|
|||||||||||||||||
|
|
EID
Home | Ahead of Print | Past
Issues | EID Search | Contact
Us | Announcements | Suggested
Citation |
|
Research European Bat Lyssavirus Infection in Spanish Bat PopulationsJordi Serra-Cobo,* Blanca Amengual, Carlos Abellán, and
Hervé Bourhy
Rabies is a worldwide zoonosis due to Lyssavirus infection; multiple host species act as reservoirs. This disease infects the central nervous system of humans and other mammals. Bats are no exception, as proved by the 630 positive cases detected in Europe from 1977 to 2000 (1,2). Recent molecular studies have shown genetic differentiation in lyssaviruses that cause rabies among European bats, leading to a classification into two new genotypes, 5 and 6, which correspond to European bat lyssavirus 1 (EBL1) and EBL2, respectively (3,4). As a result of a recent molecular study, two new lineages within genotype 5 have been identifiedEBL1a and EBL1b; the latter is potentially of African origin, which suggests south-to-north transmission (5). However, despite molecular advances and many European cases verified to date, knowledge of the prevalence and epidemiology of EBL is limited. 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 suggests an alternative viral strategy for Lyssavirus infection of European insectivorous bats compared with the terminal infection commonly associated with rabies infection. To investigate these observations, a 9-year study was undertaken in Spain to locate and determine the colonies and species of bats carrying EBL or Lyssavirus antibodies, monitor the prevalence of seropositive bats, and characterize circulating lyssaviruses. |
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Figure 2 | ||
Click to view
enlarged image |
During 1995 through 1996, 12 brain samples were only analyzed by FAT. After 1996, the brain samples (n=79) were also analyzed by nested RT-PCR (Table 1). All brains (n=91) analyzed by FAT were negative. In contrast, brains of 1 M. myotis, 1 M. nattereri, and 1 M. schreibersii (No. 140) of Location No. 4 and 1 R. ferrumequinum (No. 128) of Location No. 1 (all collected in 2000) were positive by nested RT-PCR. Four animals (M. schreibersii [No. 140] and R. ferrumequinum [No. 128], whose brains were positive by nested RT-PCR, and two R. ferrumequinum [No. 123 and No. 135], whose brains were negative) were completely necropsied. Various organs and tissues (medulla, liver, kidney, spleen, heart, tongue, esophagus-larynx-pharynx, and lung) were collected and subjected to nRT-PCR. Esophagus-larynx-pharynx and lung of bat No. 135 and tongue, lung, and heart of bat No. 128 were positive (Figure 2).
Twenty-seven blood pellets of bats collected in 2000 were also analyzed by nRT-PCR. These samples were obtained from 8 R. ferrumequinum (location No. 1), 1 R. ferrumequinum (Location No. 3), 1 M. myotis (Location No. 5), 14 M. myotis (Location No. 4), and 3 M. schreibersii (Location No. 4). The blood pellets of three M. myotis from Location No. 4 were found positive by nRT-PCR. None of the blood samples showing positive RT-PCR results on the pellet were found positive by seroneutralization.
The threshold of detection of the nRT-PCR for the amplification of the EBL1a genomic and antigenomic RNAs of the N gene was 5 x 10-2 fluorescent forming units of EBL1a/mL. In all these experiments, negative controls performed individually for each step (extraction, RT, primary, and secondary PCR) were negative. Furthermore, nRT-PCR performed on positive tissues without previous reverse transcription gave negative results, demonstrating the absence of complementary DNA contamination.
Nucleotide (nt) sequences were determined by using the positive nRT-PCR products obtained from the four brains and from one blood sample. These 122-nt long sequences of the nucleoprotein gene were strictly similar to the sequence of two EBL1b Spanish isolates (94285SPA and 9483 SPA) described previously (5), except that the sequence obtained from the positive blood pellet exhibited a TA mutation in position 145 of the coding region of the nucleoprotein gene. Four mutations distinguished the sequence of the positive control corresponding to a French bat (No. 2002FRA) from the different sequences obtained from Spanish bats (not shown). This further confirms the specificity of the products amplified from the Spanish bat samples.
This is the first report of the presence of EBL1-specific neutralizing antibodies in four European insectivorous bat species (M. myotis, M. schreibersii, T. teniotis, and R. ferrumequinum). These findings lead to the following observations on the circulation and possible bat species involved in the dispersion of EBL1 in southern Europe. First, the identification of EBL1 antibodies in 24% of the M. myotis analyzed in Locations No. 4 and No. 5 in 1995 through 2000 (n=276) indicates that bats of this genus are infected with EBL1. Second, the distribution of T. teniotis and M. schreibersii in southern Europe and northern Africa (13,27) could contribute to the dispersion of EBL1 in southern Europe and is concordant with the possible African origin of EBL1, as suggested by Amengual et al. (5).
Although the seasonal movements of T. teniotis are scarcely known, the quick, straight flight of this species suggests that such movements are long, as is the case with the American bat (T. brasiliensis mexicana), which is capable of performing annual migrations of more than 1,000 km. Since M. schreibersii makes seasonal migrations (some of them >350 km) (16), this species could also be one of the dispersion vectors of the disease in southern Europe, where it abounds. M. schreibersii dwells in five out of the six sites where seropositive bats have been found. In three of them, M. schreibersii forms mixed colonies with M. myotis, in one it shelters next to R. ferrumequinum, and in the fifth it shelters alone. M. schreibersii and M. myotis have direct physical contact in the mixed colonies. However, it is unlikely that Pipistrellus nathusii is a dispersion vector of the lyssaviruses in Spain, as Brosset (6) suggests, since this is a very rare bat in the Iberian Peninsula.
