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European Bat Lyssavirus Infection in Spanish Bat Populations

European Bat Lyssavirus Infection in Spanish Bat Populations RESEARCH European Bat Lyssavirus Infection in Spanish Bat Populations Jordi Serra-Cobo,* Blanca Amengual,† Carlos Abellán,‡ and Hervé Bourhy† From 1992 to 2000, 976 sera, 27 blood pellets, and 91 brains were obtained from 14 bat species in 37 localities in Spain. Specific anti-European bat lyssavirus 1 (EBL1)-neutralizing antibodies have been detected in Myotis myotis, Miniopterus schreibersii, Tadarida teniotis, and Rhinolophus ferrumequinum in the region of Aragon and the Balearic Islands. Positive results were also obtained by nested reverse tran- scription-polymerase chain reaction on brain, blood pellet, lung, heart, tongue, and esophagus-larynx- pharynx of M. myotis, Myotis nattereri, R. ferrumequinum, and M. schreibersii. Determination of nucleotide sequence confirmed the presence of EBL1 RNA in the different tissues. In one colony, the prevalence of seropositive bats over time corresponded to an asymmetrical curve, with a sudden initial increase peaking at 60% of the bats, followed by a gradual decline. Banded seropositive bats were recovered during several years, indicating that EBL1 infection in these bats was nonlethal. At least one of this species (M. schreiber- sii) is migratory and thus could be partially responsible for the dissemination of EBL1 on both shores of the Mediterranean Sea. abies is a worldwide zoonosis due to Lyssavirus infec- of European insectivorous bats compared with the terminal tion; multiple host species act as reservoirs. This disease infection commonly associated with rabies infection. infects the central nervous system of humans and other mam- To investigate these observations, a 9-year study was mals. Bats are no exception, as proved by the 630 positive undertaken in Spain to locate and determine the colonies and cases detected in Europe from 1977 to 2000 (1,2). Recent species of bats carrying EBL or Lyssavirus antibodies, monitor molecular studies have shown genetic differentiation in lys- the prevalence of seropositive bats, and characterize circulat- saviruses that cause rabies among European bats, leading to a ing lyssaviruses. classification into two new genotypes, 5 and 6, which corre- spond to European bat lyssavirus 1 (EBL1) and EBL2, respec- Material and Methods tively (3,4). As a result of a recent molecular study, two new Selection of Bat Colonies and Banding lineages within genotype 5 have been identified—EBL1a and EBL1b; the latter is potentially of African origin, which sug- The study area consisted mainly of the Spanish Autono- gests south-to-north transmission (5). However, despite molec- mous Regions of Aragon, Balearic Islands, Catalonia, and ular advances and many European cases verified to date, knowledge of the prevalence and epidemiology of EBL is lim- ited. Of the 30 insectivorous bat species present in Europe, approximately 95% of cases occur in the species Eptesicus serotinus (2). This species, which is nonmigratory, cannot be linked to all the different foci of positive cases in Europe (6). In Spain, the first case of bat lyssaviruses was recorded in 1987 in Valencia. Sixteen more cases were reported in E. serotinus (7). The distribution of positive cases in Spain is indicated in Figure 1. Recently, clinically silent rabies infection has been reported in zoo bats (Rousettus aegyptiacus) in Denmark and the Netherlands (8). This observation, together with the results of an experimental challenge, suggests that this frugivorous bat species of African origin can survive EBL1 infection or inoculation (9). Silent infection has also been described in the American bat (Tadarida brasiliensis mexicana) (10,11) and Figure 1. Map showing the localities in Spain where bats have been suggests an alternative viral strategy for Lyssavirus infection analyzed. 1. Ciutadella; 2. El Saler; 3. Ferreries; 4. Inca; 5. Llucmajor; 6. Oliete; 7. Pollença; 8. Granada; 9. Huelva; 10. Sevilla. Points in red indicate colonies where positive results were obtained according to our *Universitat de Barcelona, Barcelona, Spain; †Institut Pasteur, Paris, study (Localities Nos. 1, 3, 4, 5, 6, and 7) and previous studies (Locali- France; and ‡Ministerio de Sanidad y Consumo, Madrid, Spain ties Nos. 2, 8, 9, and 10) (7). Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 413 RESEARCH Valencia (Figure 1) (12-15). The region of Ceuta (North citizens. Dead bats found in the studied refuges were also Africa, near the Straits of Gibraltar) was also studied because gathered. The bats found dead from 1994 to 1996 were ana- of its proximity to Europe. Bat colonies were selected accord- lyzed by direct immunofluorescence technique (17,18). The ing to the following criteria: colony behavior (anthropophilic, bats found dead from 1997 to 2000 were analyzed by nested migratory, gregarious) and proximity of the colonies to urban reverse transcription-polymerase chain reaction (RT-PCR) (9). areas. The Valencia bat colony was widely sampled because To eliminate cross-contamination at necropsy, sterilized instru- the first case of bat Lyssavirus in Spain was reported there (7). ments were used. Colonies exhibiting positive sera were more intensively Detection of EBL Antigens explored during the years after the first detection. From 1996 to 2000, bats from the locations Nos. 4, 5, and 7 were banded The standard fluorescent antibody test (FAT) was per- in the forearm to facilitate monitoring of their movements formed on brain tissue specimens of the bats by using the poly- between colonies (16). clonal fluorescein isothiocyanate-labeled rabbit anti-rabies nucleocapsid immunoglobulin G, as described by the manufac- Blood Sampling turer (Bio-Rad). Brain smears obtained from noninfected and To draw blood, we set the bat face upward with a stretched CVS-infected mice were incorporated as controls in each FAT wing. The patagium was wiped clean and locally disinfected test run. with a sanitary towel soaked in 96% alcohol to prevent infec- Detection of EBL1 RNA tions. Immediately afterwards, a small puncture was made next to the radius proximal epiphysis. Blood was collected in Total RNA was extracted from tissue samples (50 mg -100 an Eppendorf vial by using a Pasteur pipette. The amount of mg) by using the TRIzol method (Invitrogen, Groningen, the blood sampled varied from 0.2 mL to 0.5 mL, according to the Netherlands), purified with chloroform and precipitated with size of the animal. A sterilized absorbent hemostatic sponge iso-propanol (Merck, Darmstadt, Germany). After being impregnated with gelatin was administered to prevent bleeding washed with 70% ethanol, the RNA pellet was dried, resus- and facilitate healing. Pressure was applied to the wound with pended in a volume of 50 mL bidistilled water and stored at a sanitary towel for 30 seconds. The bats were given 10% glu- –20ºC. cDNA synthesis of the genomic and antigenomic sense cose water to drink to prevent dehydration and provide rapidly of the EBL1a nucleoprotein RNA was performed by anneal- assimilated compounds for energy. Once bleeding ceased, the ing, at 70ºC for 3 minutes, 2 mL of total RNA extract with 15 bat was released. Vials containing blood were stored at 4ºC for pmol of primers N60 (5'-TCCATAATCAGCTGGTCTCG-3', a few hours. Samples were centrifuged for 20 minutes at 5,000 positions 98-117, relative to rabies genome) (19) and N41, as rpm, and the serum was extracted with a pipette. Serum sam- described previously (5). ples and blood pellets were stored at –20°C. Amplification of 5 mL of the cDNA template was per- formed in a final volume of 50 mL containing 1x magnesium- Detection of EBL Antibodies free PCR buffer (Invitrogen), 5 mM deoxynucleoside triphos- The technique used for the detection of EBL antibodies is phate (NTP) mix (containing 1.25 mM each of dATP, dCTP, an adaptation of the Rapid Fluorescent Focus Inhibition Test dGTP, and dTTP), 5 mM magnesium chloride (Invitrogen), 2 (17). A constant dose of a previously titrated, cell culture- U Taq DNA polymerase (Invitrogen), and 30 pmol of primers adapted EBL1 challenge virus 8918FRA (5) was incubated N60 and N41. The amplification was performed on a Gene- with threefold dilutions of the sera to be titrated. After incuba- Amp PCR System 9700 Thermal cycler. The program started tion of the serum/virus mixtures, a suspension of BSR (a clone with one denaturation step at 94ºC for 5 minutes, followed by of BHK-21) cells was added. After 24 hours’ incubation, the 30 cycles of 94ºC for 30 sec, 60ºC for 30 sec, and 72ºC for 40 cell monolayer was acetone-fixed and stained with a fluores- sec. The amplification was finalized by an ultimate elongation cent anti-nucleocapsid antibody (Bio-Rad, Marnes-la- step at 72ºC for 5 min. The primary amplification products