabies incidence and eradicate wildlife reservoirs, a national rabies eradicationprogram has been in place. Since 1970 a vaccination program in dogs and cats has been executed, and in 1991 oralvaccination of foxes was started. However, due to an insufficient budget, the latter was not done regularly andnationwide before 2000. Now, the program in force consists of compulsory vaccination of all dogs and cats, and atetracycline marker vaccine oral vaccination program of foxes in the whole country. In 2001, 151 of 285 (53%) foxjaws were tested positive for tetracycline. All animals showing rabies-like symptoms were killed and tested forrabies. In this way, 250–400 cases of rabies per year were diagnosed in wildlife. To molecularly characterize theprevalent lyssaviruses in wildlife, lyssavirus RNA of 25 recent rabies positive samples, collected in the year 1999,was amplified by RT-PCR. Direct sequencing of the RT-PCR-amplified products of the virus’ nucleoprotein en-coding region and subsequent sequence analyses resulted in a 99.3–100% homology between isolates and a99.0–100% similarity with a 1995 genotype I, classical rabies virus (RABV) raccoon dog isolate from Estonia. Theseresults confirmed that RABV is endemic in wildlife in Latvia and should be considered a serious public healththreat. To successfully eradicate the wildlife reservoirs, the national rabies eradication program must be contin-ued, and it may need to be intensified. Key Words: Latvia—Lyssavirus—Rabies—Wildlife. Vector-Borne ZoonoticDis. 3, 117–124.

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INTRODUCTION

RABIES VIRUS is the type species of theLyssavirus genus, a group of negative

stranded RNA viruses with an almost world-wide distribution. The Lyssavirus genus be-longs to the family of Rhabdoviridae and can bedivided into seven genotypes, rabiesvirus be-ing genotype I (Badrane et al. 2001). All terres-trial mammals are susceptible to classical ra-bies virus (RABV). Without vaccination, thebite of an infected animal can cause an acutefatal encephalitis in humans. Therefore, rabies

is considered a major public health threat andan important zoonosis in countries where thevirus is endemic (Rupprecht et al. 1995). InLatvia, rabies is regularly observed in animals,and the isolated lyssaviruses in terrestrialwildlife have always been designated intogenotype 1, classical rabies virus (RABV).Dogs, cats and wildlife are considered the mainreservoirs of rabies in this country. In order todecrease rabies incidence and eradicate wildlifereservoirs, a national rabies eradication pro-gram has been installed. In this report the pres-ent situation will be described. To molecularly

1Department of Virology, National Veterinary Laboratory, Latvia.2Microbiological Laboratory for Health Protection, National Institute for Public Health and the Environment,

Bilthoven, The Netherlands.

characterize the circulating RABVs, RT-PCRamplification products of RABV RNAs weresequenced and analyzed phylogenetically.

MATERIALS AND METHODS

Specimens

Brain tissue specimen were collected fromanimals clinically suspected of rabies. In Latvia,

animals showing rabies-like symptoms, that is,aggressive/biting behavior or loss of naturalfear for humans, as well as stray dogs and catsare killed by police officers as well as civilians,and are sent in for rabies testing at the DistrictVeterinary Laboratories and the Department ofVirology of the National Veterinary Laboratoryin Riga. Each year, 500–1500 of these animalsare tested for rabies (Fig. 1). In Latvia, bats areprotected animals and not tested for rabies sur-veillance.

Rabies testing

Rabies testing was performed by standardfluorescent antibody test (FAT) as described byDean et al. (1996) with modifications, using thepolyclonal fluorescein isothiocyanate-labeledrabbit anti-rabies nucleocapsid IgG (Diagnos-tics Pasteur, Marnes-la-Coquette, France). TheFAT was performed as described by the man-ufacturer. Positive controls—brain tissuesmears (from mice infected with RABV)—wereincorporated in each test run. To check thespecificity of the FAT, a second impression of

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FIG. 1. RABV detections performed in animals in Latvia1997–2001: fluorescent antibody test (FAT) and mouse in-oculation test (MIT).