Figure 3 | ||
Click to view
enlarged image |
The results obtained in 1995-2000 in Location No. 4 show that the evolution in the number of seropositive bats after a Lyssavirus infection corresponded to an asymmetrical curve, with a sudden initial increase reaching more than 60% of the colony and a gradual decline over subsequent years (24)unless a new episode took place (Figure 3). Because of the gregarious behavior of this species, a quick increase and a high seropositive percentage (almost 60% in this location) after a Lyssavirus episode are not unusual. The intimate contact that always exists among bats must facilitate viral transmission and antibody development. A high seropositive percentage also occurs in colonies of T. brasiliensis mexicana, where percentages >80% have been observed (10,11). The transmission of lyssaviruses between bats from mixed colonies could take place through breathing or biting but is currently not documented.
The low prevalence (0 of 91, <1.1%) of active infection as determined by FAT is concordant with previous results obtained in America, which show a prevalence of active rabies infection in bats between 0.1 and 2.9% (10,28,29). However, we report the first detection of EBL1 RNA by nRT-PCR in several tissues (brain, blood pellet, lung, heart, tongue, and esophagus-larynx-pharynx) of four M. myotis, one M. nattereri, one M. schreibersii, and two R. ferrumequinum. These isolates show the existence of a low or nonproductive infection in these species, although some small remnant of RNA remaining in a clinically normal bat as a result of an earlier nonlethal exposure to a Lyssavirus is also possible. This low amount of viral DNA present in the tissues underscores the need to use nRT-PCR as a very sensitive technique for epidemiologic studies of EBL1 in bat populations. Rønsholt et al. (8) also comment on the difficulty of detecting Lyssavirus infection by immunofluorescence 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 finding. This technique would be an easy test for screening positive 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 virus species involved. Therefore, we have summarized in Table 5 (2,5,24,30,31) the bat species in which either Lyssavirus or antibodies against Lyssavirus have been detected. Further studies are needed to determine which of the European bat species are the reservoir of EBL infection and if different species act as sentinels for the presence of the virus in the colony.
The presence of EBL1 RNA and immunity to EBL1 in several wild bat colonies also has important implications for bat management and public health. The probability of humans having contact with these colonies should be reduced and controlled. In our study, most bat colonies were found in sites that are frequently visited by speleologists, tourists, and bat-lovers. As a consequence of our findings, the entry to these caves is now controlled and limited during the periods when bats are present (in spring, summer, and autumn for Location No. 4). Entry is limited by horizontal bars that allow the bats to fly across them but prevent access to people without obscuring the view.
We wish to acknowledge Josep Márquez,Catalina Massuti, Joan Oliver, and Antonia Sánchez for their cooperation and logistical support in the field work.
The Spanish Ministerio de Sanidad y Consumo and the Conselleria de Sanitat I Consum (Govern de les Illes Balear) financed this study.
Jordi Serra-Cobo is a member of the Quality Research Team (Biology of Vertebrates, 96-SGR0072) of the Universitat de Barcelona and a contracted doctor by the Instituto Pirenaico de Ecología (CSIC). His areas of expertise are vertebrates, population ecology, and bat lyssaviruses. Since 1990 he has been working in the research of Spanish bat lyssaviruses for the Ministerio de Sanidad y Consumo and the Conselleria de Sanitat of the Balearic Autonomous Government.
Address for correspondence: Jordi Serra-Cobo, Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain; fax: 34-93-403-57-40; e-mail: bamengua@pie.xtec.es
Table 5. Bat species positive for Lyssavirus, Europe, 19542000a | |||
|
|||
Family |
Species |
Lyssavirusb |
Antibodiesc |
|
|||
Vespertilionidae |
Eptesicus serotinus |
EBL1a & b |
EBL1 |
Pipistrellus pipistrellus |
NC |
ND |
|
Pipistrellus nathusii |
NC |
ND |
|
Vespertilio murinus |
EBL1a |
ND |
|
Myotis dasycneme |
EBL2a |
ND |
|
Myotis daubentonii |
EBL2a & b |
ND |
|
Myotis myotis |
EBL1b |
EBL1 |
|
Myotis nattereri |
EBL1b |
ND |
|
Nyctalus noctula |
NC |
ND |
|
Miniopterus schreibersii |
EBL1b |
EBL1 |
|
Molossidae |
Tadarida teniotis |
NC |
EBL1 |
Rhinolophidae |
Rhinolophus ferrumequinum |
EBL1b |
EBL1 |
|
|||
aThe additional information was obtained
from Kappeler (29), Pérez-Jordá et al. (24),
Amengual et al. (5), Bulletin épidemiologique mensuel
de la rage en France (30), and Muller (2).
|
|
|||||
|
|||||
|
EID Home | Top of Page | Ahead-of-Print | Past Issues | Suggested Citation | EID Search | Contact Us | Accessibility | Privacy Policy Notice | CDC Home | CDC Search | Health Topics A-Z |
||
This page last reviewed March 28, 2002 |
||
Emerging
Infectious Diseases Journal |
||