Coquette, France) to detect the presence of non-neutralized were stored at –20ºC. For nested RT-PCR, the amplified prod- virus (fluorescent foci). Titers are presented as an arithmetic uct was diluted 10 times in distilled water. Then the second mean of two independent repetitions. Serum samples with amplification was performed as described above with the fol- antibody titers <27 are considered negative for EBL1-neutral- lowing modifications: 30 pmol of primers N62 and N63 (N62: izing antibodies. The percentages of seropositive bats and the 5'-AAACCAAGCATCACTCTCGG-3', position 181-200; years in which bats were analyzed (from 1996 to 2000) were N63: 5'-ACTAGTCCAATCTTCCGGGC-3', position 342-323 correlated, and regression curves were obtained. To confirm relative to the Rabies virus genome) (19) were used, and the the specificity of the reaction, the same test was performed on elongation steps were performed at 72ºC for 30 sec. Aliquots selected sera by using the challenge virus strain (CVS) (17) (5 µL) of nRT-PCR products were analyzed by horizontal aga- and 9007FIN EBL2 challenge viruses (5). rose (1.5%) gel electrophoresis. Gels were stained with 1 µg/ mL ethidium bromide and photographed under UV light. Brain Sampling Extraction of RNA was performed in a level-2 biosafety Brain samples were obtained from dead bats, submitted by laboratory. Then we prepared the template and RT-PCR mix 414 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 RESEARCH and added DNA to the mix with aerosol-resistant tips in two tions of the seroneutralization also reinforced the accuracy of different rooms. We also performed nRT-PCR on tissue RNA, our results. omitting reverse transcriptase. Positive (isolate no. 2002FRA) Throughout the 9-year study, 976 sera obtained from 14 and negative (H O) controls were incorporated into each of the bat species in 37 different locations were analyzed (Table 1); following steps: total RNA extraction, cDNA synthesis, and 76 (7.8%) were positive (Table 2). Lyssavirus antibodies were each of the two steps of the amplification program. To avoid detected in four bat species (Myotis myotis, Miniopterus false-positive results, usual precautions for PCR were strictly schreibersii, Tadarida teniotis, and Rhinolophus ferrumequi- followed in the laboratory (20,21). num). Sixteen positive sera and 5 negative sera against EBL1 The threshold of detection of the nRT-PCR method was (genotype 5 of lyssaviruses) were further tested against stan- determined by preparing 10-fold dilutions of a pretitrated sus- dard strains of genotypes 1 (CVS), and 6 (EBL2). These sera pension of Strain 8918FRA (4) in TRIzol (GIBCO-BRL). were obtained from the four EBL1-seropositive bat species Total RNA extraction, cDNA synthesis, and the RT-PCR pro- and from another bat species that remained negative (R. eury- cedures were performed as described above. ale). None of them reacted positively against CVS and EBL2, Sequencing of amplified products was performed by using confirming the specificity of the positive reactions against the primers N62 and N63 and an Applied Biosystems 373A EBL1 obtained in these species (Table 3). sequencer (Foster City, CA), according to the Applied Biosys- The highest percentages of seropositive bats, 22.7% and tems protocol. Multiple sequence alignments were generated 20.8%, were observed in the Balearic Islands in the locations with the Clustal W 1.60 program (22). of Inca (No. 4) and Llucmajor (No. 5), respectively (Table 2). From spring to autumn, location No. 4 shelters a plurispecific Results colony of approximately 1,000 bats belonging to the following species: M. myotis (25% of seropositives), M. schreibersii, R. Presence of EBL1 Antibodies in Six Bat Colonies ferrumequinum, M. capaccinii, and M. nattereri. At the begin- We describe here a very efficient technique of blood col- ning of summer, M. myotis, M. nattereri, and M. schreibersii lection, which is more humane than collection by cardiac species form breeding pairs. Location No. 5 shelters a sum- puncture (23,24). The bats recaptured 1 week after the blood mer-breeding colony of approximately 500 bats of the species extraction did not show any trace of a scar. Furthermore, our M. myotis (22.5% of seropositives), M. schreibersii (7.1% of technique is easier than collection by puncture of the uropat- seropositives), and M. capaccinii. In both sites the most abun- agium or the propatagium cardiac veins (25). To eliminate any dant species is M. myotis. false- or doubtful positive reactions in seroneutralization, the Seropositive bats were also found in four other locations, threshold of positivity (titer=27) was chosen higher than the Nos. 1, 3, 6, and 7. Location No. 1 (5.5% of seropositive R. fer- one adopted by other authors (24). Two independent repeti- rumequinum) shelters a breeding colony of R. ferrumequinum. Table 1. Number of bat samples analyzed per species, 1992–2000 Species 1992 1993 1994 1995 1996 1997 1998 1999 2000 Total R. ferrumequinum 8 9/3 11/1 30/3 58/7 R. euryale 610 16 R. hipposideros 16 0/1 16/1 P. pipistrellus 61 64 75 18 13/5 0/16 0/14 3/15 234/50 P. kulhii 11 E. serotinus 21 44 33/1 1 99/1 M. myotis 1 63 65/2 44 29/2 58/8 35/3 295/15 M. blythi 20 1 2 23 M. nattereri 1 0/1 0/1 1/2 M. capaccinii 33 M. emarginatus 9 7/2 16/2 P. austriacus 3 6 2/4 1 12/4 Mi. schreibersii 8 18 14 8 9/2 70/6 41/1 168/9 T. teniotis 22 12 34 Total 90 118 123 148/1 127/11 83/22 38/19 143/30 106/8 976/91 Where fractions (x/y) are shown, the numerator (x) corresponds to the number of sera analyzed and the denominator (y) to the number of brains analyzed. E = Eptesicus; M = Myotis; Mi=Miniopterus P = Plecotus; R = Rhinolophus; T = Tadarida. Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 415 RESEARCH Table 2. Positive serologic results in bat populations, the Spanish Autonomous Regions of Balearic Islands and Aragon, 1995–2000 Years Location and coordinates Variables analyzed 1995 1996 1997 1998 1999 2000 No. 1 A/B - - 1/5 - 0/11 1/20 X±SD -- 515 - - 34 39°58’N,3°58’E Species Rf Rf Rf Rf Rf Rf No. 3 A/B 1/34 0/31 X±SD 215 39°58’N,3°59’E Species Ms Ms Ms Ms Ms Ms No. 4 A/B 1/30 16/27 11/27 7/22 3/30 3/29 X±SD 90 348±237 191±225 718±657 78±27 58±42 39°44’N,2°58’E Range 49–908 29-783 79-1677 47-95 29–107 Species Mm Mm Mm Mm Mm Mm No. 5 A/B 7/21 7/32 3/17 0/6 3/7 1/8 5/28 0/6 X±SD 122±45 207±159 218±136 412±454 8,508 106±61 39°25’N,2°55’E Range 83-195 53-442 129-374 87-930 29-176 Species Mm Mm Mm Ms Mm Ms Mm Mm No. 6 A/B 0/22 2/12 X±SD 243±284 41°01’N,0°39’W Range 420-444 Species Tt Tt Tt Tt Tt Tt No. 7 A/B 2/14 2/19 X±SD 93±68 35±6 39°50’N,3°00’E Range 45-141 31-40 Species Ms Ms Ms Ms Ms Ms A = no. bats positive, B = no. bats analyzed. X = seroneutralization average; SD = standard deviation. Species analyzed: Rf = Rhinolophus ferrumequinum; Ms = Miniopterus schreibersii; Mm = Myotis myotis; Tt = Tadarida teniotis. In spring, the colony also includes some M. schreibersii. Loca- remained stable in 2000. The percentage of seropositive bats tion No. 3 (2.9% of seropositive M. schreibersii) is a hiberna- remained stable in Location No. 5 from 1995 to 2000. tion refuge for approximately 2,200 M. schreibersii; some M. Exchange of Animals Between Colonies capaccinii are also present. Location No. 6 (5.8% of seroposi- and Survival of Seropositive Bats tive T. teniotis) is a big sinkhole with a resident bat colony belonging to the following species: T. teniotis, M. blythii, M. During the period 1996-2000, 355 and 87 M. myotis were daubentonii, Pipistrellus pipistrellus, Pipistrellus kuhlii, Hyp- banded in Locations Nos. 4 and 5, respectively (Table 4). sugo savii, E. serotinus, Plecotus austriacus, and Barbastella Recapture of the banded M. myotis allowed us to prove a few barbastellus (26). Location No. 7 (12% of seropositive M. exchange of bats between the colonies. Two percent of M. schreibersii) shelters a colony of M. schreibersii, M. capacci- myotis banded in Location No. 5 moved to Location No. 4 (the nii, and M. myotis. refuges are about 35 km apart). During the same period, 13 and 33 M. schreibersii were banded in Locations Nos. 5 and 7, Evolution of the Percentage of Seropositive respectively. One of the 33 M. schreibersii moved to Location Bats in Colonies Nos. 4 and 5 No. 5 (the refuges are approximately 47 km apart); another In Location 4, the percentage of seropositive bats rose moved to Location No. 4 (a distance of 11 km) (Figure 1). from 3.3% in 1995 to 59.3% in 1996 (Table 2). Then it Banding also allowed us to follow the seroneutralization decreased significantly (Y=-15.6X + 31,196.5, r=-0.989, titer of some bats during the study period. The serum of a M. p<0.05) until 1999, when it reached 10%. This percentage schreibersii captured in Location No. 7 in 1996 was negative; 416 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 RESEARCH Table 3. Specificity of results from serologic studies of bat popula- two R. ferrumequinum [No. 123 and No. 135], whose brains tions, Spanish Autonomous Regions of Balearic Islands and Aragon, were negative) were completely necropsied. Various organs 1995–2000 and tissues (medulla, liver, kidney, spleen, heart, tongue, Location Species CVS EBL1 EBL2 esophagus-larynx-pharynx, and lung) were collected and sub- No. 1 Rhinolophus 0 51 ND jected to nRT-PCR. Esophagus-larynx-pharynx and lung of bat ferrumequinum 0 <27 ND No. 135 and tongue, lung, and heart of bat No. 128 were posi- No. 4 Myotis myotis 0 588 <27 tive (Figure 2). 