TABLE 1. ANIMAL SAMPLES SELECTED FOR RT-PCR AND LYSSAVIRUS RNA SEQUENCE ANALYSIS

SamplingSample dateserial no. (day/month/year) Area of origin Species

LAT01 18.03.99 Ludzas raj. Istras pag. s.Belejeva FoxLAT02 24.03.99 Ludzas raj. L õ¯dumnieku pag. s. Babrova DogLAT03 12.08.99 R õ gas raj. Sejas pag. “ZÆagatas” FoxLAT04 21.12.99 Aluksnes raj. ViresÆu pag. “Vanagi” Raccoon dogLAT05 21.04.99 Dobeles raj. Krimunu pag. “Vidzemes strelnieki” FoxLAT06 22.04.99 Preilu raj. Upmalas pag. s. Vepri FoxLAT07 22.04.99 Preilu raj. Aglonas pag. s.Leitani FoxLAT08 07.05.99 Dobeles raj. Tervetes pag. “Kuras” FoxLAT09 18.05.99 Tukuma raj. Pures pag. “Kalnenieki” FoxLAT10 24.05.99 Gulbenes raj. Stradu pag. “Kalnenieki” FoxLAT11 25.05.99 Kraslavas raj. Graveru pag. s.Belogrudova FoxLAT12 31.05.99 Gulbenes raj. Lizuma pag. “Mazbrenguli” FoxLAT13 17.06.99 Tukuma raj. Slampes pag. “V õ¯ksele” Raccoon dogLAT14 03.05.99 Ventspils raj. Usmas pag. “BriezÆkalni” FoxLAT15 05.05.99 Talsu raj. Laucienas pag. “Dzerves” FoxLAT16 18.06.99 Dobeles raj. “Jaunstrautnieki” CatLAT17 20.12.99 R õ gas raj. Malpils ag. “Kraukli” CatLAT18 07.10.99 Aluksnes raj. Veclaicenes pag. “Miez õ¯sÆi” DogLAT08 28.09.99 Dobeles raj. Tervetes pag. “Gredzeni” FoxLAT20 14.09.99 Preilu raj. RozÆkalnu pag. Krauklis FoxLAT21 16.09.99 R õ gas raj. RopazÆu pag. “Rotkali” FoxLAT22 17.09.99 R õ gas raj. Baldones pag. “Zeltkalni” BadgerLAT23 22.09.99 Preilu raj. RozÆupes pag. “Malkalnes” PolecatLAT24 10.11.99 Dobeles raj. Auce Benes iela 45a Raccoon dogLAT25 10.01.00 Cesu raj. N õ¯taures pag. Dog

aSerial numbers refer to Figure 4.

each sample was stained with negative controlserum. Negative control serum was obtainedby adsorbing the anti-rabies serum with rabies-infected mouse brain tissue. Virus detectionwas performed on brain tissue samples by stan-dard mouse inoculation test (MIT) as describedby Koprowski et al. (1996). All samples testednegative by FAT were re-tested by MIT.

RT-PCR analyses

For molecular characterization of RABV inwildlife in Latvia, 25 specimen were selected:15 fox, three raccoon, dogs, two cats, threedogs, one polecat, and one badger (Table 1 andFigs. 2 and 3). For amplification of RABV-spe-cific RNA, brain tissue specimens (3 mm3) wereput in 0.5 mL RNA extraction buffer. The RNAextraction was performed as essentially de-scribed by Chomczynski et al. (1987). Reversetranscription, PCR amplification, and probehybridizations were performed as described byVan der Poel et al. (2000).

RABV RNA sequence analyses

Direct sequencing of the RT-PCR-amplifiedproducts of a 400-nucleotide coding region ofthe amino terminus of the nucleoprotein ofRABV and analyses of the nucleotide se-quences were performed as described byAmengual et al. (1997). PCR fragments werepurified by QIAquick PCR Purification Kit (QIAgen, Hilden, Germany) and then se-

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FIG. 2. Animals in Latvia tested positive for rabies1997–2001: fluorescent antibody test (FAT) and mouse in-oculation test (MIT).