0 222 <27 Twenty-seven blood pellets of bats collected in 2000 were 0 350 <27 0 246 <27 also analyzed by nRT-PCR. These samples were obtained from 0 709 <27 8 R. ferrumequinum (location No. 1), 1 R. ferrumequinum 0 <27 ND 0 537 <27 (Location No. 3), 1 M. myotis (Location No. 5), 14 M. myotis 0 95 <27 (Location No. 4), and 3 M. schreibersii (Location No. 4). The No. 5 M. myotis 0 53 <27 blood pellets of three M. myotis from Location No. 4 were ND 421 <27 0 97 <27 found positive by nRT-PCR. None of the blood samples show- 0 188 <27 ing positive RT-PCR results on the pellet were found positive No. 6 Tadarida teniotis 0 42 ND by seroneutralization. 0 444 ND The threshold of detection of the nRT-PCR for the amplifi- 0 <27 ND cation of the EBL1a genomic and antigenomic RNAs of the N No. 7 Miniopterus schreibersii 0 45 <27 -2 gene was 5 x 10 fluorescent forming units of EBL1a/mL. In 0 141 <27 0 <27 <27 all these experiments, negative controls performed individu- ally for each step (extraction, RT, primary, and secondary Rhinolophus euryale 0<27 <27 PCR) were negative. Furthermore, nRT-PCR performed on ND = not done; CVS = challenge virus strain; EBL1 = European bat lys- savirus 1. positive tissues without previous reverse transcription gave negative results, demonstrating the absence of complementary another serologic sample obtained from the same bat 2 years DNA contamination. later in Location No. 5 yielded a titer of 8,508. During spring Nucleotide (nt) sequences were determined by using the 2000, 12 M. myotis previously banded and analyzed were positive nRT-PCR products obtained from the four brains and recaptured in Location No. 4. Four (33%) of them had already from one blood sample. These 122-nt long sequences of the been shown to be seropositive in preceding years: two in sum- nucleoprotein gene were strictly similar to the sequence of two mer 1997 (titers 29 and 145, respectively), one in summer EBL1b Spanish isolates (94285SPA and 9483 SPA) described 1998 (titer 303), and one in summer 1999 (titer 95). This indi- previously (5), except that the sequence obtained from the pos- cates that some seropositive bats may survive at least 3 years itive blood pellet exhibited a TJ A mutation in position 145 after Lyssavirus infection. of the coding region of the nucleoprotein gene. Four mutations distinguished the sequence of the positive control correspond- Detection and Characterization of EBL1 RNA in Bats ing to a French bat (No. 2002FRA) from the different During 1995 through 1996, 12 brain samples were only sequences obtained from Spanish bats (not shown). This fur- analyzed by FAT. After 1996, the brain samples (n=79) were ther confirms the specificity of the products amplified from the also analyzed by nested RT-PCR (Table 1). All brains (n=91) Spanish bat samples. analyzed by FAT were negative. In contrast, brains of 1 M. myotis, 1 M. nattereri, and 1 M. schreibersii (No. 140) of Discussion Location No. 4 and 1 R. ferrumequinum (No. 128) of Location This is the first report of the presence of EBL1-specific neu- No. 1 (all collected in 2000) were positive by nested RT-PCR. tralizing antibodies in four European insectivorous bat species Four animals (M. schreibersii [No. 140] and R. ferrumequinum (M. myotis, M. schreibersii, T. teniotis, and R. ferrumequi- [No. 128], whose brains were positive by nested RT-PCR, and num). These findings lead to the following observations on the circulation and possible bat species involved in the dispersion Table 4. No. of recaptured and analyzed bats in Locations 4, 5, and 7, of EBL1 in southern Europe. First, the identification of EBL1 Spain, 1996–2000 antibodies in 24% of the M. myotis analyzed in Locations No. a b c d e f Species BB BA BR BRD ATT 4 and No. 5 in 1995 through 2000 (n=276) indicates that bats of this genus are infected with EBL1. Second, the distribution Mm 442 221 25 2 4 of T. teniotis and M. schreibersii in southern Europe and north- Ms 46 46 0 2 1 ern Africa (13,27) could contribute to the dispersion of EBL1 Species studied: Mm = Myotis myotis; Ms = Miniopterus schreibersii. in southern Europe and is concordant with the possible African BB = No.of bats banded. BA = No. of bats banded and analyzed. origin of EBL1, as suggested by Amengual et al. (5). BR = No. of bats banded and recaptured in the same location. Although the seasonal movements of T. teniotis are BRD = No. of bats banded and recaptured in different localities. ATT = No. of bats analyzed twice at interval of >1 year. scarcely known, the quick, straight flight of this species Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 417 RESEARCH Figure 2. Detection of European bat Lyssavirus 1 RNA in bats by nested reverse transcription-polymerase chain reaction (PCR). Lanes: 1, brain of Miniopterus schreibersii No. 140; 2- 5, medulla, tongue, esophagus-larynx-pharynx, and lung of Rhinolophus ferrumequinum No. 135, respectively; 6-14, brain, medulla, esophagus-larynx-pharynx, liver, lung, heart, tongue, spleen, and kidney of R. ferrumequinum No. 128, respectively; 15, neg- ative control of RNA extraction of bat No. 135; 16, negative control of RNA extraction of bat No. 128; 17, negative control of RNA extraction of bat No. 140; 18, positive control; 19, negative control of first PCR; 20, negative control of second PCR. suggests that such movements are long, as is the case with the always exists among bats must facilitate viral transmission and American bat (T. brasiliensis mexicana), which is capable of antibody development. A high seropositive percentage also performing annual migrations of more than 1,000 km. Since occurs in colonies of T. brasiliensis mexicana, where percent- M. schreibersii makes seasonal migrations (some of them ages >80% have been observed (10,11). The transmission of >350 km) (16), this species could also be one of the dispersion lyssaviruses between bats from mixed colonies could take vectors of the disease in southern Europe, where it abounds. place through breathing or biting but is currently not M. schreibersii dwells in five out of the six sites where seropos- documented. itive bats have been found. In three of them, M. schreibersii The low prevalence (0 of 91, <1.1%) of active infection as forms mixed colonies with M. myotis, in one it shelters next to determined by FAT is concordant with previous results R. ferrumequinum, and in the fifth it shelters alone. M. obtained in America, which show a prevalence of active rabies schreibersii and M. myotis have direct physical contact in the infection in bats between 0.1 and 2.9% (10,28,29). However, mixed colonies. However, it is unlikely that Pipistrellus we report the first detection of EBL1 RNA by nRT-PCR in nathusii is a dispersion vector of the lyssaviruses in Spain, as several tissues (brain, blood pellet, lung, heart, tongue, and Brosset (6) suggests, since this is a very rare bat in the Iberian esophagus-larynx-pharynx) of four M. myotis, one M. nattereri, Peninsula. one M. schreibersii, and two R. ferrumequinum. These isolates The results obtained in 1995-2000 in Location No. 4 show show the existence of a low or nonproductive infection in these that the evolution in the number of seropositive bats after a species, although some small remnant of RNA remaining in a Lyssavirus infection corresponded to an asymmetrical curve, clinically normal bat as a result of an earlier nonlethal exposure with a sudden initial increase reaching more than 60% of the to a Lyssavirus is also possible. This low amount of viral DNA colony and a gradual decline over subsequent years (24)— present in the tissues underscores the need to use nRT-PCR as a unless a new episode took place (Figure 3). Because of the gre- very sensitive technique for epidemiologic studies of EBL1 in garious behavior of this species, a quick increase and a high bat populations. Rønsholt et al. (8) also comment on the diffi- seropositive percentage (almost 60% in this location) after a culty of detecting Lyssavirus infection by immunofluorescence Lyssavirus episode are not unusual. The intimate contact that in bats when a clinically silent infection exists. EBL1 are known to actively infect the brain, lung, and tongue of E. serotinus (3). However, this is the first report that EBL1 RNA can be detected in various organs and tissues in the absence of active infection, as demonstrated by negative results obtained by FAT. Most of these bats were dead when collected but were kept in conditions that allowed the classic diagnosis by FAT to be performed properly. These negative FAT results indicate that these bats died of causes other than their low productive Lyssavirus infection. The recapture of seropositive bats over several years also shows that some of these bats survived EBL1 infection. The detection of EBL1b sequences in the blood pellet of bats (3/27) is also a new find- ing. This technique would be an easy test for screening posi- tive bats. However, further studies are needed to establish the interest and sensitivity of this sample. The sensitivity of the different European bat species to EBL infection probably varies according to the animal and Figure 3. Incidence of seropositive bats observed in Myotis myotis col- virus species involved. Therefore, we have summarized in onies, Spanish Locations No. 4 and No. 5, 1995–2000 (95% confi- dence intervals shown). 418 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 RESEARCH of Spanish bat lyssaviruses for the Ministerio de Sanidad y Consumo Table 5. Bat species positive for Lyssavirus, Europe, 1954–2000 and the Conselleria de Sanitat of the Balearic Autonomous b c Family Species Lyssavirus Antibodies Government. Vespertilionidae Eptesicus serotinus EBL1a & b EBL1 Pipistrellus pipistrellus NC ND References 1. Kuzmin IV, Botvinkin AD. The behaviour of bats Pipistrellus pipistrellus Pipistrellus nathusii NC ND after experimental inoculation with rabies and rabies-like viruses and Vespertilio murinus EBL1a ND some aspects of the pathogenesis. Myotis 1996;34:93-9. 2. Muller WW. Review of reported rabies cases data in Europe to the WHO Myotis dasycneme EBL2a ND Collaborative Centre Tübingen from 1977 to 2000. Rabies Bulletin Myotis daubentonii EBL2a & b ND Europe 2000;24:11-19. 3. Bourhy H, Kissi B, Lafon M, Sacramento D, Tordo N. Antigenic and Myotis myotis EBL1b EBL1 molecular characterization of bat rabies virus in Europe. J Clin Microbiol Myotis nattereri EBL1b ND 1992;30:2419-26. 4. Bourhy H, Kissi B, Tordo N. Molecular diversity of the lyssavirus genus. Nyctalus noctula NC ND Virology 1993;194:70-81. Miniopterus schreibersii EBL1b EBL1 5. Amengual B, Whitby JE, King A, Serra-Cobo J, Bourhy H. Evolution of European bat lyssaviruses. J Gen Virol 1997;78:2319-28. Molossidae Tadarida teniotis NC EBL1 6. Brosset A. Les migrations de la pipistrelle de Nathusius, Pipistrellus Rhinolophidae Rhinolophus EBL1b EBL1 nathusii, en France. Ses incidences possibles sur la propagation de la ferrumequinum rage. Mammalia 1990;54:207-12. The additional information was obtained from Kappeler (29), Pérez-Jordá et al. (24), 7. Sánchez Serrano LP. Rabia transmitida por murciélagos insectívoros en Amengual et al. (5), Bulletin épidemiologique mensuel de la rage en France (30), and España. Boletín Epidemiológico Instituto de Salud Carlos III 1999;7:149- Muller (2). NC = not characterized. 8. Rønsholt L, Sorensen KJ, Bruschke CIM, Wellenberg GJ, Oirschot JT ND = not done. van, Johnstone P, et al. Clinical silent rabies infection in (zoo) bats. Vet Rec 1998;142:519-20. Table 5 (2,5,24,30,31) the bat species in which either Lyssavi- 9. Poel WHM van der, Heide R van der, Amerongen G van, Keulen LJM rus or antibodies against Lyssavirus have been detected. Fur- van, Bourhy H, Schaftenaar W, et al. Characterization of recently isolated lyssavirus in frugivorous zoo bats. Arch Virol 2000;145:1919-31. ther studies are needed to determine which of the European bat 10. Steece R, Altenbach IS. Prevalence of rabies specific antibodies in the species are the reservoir of EBL infection and if different spe- Mexican free-tailed bat (Tadarida brasiliensis mexicana) at Lava Cave, cies act as sentinels for the presence of the virus in the colony. New Mexico. J Wildl Dis 1989;25:490-6. The presence of EBL1 RNA and immunity to EBL1 in 11. Baer GM. The natural history of rabies. Boca Raton (FL): CRC Press; several wild bat colonies also has important implications for 1991. 12. Alcover A, Muntaner J. El registre quiropterològic de Les Balears i bat management and public health. The probability of humans’ Pitiuses: una revisió. Endins 1986;12:51-63. having contact with these colonies should be reduced and con- 13. Serra-Cobo J. Biological and ecological study of the Miniopterus trolled. In our study, most bat colonies were found in sites that schreibersii. [PhD thesis]. Barcelona: University of Barcelona; 1989. are frequently visited by speleologists, tourists, and bat-lovers. 14. Serra-Cobo J, Faus V. Nuevas citas y comentarios faunísticos sobre los As a consequence of our findings, the entry to these caves is quirópteros de la Comunidad Valenciana. Serie de Estudios Biológicos 1989;11:59-76. now controlled and limited during the periods when bats are 15. Serra-Cobo J, Amengual-Pieras B, Estrada-Peña A. Nuevos datos sobre present (in spring, summer, and autumn for Location No. 4). los quirópteros de Aragón. In: Alemany A, editor. Historia natural '91. Entry is limited by horizontal bars that allow the bats to fly Palma de Mallorca, Spain: Universitat Illes Balears; 1991. p. 229-36. across them but prevent access to people without obscuring the 16. Serra-Cobo J, Sanz-Trullén V, Martínez-Rica JP. Migratory movements view. of Miniopterus schreibersii in the north-east of Spain. Acta Theriologica 1998;43:271-83. 17. Bourhy H, Sureau P. Rapid fluorescent focus inhibition test (RFFIT). In: Acknowledgments Commission des Laboratories de Référence et d’Expertise, editor. Meth- We wish to acknowledge Josep Márquez, Catalina Massuti, Joan odes de laboratoire pour le diagnostique de la rage. Paris: Institut Pasteur; Oliver, and Antonia Sánchez for their cooperation and logistical sup- 1990. p. 191-3. port in the field work. 18. Bourhy H, Rollin PE, Vincent J, Sureau P. Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and The Spanish Ministerio de Sanidad y Consumo and the Conselle- enzyme immunodiagnosis for rapid laboratory diagnosis of rabies. J Clin ria de Sanitat I Consum (Govern de les Illes Balear) financed this Microbiol 1989;27:519-23. 19. Tordo N, Poch O, Ermine A, Keith G, Rougeon F. Walking along the study. rabies genome: is the large G-L intergenic region a remnant gene? Proc Jordi Serra-Cobo is a member of the Quality Research Team Natl Acad Sci U S A 1986;83:3914-18. 20. Crepin P, Audry L, Rotivel Y, Gacoin A, Caroff C, Bourhy H. Intravitam (Biology of Vertebrates, 96-SGR0072) of the Universitat de Barce- diagnosis of human rabies by PCR on saliva and cerebrospinal fluid. J lona and a contracted doctor by the Instituto Pirenaico de Ecología Clin Microbiol 1998;36:1117-21. (CSIC). His areas of expertise are vertebrates, population ecology, 21. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature and bat lyssaviruses. Since 1990 he has been working in the research 1989;339:237-8. Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 419 RESEARCH 22. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sen- 27. Aulagnier S, Thevenot M. Catalogue des Mammifères sauvages du sitivity of progressive multiple alignment through sequence weighting, Maroc. Rabat-Agdal, Morocco: Travaux de l’Institut Scientifique, Série position-specific gap penalties and weight matrix choice. Nucleic Acids Zoologique; 1986. Res 1994;22:4673-80. 28. Trimarchi CV, Debbie JG. Naturally occurring rabies virus and neutraliz- 23. La Motte LC. Japanese B encephalitis in bats during simulated hiberna- ing antibody in 2 species of insectivorous bats of New York State USA. J tion. Am J Hyg 1958;67: 101-8. Wildl Dis 1977;13:366-9. 24. Pérez-Jordá JL, Ibañez C, Muñoz-Cervera M, Téllez A. Lyssavirus in 29. Pybus MJ. Rabies in insectivorous bats of Western Canada, 1979 to 1983. Eptesicus serotinus (Chiroptera: Vespertilionidae). J Wildl Dis J Wildl Dis 1986;22:303-13. 1995;31:372-7. 30. Kappeler A. Bat rabies surveillance in Europe. Rabies Bulletin Europe 25. Kunz TH, Nagy K. Methods of energy budget analysis. In: Kunz TH, edi- 1989;13:12-13. tor. Ecological and behavioral methods for the study of bats. Washington: 31. Bruyére V, Janot C. La France bientôt indemne de rage. Bulletin Epidémi- Smithsonian Institution Press; 1988. p. 277-302. ologique Mensuel de la Rage en France 2000;30:1-7. 26. Serra-Cobo J, Barbault R, Estrada-Peña A. Le gouffre de San Pedro de los Griegos (Oliete, Teruel, Espagne) un refuge de biodiversité sans équiva- lent en Europe. Revue Ecologie (Terre Vie) 1993;48:341-8. Address for correspondence: Jordi Serra-Cobo, Departament de Biologia Ani- mal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain; fax: 34-93-403-57-40; e-mail: [email protected] 420 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Emerging Infectious Diseases Pubmed Central

European Bat Lyssavirus Infection in Spanish Bat Populations