FIG. 3. Sites where animal samples selected for lyssavirus molecular characterization originated from.

quenced directly on both strands. Sequencingwas performed on a Biosystems (ABI) 3700DNA automated sequencer (Perkin Elmer, Ap-plied Biosystems, Foster City, CA) using fluo-rescent dye-labeled dideoxynucleo terminators(BigDye™ Terminator Cycle Sequencing ReadyReaction, Perkin Elmer Applied Biosystems,U.K.). Nucleotide sequences were edited usingSeq Ed (V1.03, Applied Biosystems), and alignedusing Bionumerics (V3.0; Applied Maths, Kor-trijk, Belgium). Distance calculations were doneusing Kimura 2 parameter correction for evolu-tionary rate. The confidence values of the inter-nal nodes were calculated performing 1000 boot-strap analyses. Evolutionary trees for nucleotidesequences were drawn using the Neighbor Join-ing method, with the CVS vaccine strain (Gen-bank Accession No. D42112) bp 54–442 (N-gene)as reference.

Eradication strategy

According to the national rabies eradicationplan, vaccination of dogs and cats is compul-sory, and all domestic animals have to be vac-cinated after a contact with rabies affected orsuspected animals. However, vaccination cov-erage is far from complete. It is estimated bythe Latvian Veterinary Laboratory that about25–30% of dogs and cats are not vaccinated reg-ularly.

A first oral vaccination campaign of foxeswas started in 1991. At that time, it was not per-formed periodically and accurately due to re-stricted budget. Until 1998, unmarked vaccines(produced in Russia, no details available) wereused, and it was not possible to evaluate effec-tiveness of the campaign. In 1998, a new effortwas made to decrease rabies incidence and

eradicate the virus in the wildlife reservoirs; theState Veterinary Service restarted oral vaccina-tion of foxes in 14 of 26 administrative districts.In this campaign, marker vaccines produced inGermany (Rabifox®) and the Czech Republic(Lysvulpen®) were used. Vaccine-laden baitswere distributed by veterinarians and hunters,twice with a 7–8-day interval in spring and au-tumn. The numbers of vaccine doses per squarekilometer of forest are listed in Table 2. Baitswere distributed by hand and placed next tofox caves and on fox paths. Vaccine spots weremarked on a geographical map. After 7–10days, bait uptake was checked by the distribu-tors. Counts of foxes were performed in everyseason, by veterinarians and hunters. For eachdistrict, results were reported to and recordedby the National Veterinary Laboratory.

The amount of vaccine uptake was checkedby testing fox jaws for tetracycline marker. Jawsof shot foxes were collected, cut in 0.05-mmslices and investigated using a luminescent mi-croscope. Raccoon dogs were not tested. Fromthe year 2000 on, the oral vaccination programcovers the whole country. These activities areperformed according to the National Program0402 on eradication of animal infectious dis-eases. Annually, the National Food and Vet-erinary Service composes an action plan forsurveillance of infectious diseases in animalsthat is sent to all 26 country districts. This ac-tion plan describes for each disease, the testsystem, the surveillance methods and the num-ber of animals that have to be investigated.Most of the wildlife samples are submitted inthe spring and autumn during the hunting sea-son. In the year 2002, according to this pro-gram, 500 fox jaws will have to be tested forthe tetracycline marker.

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TABLE 2. ORAL VACCINATION OF FOXES. NUMBER OF VACCINE DOSES PER SQUARE km

Area of forests, Number ofYear Number of districts km2 vaccine doses

1991 1 403 3331992 11 8,882 14,2101995 8 7,993 10,0001996 4 4,456 3,4001998 autumn 14 14,692 56,1001999 spring 14 14,692 30,0001999 autumn 14 14,692 30,0002002 spring and autumn 26 26,570 300,000

RESULTS

Rabies testing

From 1997 to 2001, each year, 529–1417 ani-mals were tested for rabies, including sevenspecies: raccoon-dog (Nyctereutes procyonoides),red fox (Vulpes vulpes), badger (Meles meles), fer-ret (Mustela nigripes), polecat (Mustela putorius),and domestic dogs and cats. Between 141 and516 (23–43%) animals per year, were tested pos-itive according to the described test scheme. Ofall samples tested negative by FAT, 1–3% wastested positive by MIT subsequently. Test re-sults per year are depicted in Figure 1. Inwildlife in Latvia, the highest RABV incidenceswere found in foxes and raccoon dogs (Fig. 2).