Serra-Cobo, Jordi; Amengual, Blanca; Abellán, Carlos; Bourhy, Hervé
Emerging Infectious Diseases , Volume 8 (4) – Apr 1, 2002
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Abstract

RESEARCH European Bat Lyssavirus Infection in Spanish Bat Populations Jordi Serra-Cobo,* Blanca Amengual,† Carlos Abellán,‡ and Hervé Bourhy† From 1992 to 2000, 976 sera, 27 blood pellets, and 91 brains were obtained from 14 bat species in 37 localities in Spain. Specific anti-European bat lyssavirus 1 (EBL1)-neutralizing antibodies have been detected in Myotis myotis, Miniopterus schreibersii, Tadarida teniotis, and Rhinolophus ferrumequinum in the region of Aragon and the Balearic Islands. Positive results were also obtained by nested reverse tran- scription-polymerase chain reaction on brain, blood pellet, lung, heart, tongue, and esophagus-larynx- pharynx of M. myotis, Myotis nattereri, R. ferrumequinum, and M. schreibersii. Determination of nucleotide sequence confirmed the presence of EBL1 RNA in the different tissues. In one colony, the prevalence of seropositive bats over time corresponded to an asymmetrical curve, with a sudden initial increase peaking at 60% of the bats, followed by a gradual decline. Banded seropositive bats were recovered during several years, indicating that EBL1 infection in these bats was nonlethal. At least one of this species (M. schreiber- sii) is migratory and thus could be partially responsible for the dissemination of EBL1 on both shores of the Mediterranean Sea. abies is a worldwide zoonosis due to Lyssavirus infec- of European insectivorous bats compared with the terminal tion; multiple host species act as reservoirs. This disease infection commonly associated with rabies infection. infects the central nervous system of humans and other mam- To investigate these observations, a 9-year study was mals. Bats are no exception, as proved by the 630 positive undertaken in Spain to locate and determine the colonies and cases detected in Europe from 1977 to 2000 (1,2). Recent species of bats carrying EBL or Lyssavirus antibodies, monitor molecular studies have shown genetic differentiation in lys- the prevalence of seropositive bats, and characterize circulat- saviruses that cause rabies among European bats, leading to a ing lyssaviruses. classification into two new genotypes, 5 and 6, which corre- spond to European bat lyssavirus 1 (EBL1) and EBL2, respec- Material and Methods tively (3,4). As a result of a recent molecular study, two new Selection of Bat Colonies and Banding lineages within genotype 5 have been identified—EBL1a and EBL1b; the latter is potentially of African origin, which sug- The study area consisted mainly of the Spanish Autono- gests south-to-north transmission (5). However, despite molec- mous Regions of Aragon, Balearic Islands, Catalonia, and ular advances and many European cases verified to date, knowledge of the prevalence and epidemiology of EBL is lim- ited. Of the 30 insectivorous bat species present in Europe, approximately 95% of cases occur in the species Eptesicus serotinus (2). This species, which is nonmigratory, cannot be linked to all the different foci of positive cases in Europe (6). In Spain, the first case of bat lyssaviruses was recorded in 1987 in Valencia. Sixteen more cases were reported in E. serotinus (7). The distribution of positive cases in Spain is indicated in Figure 1. Recently, clinically silent rabies infection has been reported in zoo bats (Rousettus aegyptiacus) in Denmark and the Netherlands (8). This observation, together with the results of an experimental challenge, suggests that this frugivorous bat species of African origin can survive EBL1 infection or inoculation (9). Silent infection has also been described in the American bat (Tadarida brasiliensis mexicana) (10,11) and Figure 1. Map showing the localities in Spain where bats have been suggests an alternative viral strategy for Lyssavirus infection analyzed. 1. Ciutadella; 2. El Saler; 3. Ferreries; 4. Inca; 5. Llucmajor; 6. Oliete; 7. Pollença; 8. Granada; 9. Huelva; 10. Sevilla. Points in red indicate colonies where positive results were obtained according to our *Universitat de Barcelona, Barcelona, Spain; †Institut Pasteur, Paris, study (Localities Nos. 1, 3, 4, 5, 6, and 7) and previous studies (Locali- France; and ‡Ministerio de Sanidad y Consumo, Madrid, Spain ties Nos. 2, 8, 9, and 10) (7). Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 413 RESEARCH Valencia (Figure 1) (12-15). The region of Ceuta (North citizens. Dead bats found in the studied refuges were also Africa, near the Straits of Gibraltar) was also studied because gathered. The bats found dead from 1994 to 1996 were ana- of its proximity to Europe. Bat colonies were selected accord- lyzed by direct immunofluorescence technique (17,18). The ing to the following criteria: colony behavior (anthropophilic, bats found dead from 1997 to 2000 were analyzed by nested migratory, gregarious) and proximity of the colonies to urban reverse transcription-polymerase chain reaction (RT-PCR) (9). areas. The Valencia bat colony was widely sampled because To eliminate cross-contamination at necropsy, sterilized instru- the first case of bat Lyssavirus in Spain was reported there (7). ments were used. Colonies exhibiting positive sera were more intensively Detection of EBL Antigens explored during the years after the first detection. From 1996 to 2000, bats from the locations Nos. 4, 5, and 7 were banded The standard fluorescent antibody test (FAT) was per- in the forearm to facilitate monitoring of their movements formed on brain tissue specimens of the bats by using the poly- between colonies (16). clonal fluorescein isothiocyanate-labeled rabbit anti-rabies nucleocapsid immunoglobulin G, as described by the manufac- Blood Sampling turer (Bio-Rad). Brain smears obtained from noninfected and To draw blood, we set the bat face upward with a stretched CVS-infected mice were incorporated as controls in each FAT wing. The patagium was wiped clean and locally disinfected test run. with a sanitary towel soaked in 96% alcohol to prevent infec- Detection of EBL1 RNA tions. Immediately afterwards, a small puncture was made next to the radius proximal epiphysis. Blood was collected in Total RNA was extracted from tissue samples (50 mg -100 an Eppendorf vial by using a Pasteur pipette. The amount of mg) by using the TRIzol method (Invitrogen, Groningen, the blood sampled varied from 0.2 mL to 0.5 mL, according to the Netherlands), purified with chloroform and precipitated with size of the animal. A sterilized absorbent hemostatic sponge iso-propanol (Merck, Darmstadt, Germany). After being impregnated with gelatin was administered to prevent bleeding washed with 70% ethanol, the RNA pellet was dried, resus- and facilitate healing. Pressure was applied to the wound with pended in a volume of 50 mL bidistilled water and stored at a sanitary towel for 30 seconds. The bats were given 10% glu- –20ºC. cDNA synthesis of the genomic and antigenomic sense cose water to drink to prevent dehydration and provide rapidly of the EBL1a nucleoprotein RNA was performed by anneal- assimilated compounds for energy. Once bleeding ceased, the ing, at 70ºC for 3 minutes, 2 mL of total RNA extract with 15 bat was released. Vials containing blood were stored at 4ºC for pmol of primers N60 (5'-TCCATAATCAGCTGGTCTCG-3', a few hours. Samples were centrifuged for 20 minutes at 5,000 positions 98-117, relative to rabies genome) (19) and N41, as rpm, and the serum was extracted with a pipette. Serum sam- described previously (5). ples and blood pellets were stored at –20°C. Amplification of 5 mL of the cDNA template was per- formed in a final volume of 50 mL containing 1x magnesium- Detection of EBL Antibodies free PCR buffer (Invitrogen), 5 mM deoxynucleoside triphos- The technique used for the detection of EBL antibodies is phate (NTP) mix (containing 1.25 mM each of dATP, dCTP, an adaptation of the Rapid Fluorescent Focus Inhibition Test dGTP, and dTTP), 5 mM magnesium chloride (Invitrogen), 2 (17). A constant dose of a previously titrated, cell culture- U Taq DNA polymerase (Invitrogen), and 30 pmol of primers adapted EBL1 challenge virus 8918FRA (5) was incubated N60 and N41. The amplification was performed on a Gene- with threefold dilutions of the sera to be titrated. After incuba- Amp PCR System 9700 Thermal cycler. The program started tion of the serum/virus mixtures, a suspension of BSR (a clone with one denaturation step at 94ºC for 5 minutes, followed by of BHK-21) cells was added. After 24 hours’ incubation, the 30 cycles of 94ºC for 30 sec, 60ºC for 30 sec, and 72ºC for 40 cell monolayer was acetone-fixed and stained with a fluores- sec. The amplification was finalized by an ultimate elongation cent anti-nucleocapsid antibody (Bio-Rad, Marnes-la- step at 72ºC for 5 min. The primary amplification products Coquette, France) to detect the presence of non-neutralized were stored at –20ºC. For nested RT-PCR, the amplified prod- virus (fluorescent foci). Titers are presented as an arithmetic uct was diluted 10 times in distilled water. Then the second mean of two independent repetitions. Serum samples with amplification was performed as described above with the fol- antibody titers <27 are considered negative for EBL1-neutral- lowing modifications: 30 pmol of primers N62 and N63 (N62: izing antibodies. The percentages of seropositive bats and the 5'-AAACCAAGCATCACTCTCGG-3', position 181-200; years in which bats were analyzed (from 1996 to 2000) were N63: 5'-ACTAGTCCAATCTTCCGGGC-3', position 342-323 correlated, and regression curves were obtained. To confirm relative to the Rabies virus genome) (19) were used, and the the specificity of the reaction, the same test was performed on elongation steps were performed at 72ºC for 30 sec. Aliquots selected sera by using the challenge virus strain (CVS) (17) (5 µL) of nRT-PCR products were analyzed by horizontal aga- and 9007FIN EBL2 challenge viruses (5). rose (1.5%) gel electrophoresis. Gels were stained with 1 µg/ mL ethidium bromide and photographed under UV light. Brain Sampling Extraction of RNA was performed in a level-2 biosafety Brain samples were obtained from dead bats, submitted by laboratory. Then we prepared the template and RT-PCR mix 414 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 RESEARCH and added DNA to the mix with aerosol-resistant tips in two tions of the seroneutralization also reinforced the accuracy of different rooms. We also performed nRT-PCR on tissue RNA, our results. omitting reverse transcriptase. Positive (isolate no. 2002FRA) Throughout the 9-year study, 976 sera obtained from 14 and negative (H O) controls were incorporated into each of the bat species in 37 different locations were analyzed (Table 1); following steps: total RNA extraction, cDNA synthesis, and 76 (7.8%) were positive (Table 2). Lyssavirus antibodies were each of the two steps of the amplification program. To avoid detected in four bat species (Myotis myotis, Miniopterus false-positive results, usual precautions for PCR were strictly schreibersii, Tadarida teniotis, and Rhinolophus ferrumequi- followed in the laboratory (20,21). num). Sixteen positive sera and 5 negative sera against EBL1 The threshold of detection of the nRT-PCR method was (genotype 5 of lyssaviruses) were further tested against stan- determined by preparing 10-fold dilutions of a pretitrated sus- dard strains of genotypes 1 (CVS), and 6 (EBL2). These sera pension of Strain 8918FRA (4) in TRIzol (GIBCO-BRL). were obtained from the four EBL1-seropositive bat species Total RNA extraction, cDNA synthesis, and the RT-PCR pro- and from another bat species that remained negative (R. eury- cedures were performed as described above. ale). None of them reacted positively against CVS and EBL2, Sequencing of amplified products was performed by using confirming the specificity of the positive reactions against the primers N62 and N63 and an Applied Biosystems 373A EBL1 obtained in these species (Table 3). sequencer (Foster City, CA), according to the Applied Biosys- The highest percentages of seropositive bats, 22.7% and tems protocol. Multiple sequence alignments were generated 20.8%, were observed in the Balearic Islands in the locations with the Clustal W 1.60 program (22). of Inca (No. 4) and Llucmajor (No. 5), respectively (Table 2). From spring to autumn, location No. 4 shelters a plurispecific Results colony of approximately 1,000 bats belonging to the following species: M. myotis (25% of seropositives), M. schreibersii, R. Presence of EBL1 Antibodies in Six Bat Colonies ferrumequinum, M. capaccinii, and M. nattereri. At the begin- We describe here a very efficient technique of blood col- ning of summer, M. myotis, M. nattereri, and M. schreibersii lection, which is more humane than collection by cardiac species form breeding pairs. Location No. 5 shelters a sum- puncture (23,24). The bats recaptured 1 week after the blood mer-breeding colony of approximately 500 bats of the species extraction did not show any trace of a scar. Furthermore, our M. myotis (22.5% of seropositives), M. schreibersii (7.1% of technique is easier than collection by puncture of the uropat- seropositives), and M. capaccinii. In both sites the most abun- agium or the propatagium cardiac veins (25). To eliminate any dant species is M. myotis. false- or doubtful positive reactions in seroneutralization, the Seropositive bats were also found in four other locations, threshold of positivity (titer=27) was chosen higher than the Nos. 1, 3, 6, and 7. Location No. 1 (5.5% of seropositive R. fer- one adopted by other authors (24). Two independent repeti- rumequinum) shelters a breeding colony of R. ferrumequinum. Table 1. Number of bat samples analyzed per species, 1992–2000 Species 1992 1993 1994 1995 1996 1997 1998 1999 2000 Total R. ferrumequinum 8 9/3 11/1 30/3 58/7 R. euryale 610 16 R. hipposideros 16 0/1 16/1 P. pipistrellus 61 64 75 18 13/5 0/16 0/14 3/15 234/50 P. kulhii 11 E. serotinus 21 44 33/1 1 99/1 M. myotis 1 63 65/2 44 29/2 58/8 35/3 295/15 M. blythi 20 1 2 23 M. nattereri 1 0/1 0/1 1/2 M. capaccinii 33 M. emarginatus 9 7/2 16/2 P. austriacus 3 6 2/4 1 12/4 Mi. schreibersii 8 18 14 8 9/2 70/6 41/1 168/9 T. teniotis 22 12 34 Total 90 118 123 148/1 127/11 83/22 38/19 143/30 106/8 976/91 Where fractions (x/y) are shown, the numerator (x) corresponds to the number of sera analyzed and the denominator (y) to the number of brains analyzed. E = Eptesicus; M = Myotis; Mi=Miniopterus P = Plecotus; R = Rhinolophus; T = Tadarida. Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 415 RESEARCH Table 2. Positive serologic results in bat populations, the Spanish Autonomous Regions of Balearic Islands and Aragon, 1995–2000 Years Location and coordinates Variables analyzed 1995 1996 1997 1998 1999 2000 No. 1 A/B - - 1/5 - 0/11 1/20 X±SD -- 515 - - 34 39°58’N,3°58’E Species Rf Rf Rf Rf Rf Rf No. 3 A/B 1/34 0/31 X±SD 215 39°58’N,3°59’E Species Ms Ms Ms Ms Ms Ms No. 4 A/B 1/30 16/27 11/27 7/22 3/30 3/29 X±SD 90 348±237 191±225 718±657 78±27 58±42 39°44’N,2°58’E Range 49–908 29-783 79-1677 47-95 29–107 Species Mm Mm Mm Mm Mm Mm No. 5 A/B 7/21 7/32 3/17 0/6 3/7 1/8 5/28 0/6 X±SD 122±45 207±159 218±136 412±454 8,508 106±61 39°25’N,2°55’E Range 83-195 53-442 129-374 87-930 29-176 Species Mm Mm Mm Ms Mm Ms Mm Mm No. 6 A/B 0/22 2/12 X±SD 243±284 41°01’N,0°39’W Range 420-444 Species Tt Tt Tt Tt Tt Tt No. 7 A/B 2/14 2/19 X±SD 93±68 35±6 39°50’N,3°00’E Range 45-141 31-40 Species Ms Ms Ms Ms Ms Ms A = no. bats positive, B = no. bats analyzed. X = seroneutralization average; SD = standard deviation. Species analyzed: Rf = Rhinolophus ferrumequinum; Ms = Miniopterus schreibersii; Mm = Myotis myotis; Tt = Tadarida teniotis. In spring, the colony also includes some M. schreibersii. Loca- remained stable in 2000. The percentage of seropositive bats tion No. 3 (2.9% of seropositive M. schreibersii) is a hiberna- remained stable in Location No. 5 from 1995 to 2000. tion refuge for approximately 2,200 M. schreibersii; some M. Exchange of Animals Between Colonies capaccinii are also present. Location No. 6 (5.8% of seroposi- and Survival of Seropositive Bats tive T. teniotis) is a big sinkhole with a resident bat colony belonging to the following species: T. teniotis, M. blythii, M. During the period 1996-2000, 355 and 87 M. myotis were daubentonii, Pipistrellus pipistrellus, Pipistrellus kuhlii, Hyp- banded in Locations Nos. 4 and 5, respectively (Table 4). sugo savii, E. serotinus, Plecotus austriacus, and Barbastella Recapture of the banded M. myotis allowed us to prove a few barbastellus (26). Location No. 7 (12% of seropositive M. exchange of bats between the colonies. Two percent of M. schreibersii) shelters a colony of M. schreibersii, M. capacci- myotis banded in Location No. 5 moved to Location No. 4 (the nii, and M. myotis. refuges are about 35 km apart). During the same period, 13 and 33 M. schreibersii were banded in Locations Nos. 5 and 7, Evolution of the Percentage of Seropositive respectively. One of the 33 M. schreibersii moved to Location Bats in Colonies Nos. 4 and 5 No. 5 (the refuges are approximately 47 km apart); another In Location 4, the percentage of seropositive bats rose moved to Location No. 4 (a distance of 11 km) (Figure 1). from 3.3% in 1995 to 59.3% in 1996 (Table 2). Then it Banding also allowed us to follow the seroneutralization decreased significantly (Y=-15.6X + 31,196.5, r=-0.989, titer of some bats during the study period. The serum of a M. p<0.05) until 1999, when it reached 10%. This percentage schreibersii captured in Location No. 7 in 1996 was negative; 416 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 RESEARCH Table 3. Specificity of results from serologic studies of bat popula- two R. ferrumequinum [No. 123 and No. 135], whose brains tions, Spanish Autonomous Regions of Balearic Islands and Aragon, were negative) were completely necropsied. Various organs 1995–2000 and tissues (medulla, liver, kidney, spleen, heart, tongue, Location Species CVS EBL1 EBL2 esophagus-larynx-pharynx, and lung) were collected and sub- No. 1 Rhinolophus 0 51 ND jected to nRT-PCR. Esophagus-larynx-pharynx and lung of bat ferrumequinum 0 <27 ND No. 135 and tongue, lung, and heart of bat No. 128 were posi- No. 4 Myotis myotis 0 588 <27 tive (Figure 2). 