RABV RT-PCR and RNA sequence analyses

All 25 animal brain tissue samples selectedfor RABV sequence analysis were tested posi-tive by PCR. Direct sequencing of the RT-PCR-amplified products of a 400-nucleotides codingregion of the amino terminus of the nucleopro-tein and subsequent sequence analyses resultedin a 99.3–100% homology between isolates anda 99.0–100% similarity with a 1995 genotype I,(Genbank Accession Nos. AY277570–277580)raccoon dog isolate from Estonia (Genbank Ac-cession No. U22476) (Fig. 4).

Eradication strategy

In the current rabies vaccination campaign,all oral vaccines are marked with tetracycline.In the year 2001, 285 fox` jaws were received atthe laboratory and tested for tetracycline. Of285 samples, 151 were tested positive (53%).Fox density per hectare increased from 0.55 in1993 to 1.02 in 2002. Vaccination coverage ofdogs and cats is estimated at 70–75%. Vaccina-tions in dogs and cats are not recorded inLatvia.

DISCUSSION

In this paper, the present rabies situation inLatvia is reported. Rabies incidences in animalsand molecular characterizations of circulatingRABVs are described. Rabies virus incidence in

terrestrial animals showing neurological signswas found to be around 35% (23–43%) over the5-year period 1997–2001. Foxes and raccoondogs seem to be the main wildlife reservoirs.These findings are in agreement with reportsfrom other Baltic States and surrounding coun-tries (Gylys et al. 1998; Lyczak et al. 2001).

Since 1970, a vaccination program in dogsand cats is executed in Latvia, and in 1991 oralvaccination of foxes was started in 14 of 26 dis-tricts. However, due to insufficient budget, thelatter was not done periodically before 2000.Nowadays, the rabies control program in forceconsists of compulsory vaccination of all dogsand cats, and of domestic animals that mayhave been in contact with infected animals orshow rabies-like symptoms. The latest programincludes a tetracycline marker vaccine oral vac-cination program of foxes in the whole coun-try. Nevertheless, rabies infections in pets arestill regularly detected, most likely becausethere are quite a number of stray dogs and catsand because some owners do not vaccinatetheir pet animals in a timely manner. A signif-icant decrease in rabies infections in animalsfrom 1997 to 2001 was not observed. This maybe due to the fact that the rabies control pro-gram was not executed efficiently until 2000,and because it is too early to evaluate the re-sults of the program in force after 2000.

Twenty-five animal brain tissue samples se-lected for RABV sequence analyses all testedpositive by PCR. For these samples, the usedRABV RT-PCR assay proved to be at least assensitive as the FAT /MIT test system that isrun for rabies surveillance. Sequencing of theRT-PCR-amplified products of the nucleopro-tein encoding region and subsequent sequenceanalyses resulted in a 99.3–100% homology be-tween isolates and a 99.0–100% similarity witha 1995 genotype I, raccoon dog isolate from Es-tonia (Fig. 4). All latvian RABV sequences clus-tered within the group of northeastern Euro-pean isolates (Fig. 5). This is consistent with thefindings of Bourhy et al. (1993). No indicationswere found for clustering of RABV strainswithin a specific animal species or within a spe-cific geographical region of the country. All se-quences were obtained from samples collectedwithin 1-year time, and though these were allcollected after March 1999, all sequences were

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FIG. 4. Phylogenetic relationships between a nucleoprotein encoding gene fragment (400 nt) of 25 Latvian RABVisolates and a selection of RABV sequences from (North) East Europe. Genbank accession numbers: AF033870-AF033876 and AF033878-033880 (Poland); AY062071 (Russia); U224476, U42707, U42714, U42715, U43432, (Estonia);U42716 (Finland); U3002 (Lithuania); and U3007 (Slowakia). The CVS vaccine strain (bp 43-442) was used as a com-parison (Acc. Nr: D42112). Alignment: global similarities, correction: Kimura 2, Clustering Neighbor Joining (BioN-umerics 3.0, Applied Maths, Kortrijk, Belgium). The digits on top of the figure are percentages of nucleotide differ-ence.

similar genotype I sequences from North EastEurope isolated over the last 10 years. The phy-logenetic analysis data shows a single clade ofviruses in the region. This is supported bystrong boot-strap analysis (Fig. 4). These find-ings confirm that RABV is endemic in wildlife

in Latvia. As expected, wildlife seems to be animportant virus reservoir. Infections in pet an-imals regularly occur, and large domestic ani-mals are sometimes infected also. IsolatedRABV strains from different species wereclosely related, indicating a spillover from