0 222 <27 Twenty-seven blood pellets of bats collected in 2000 were 0 350 <27 0 246 <27 also analyzed by nRT-PCR. These samples were obtained from 0 709 <27 8 R. ferrumequinum (location No. 1), 1 R. ferrumequinum 0 <27 ND 0 537 <27 (Location No. 3), 1 M. myotis (Location No. 5), 14 M. myotis 0 95 <27 (Location No. 4), and 3 M. schreibersii (Location No. 4). The No. 5 M. myotis 0 53 <27 blood pellets of three M. myotis from Location No. 4 were ND 421 <27 0 97 <27 found positive by nRT-PCR. None of the blood samples show- 0 188 <27 ing positive RT-PCR results on the pellet were found positive No. 6 Tadarida teniotis 0 42 ND by seroneutralization. 0 444 ND The threshold of detection of the nRT-PCR for the amplifi- 0 <27 ND cation of the EBL1a genomic and antigenomic RNAs of the N No. 7 Miniopterus schreibersii 0 45 <27 -2 gene was 5 x 10 fluorescent forming units of EBL1a/mL. In 0 141 <27 0 <27 <27 all these experiments, negative controls performed individu- ally for each step (extraction, RT, primary, and secondary Rhinolophus euryale 0<27 <27 PCR) were negative. Furthermore, nRT-PCR performed on ND = not done; CVS = challenge virus strain; EBL1 = European bat lys- savirus 1. positive tissues without previous reverse transcription gave negative results, demonstrating the absence of complementary another serologic sample obtained from the same bat 2 years DNA contamination. later in Location No. 5 yielded a titer of 8,508. During spring Nucleotide (nt) sequences were determined by using the 2000, 12 M. myotis previously banded and analyzed were positive nRT-PCR products obtained from the four brains and recaptured in Location No. 4. Four (33%) of them had already from one blood sample. These 122-nt long sequences of the been shown to be seropositive in preceding years: two in sum- nucleoprotein gene were strictly similar to the sequence of two mer 1997 (titers 29 and 145, respectively), one in summer EBL1b Spanish isolates (94285SPA and 9483 SPA) described 1998 (titer 303), and one in summer 1999 (titer 95). This indi- previously (5), except that the sequence obtained from the pos- cates that some seropositive bats may survive at least 3 years itive blood pellet exhibited a TJ A mutation in position 145 after Lyssavirus infection. of the coding region of the nucleoprotein gene. Four mutations distinguished the sequence of the positive control correspond- Detection and Characterization of EBL1 RNA in Bats ing to a French bat (No. 2002FRA) from the different During 1995 through 1996, 12 brain samples were only sequences obtained from Spanish bats (not shown). This fur- analyzed by FAT. After 1996, the brain samples (n=79) were ther confirms the specificity of the products amplified from the also analyzed by nested RT-PCR (Table 1). All brains (n=91) Spanish bat samples. analyzed by FAT were negative. In contrast, brains of 1 M. myotis, 1 M. nattereri, and 1 M. schreibersii (No. 140) of Discussion Location No. 4 and 1 R. ferrumequinum (No. 128) of Location This is the first report of the presence of EBL1-specific neu- No. 1 (all collected in 2000) were positive by nested RT-PCR. tralizing antibodies in four European insectivorous bat species Four animals (M. schreibersii [No. 140] and R. ferrumequinum (M. myotis, M. schreibersii, T. teniotis, and R. ferrumequi- [No. 128], whose brains were positive by nested RT-PCR, and num). These findings lead to the following observations on the circulation and possible bat species involved in the dispersion Table 4. No. of recaptured and analyzed bats in Locations 4, 5, and 7, of EBL1 in southern Europe. First, the identification of EBL1 Spain, 1996–2000 antibodies in 24% of the M. myotis analyzed in Locations No. a b c d e f Species BB BA BR BRD ATT 4 and No. 5 in 1995 through 2000 (n=276) indicates that bats of this genus are infected with EBL1. Second, the distribution Mm 442 221 25 2 4 of T. teniotis and M. schreibersii in southern Europe and north- Ms 46 46 0 2 1 ern Africa (13,27) could contribute to the dispersion of EBL1 Species studied: Mm = Myotis myotis; Ms = Miniopterus schreibersii. in southern Europe and is concordant with the possible African BB = No.of bats banded. BA = No. of bats banded and analyzed. origin of EBL1, as suggested by Amengual et al. (5). BR = No. of bats banded and recaptured in the same location. Although the seasonal movements of T. teniotis are BRD = No. of bats banded and recaptured in different localities. ATT = No. of bats analyzed twice at interval of >1 year. scarcely known, the quick, straight flight of this species Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 417 RESEARCH Figure 2. Detection of European bat Lyssavirus 1 RNA in bats by nested reverse transcription-polymerase chain reaction (PCR). Lanes: 1, brain of Miniopterus schreibersii No. 140; 2- 5, medulla, tongue, esophagus-larynx-pharynx, and lung of Rhinolophus ferrumequinum No. 135, respectively; 6-14, brain, medulla, esophagus-larynx-pharynx, liver, lung, heart, tongue, spleen, and kidney of R. ferrumequinum No. 128, respectively; 15, neg- ative control of RNA extraction of bat No. 135; 16, negative control of RNA extraction of bat No. 128; 17, negative control of RNA extraction of bat No. 140; 18, positive control; 19, negative control of first PCR; 20, negative control of second PCR. suggests that such movements are long, as is the case with the always exists among bats must facilitate viral transmission and American bat (T. brasiliensis mexicana), which is capable of antibody development. A high seropositive percentage also performing annual migrations of more than 1,000 km. Since occurs in colonies of T. brasiliensis mexicana, where percent- M. schreibersii makes seasonal migrations (some of them ages >80% have been observed (10,11). The transmission of >350 km) (16), this species could also be one of the dispersion lyssaviruses between bats from mixed colonies could take vectors of the disease in southern Europe, where it abounds. place through breathing or biting but is currently not M. schreibersii dwells in five out of the six sites where seropos- documented. itive bats have been found. In three of them, M. schreibersii The low prevalence (0 of 91, <1.1%) of active infection as forms mixed colonies with M. myotis, in one it shelters next to determined by FAT is concordant with previous results R. ferrumequinum, and in the fifth it shelters alone. M. obtained in America, which show a prevalence of active rabies schreibersii and M. myotis have direct physical contact in the infection in bats between 0.1 and 2.9% (10,28,29). However, mixed colonies. However, it is unlikely that Pipistrellus we report the first detection of EBL1 RNA by nRT-PCR in nathusii is a dispersion vector of the lyssaviruses in Spain, as several tissues (brain, blood pellet, lung, heart, tongue, and Brosset (6) suggests, since this is a very rare bat in the Iberian esophagus-larynx-pharynx) of four M. myotis, one M. nattereri, Peninsula. one M. schreibersii, and two R. ferrumequinum. These isolates The results obtained in 1995-2000 in Location No. 4 show show the existence of a low or nonproductive infection in these that the evolution in the number of seropositive bats after a species, although some small remnant of RNA remaining in a Lyssavirus infection corresponded to an asymmetrical curve, clinically normal bat as a result of an earlier nonlethal exposure with a sudden initial increase reaching more than 60% of the to a Lyssavirus is also possible. This low amount of viral DNA colony and a gradual decline over subsequent years (24)— present in the tissues underscores the need to use nRT-PCR as a unless a new episode took place (Figure 3). Because of the gre- very sensitive technique for epidemiologic studies of EBL1 in garious behavior of this species, a quick increase and a high bat populations. Rønsholt et al. (8) also comment on the diffi- seropositive percentage (almost 60% in this location) after a culty of detecting Lyssavirus infection by immunofluorescence Lyssavirus episode are not unusual. The intimate contact that in bats when a clinically silent infection exists. EBL1 are known to actively infect the brain, lung, and tongue of E. serotinus (3). However, this is the first report that EBL1 RNA can be detected in various organs and tissues in the absence of active infection, as demonstrated by negative results obtained by FAT. Most of these bats were dead when collected but were kept in conditions that allowed the classic diagnosis by FAT to be performed properly. These negative FAT results indicate that these bats died of causes other than their low productive Lyssavirus infection. The recapture of seropositive bats over several years also shows that some of these bats survived EBL1 infection. The detection of EBL1b sequences in the blood pellet of bats (3/27) is also a new find- ing. This technique would be an easy test for screening posi- tive bats. However, further studies are needed to establish the interest and sensitivity of this sample. The sensitivity of the different European bat species to EBL infection probably varies according to the animal and Figure 3. Incidence of seropositive bats observed in Myotis myotis col- virus species involved. Therefore, we have summarized in onies, Spanish Locations No. 4 and No. 5, 1995–2000 (95% confi- dence intervals shown). 