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FIG 5. Phylogenetic clustering of RABV isolates from Latvia within the group of RABV isolates from northeast Eu-rope, based on nucleotide sequences of a nucleoprotein encoding gene fragment (400 nt). Clusters of RABV isolatesfrom other European countries are depicted for comparison (unrooted tree). Genbank accession numbers: U42706,U43008, U43006, U42708, U42702, U42713, U42992, U42999, AF033882, U42702, U22475, AF033894, U43010, AY062071,U42716, U43002, AF033875, U42715, and U43007. Maximum likelihood cluster method (BioNumerics 3.0, AppliedMaths, Kortrijk, Belgium).

wildlife to pet animals and large domestic an-imals. RABV in wildlife in Latvia thereforeshould be regarded a public health threat.

To successfully eradicate rabies in wildlifereservoirs (in order to reduce the veterinaryand public health threat), an effective rabiescontrol program is needed. Besides vaccinationof pet animals, rabies eradication through oralvaccination of wildlife has shown to be able toreduce rabies incidence in specific species(MacInnes et al. 2001). These approaches alsoseem to be the most practical ones in the Lat-vian situation. However, according to datapublished for Western Europe (Pastoret et al.1999), the present vaccination coverage of 53%,as found in Latvia, most likely needs to be en-hanced to successfully suppress the epizootic.To evaluate effectiveness, RABV detection andsurveillance in wildlife is absolutely essential.Therefore, the Latvian national rabies controlprogram must be continued. Future rabies sur-veillance data will show what parts of the pro-gram definitely need to be optimized or inten-sified.

REFERENCES

Amengual, B, Whitby, JE, King, A, et al. Evolution of Eu-ropean bat lyssaviruses. J Gen Virol 1997;78:2319–2328.

Badrane, H, Bahloul, C, Perrin, P, et al. Evidence of twoLyssavirus phylogroups with distinct pathogenicityand immunogenicity. J Virol 2001;75:3268–3276.

Bourhy, H, Kissi, B, Tordo, N. Molecular diversity of theLyssavirus genus. Virology 1993;194:70–81.

Chomczynski, P, Sacchi, N. Single step method of RNAisolation by guanidinum thiocyanate-phenol-chloro-form extraction. Anal Biol 1987;162:156–159.

Dean, DJ, Abelseth, MK, Athanasiu, P. The fluorescenceantibody test. In: Meslin, FX, Kaplan, MM, Koprowski,H, eds. Laboratory Techniques in Rabies, 4th ed. Geneva:World Health Organization, 1996:88–93.

Gylys, L, Chomel, BB, Gardner, IA. Epidemiological sur-veillance of rabies in lithuania from 1986 to 1996. RevSci 1998;(Tech 17):691–698.

Koprowski, H. The mouse inoculation test. In: Meslin, FX,Kaplan, MM, Koprowski, H, eds. Laboratory Techniquesin Rabies, 4th ed. Geneva: World Health Organization,1996:80–87.

Lyczak, A, Tomasiewicz, K, Krawczuk, G, et al. Epizooticsituation and risk of rabies exposure in Polish popula-tion in 2000, with special attention to Lublin province.Ann Agric Environ Med 2001;8:131–135.

MacInnes, CD, Smith, SM, Tinline, RR, et al. Eliminationof rabies from red foxes in eastern Ontario. J Wildl Dis2001;37:119–132.

Pastoret, PP, Brochier, B. Epidemiology and control of foxrabies in Europe. Vaccine 1999;17:1750–1754.

Rupprecht, CE, Smith, JS, Fekadu, M, et al. The ascensionof wildlife rabies: a cause for public health concern orintervention. Emerg Infect Dis 1995;1:107–114.

Van der Poel, WHM, Van de Heide, R, Van Amerongen,G, et al. Characterisation of a recently isolated lyssa-virus in frugivorous zoo bats. Arch Virol 2000;145:1919–1931.

Address reprint requests to:Dr. Wim H.M. van der PoelMicrobiological Laboratory

for Health Protection (MGB)National Institute of Public Health

and the Environment (RIVM)P.O. Box 1

3720 BA Bilthoven, The Netherlands

E-mail: wim.van.der.poel@rivm.nl

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