418 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 RESEARCH of Spanish bat lyssaviruses for the Ministerio de Sanidad y Consumo Table 5. Bat species positive for Lyssavirus, Europe, 1954–2000 and the Conselleria de Sanitat of the Balearic Autonomous b c Family Species Lyssavirus Antibodies Government. Vespertilionidae Eptesicus serotinus EBL1a & b EBL1 Pipistrellus pipistrellus NC ND References 1. Kuzmin IV, Botvinkin AD. The behaviour of bats Pipistrellus pipistrellus Pipistrellus nathusii NC ND after experimental inoculation with rabies and rabies-like viruses and Vespertilio murinus EBL1a ND some aspects of the pathogenesis. Myotis 1996;34:93-9. 2. Muller WW. Review of reported rabies cases data in Europe to the WHO Myotis dasycneme EBL2a ND Collaborative Centre Tübingen from 1977 to 2000. Rabies Bulletin Myotis daubentonii EBL2a & b ND Europe 2000;24:11-19. 3. Bourhy H, Kissi B, Lafon M, Sacramento D, Tordo N. Antigenic and Myotis myotis EBL1b EBL1 molecular characterization of bat rabies virus in Europe. J Clin Microbiol Myotis nattereri EBL1b ND 1992;30:2419-26. 4. Bourhy H, Kissi B, Tordo N. Molecular diversity of the lyssavirus genus. Nyctalus noctula NC ND Virology 1993;194:70-81. Miniopterus schreibersii EBL1b EBL1 5. Amengual B, Whitby JE, King A, Serra-Cobo J, Bourhy H. Evolution of European bat lyssaviruses. J Gen Virol 1997;78:2319-28. Molossidae Tadarida teniotis NC EBL1 6. Brosset A. Les migrations de la pipistrelle de Nathusius, Pipistrellus Rhinolophidae Rhinolophus EBL1b EBL1 nathusii, en France. Ses incidences possibles sur la propagation de la ferrumequinum rage. Mammalia 1990;54:207-12. The additional information was obtained from Kappeler (29), Pérez-Jordá et al. (24), 7. Sánchez Serrano LP. Rabia transmitida por murciélagos insectívoros en Amengual et al. (5), Bulletin épidemiologique mensuel de la rage en France (30), and España. Boletín Epidemiológico Instituto de Salud Carlos III 1999;7:149- Muller (2). NC = not characterized. 8. Rønsholt L, Sorensen KJ, Bruschke CIM, Wellenberg GJ, Oirschot JT ND = not done. van, Johnstone P, et al. Clinical silent rabies infection in (zoo) bats. Vet Rec 1998;142:519-20. Table 5 (2,5,24,30,31) the bat species in which either Lyssavi- 9. Poel WHM van der, Heide R van der, Amerongen G van, Keulen LJM rus or antibodies against Lyssavirus have been detected. Fur- van, Bourhy H, Schaftenaar W, et al. Characterization of recently isolated lyssavirus in frugivorous zoo bats. Arch Virol 2000;145:1919-31. ther studies are needed to determine which of the European bat 10. Steece R, Altenbach IS. Prevalence of rabies specific antibodies in the species are the reservoir of EBL infection and if different spe- Mexican free-tailed bat (Tadarida brasiliensis mexicana) at Lava Cave, cies act as sentinels for the presence of the virus in the colony. New Mexico. J Wildl Dis 1989;25:490-6. The presence of EBL1 RNA and immunity to EBL1 in 11. Baer GM. The natural history of rabies. Boca Raton (FL): CRC Press; several wild bat colonies also has important implications for 1991. 12. Alcover A, Muntaner J. El registre quiropterològic de Les Balears i bat management and public health. The probability of humans’ Pitiuses: una revisió. Endins 1986;12:51-63. having contact with these colonies should be reduced and con- 13. Serra-Cobo J. Biological and ecological study of the Miniopterus trolled. In our study, most bat colonies were found in sites that schreibersii. [PhD thesis]. Barcelona: University of Barcelona; 1989. are frequently visited by speleologists, tourists, and bat-lovers. 14. Serra-Cobo J, Faus V. Nuevas citas y comentarios faunísticos sobre los As a consequence of our findings, the entry to these caves is quirópteros de la Comunidad Valenciana. Serie de Estudios Biológicos 1989;11:59-76. now controlled and limited during the periods when bats are 15. Serra-Cobo J, Amengual-Pieras B, Estrada-Peña A. Nuevos datos sobre present (in spring, summer, and autumn for Location No. 4). los quirópteros de Aragón. In: Alemany A, editor. Historia natural '91. Entry is limited by horizontal bars that allow the bats to fly Palma de Mallorca, Spain: Universitat Illes Balears; 1991. p. 229-36. across them but prevent access to people without obscuring the 16. Serra-Cobo J, Sanz-Trullén V, Martínez-Rica JP. Migratory movements view. of Miniopterus schreibersii in the north-east of Spain. Acta Theriologica 1998;43:271-83. 17. Bourhy H, Sureau P. Rapid fluorescent focus inhibition test (RFFIT). In: Acknowledgments Commission des Laboratories de Référence et d’Expertise, editor. Meth- We wish to acknowledge Josep Márquez, Catalina Massuti, Joan odes de laboratoire pour le diagnostique de la rage. Paris: Institut Pasteur; Oliver, and Antonia Sánchez for their cooperation and logistical sup- 1990. p. 191-3. port in the field work. 18. Bourhy H, Rollin PE, Vincent J, Sureau P. Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and The Spanish Ministerio de Sanidad y Consumo and the Conselle- enzyme immunodiagnosis for rapid laboratory diagnosis of rabies. J Clin ria de Sanitat I Consum (Govern de les Illes Balear) financed this Microbiol 1989;27:519-23. 19. Tordo N, Poch O, Ermine A, Keith G, Rougeon F. Walking along the study. rabies genome: is the large G-L intergenic region a remnant gene? Proc Jordi Serra-Cobo is a member of the Quality Research Team Natl Acad Sci U S A 1986;83:3914-18. 20. Crepin P, Audry L, Rotivel Y, Gacoin A, Caroff C, Bourhy H. Intravitam (Biology of Vertebrates, 96-SGR0072) of the Universitat de Barce- diagnosis of human rabies by PCR on saliva and cerebrospinal fluid. J lona and a contracted doctor by the Instituto Pirenaico de Ecología Clin Microbiol 1998;36:1117-21. (CSIC). His areas of expertise are vertebrates, population ecology, 21. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature and bat lyssaviruses. Since 1990 he has been working in the research 1989;339:237-8. Emerging Infectious Diseases • Vol. 8, No. 4, April 2002 419 RESEARCH 22. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sen- 27. Aulagnier S, Thevenot M. Catalogue des Mammifères sauvages du sitivity of progressive multiple alignment through sequence weighting, Maroc. Rabat-Agdal, Morocco: Travaux de l’Institut Scientifique, Série position-specific gap penalties and weight matrix choice. Nucleic Acids Zoologique; 1986. Res 1994;22:4673-80. 28. Trimarchi CV, Debbie JG. Naturally occurring rabies virus and neutraliz- 23. La Motte LC. Japanese B encephalitis in bats during simulated hiberna- ing antibody in 2 species of insectivorous bats of New York State USA. J tion. Am J Hyg 1958;67: 101-8. Wildl Dis 1977;13:366-9. 24. Pérez-Jordá JL, Ibañez C, Muñoz-Cervera M, Téllez A. Lyssavirus in 29. Pybus MJ. Rabies in insectivorous bats of Western Canada, 1979 to 1983. Eptesicus serotinus (Chiroptera: Vespertilionidae). J Wildl Dis J Wildl Dis 1986;22:303-13. 1995;31:372-7. 30. Kappeler A. Bat rabies surveillance in Europe. Rabies Bulletin Europe 25. Kunz TH, Nagy K. Methods of energy budget analysis. In: Kunz TH, edi- 1989;13:12-13. tor. Ecological and behavioral methods for the study of bats. Washington: 31. Bruyére V, Janot C. La France bientôt indemne de rage. Bulletin Epidémi- Smithsonian Institution Press; 1988. p. 277-302. ologique Mensuel de la Rage en France 2000;30:1-7. 26. Serra-Cobo J, Barbault R, Estrada-Peña A. Le gouffre de San Pedro de los Griegos (Oliete, Teruel, Espagne) un refuge de biodiversité sans équiva- lent en Europe. Revue Ecologie (Terre Vie) 1993;48:341-8. Address for correspondence: Jordi Serra-Cobo, Departament de Biologia Ani- mal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain; fax: 34-93-403-57-40; e-mail: [email protected] 420 Emerging Infectious Diseases • Vol. 8, No. 4, April 2002

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