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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All kind of mammals are susceptible to rabies, a disease that is currently considered a re-emerging zoonosis. Due to adequate pet rabies vaccination campaigns in Brazil, dogs are no longer the main reservoir of rabies in this country. Meanwhile the number of reported cases of bat2–4 and bovine rabies is increasing.5 Herbivore rabies has been reported in the State of Rio de Janeiro in a crescent way (Romijn PC, Kimura LMS, unpublished data). The number of positive samples ranged from 22 of 197 received in 1988 to 147 of 241 received in 1996. An investigation of a bovine rabies epizootic involving bats in the State of Rio de Janeiro from 1994 to 1997 reported increasing numbers of cases each year (Silva RDCF and others, unpublished data).
In South America, the most common theory of herbivore rabies origin is transmission of the virus by the bite of infected vampire bats (Desmodus rotundus).6 Bat rabies in Brazil is normally diagnosed only indirectly by the detection of rabies in herbivores, and sporadically when a sick bat is sent to a laboratory for testing. Bat rabies may spread to domestic animals, mainly when bat colony populations become too large and subsequently start to migrate.7
Periodic vaccination of cattle herds is beneficial in those areas where sylvatic rabies occurs.8 In one study, only three cases occurred in unvaccinated animals with documented vampire bat bites in a controlled area (Cattaneo CAM and others, unpublished data).
In addition to domestic animal vaccination in endemic and epidemic areas, the monitoring of bat colonies to determine their colony size and sanitary state is essential in controlling the spread of rabies. A reduction in the number of vampire bats in colonies in a microhydrobasin in Itaperuna, Rio de Janeiro State reduced herbivore rabies from eight foci in 1996 to none in 1997; currently, no cases have been detected (Romijn PC and others, unpublished data).
The Empresa de Pesquisa Agropecuaria do Rio de Janeiro (PESAGRO-RIO) (State Agricultural Research Institute) has been monitoring rabies among domestic animals, and its spread to new regions possibly by infected vampire bats, for more then 20 years by using the direct immunofluorescent test (DIFT) and the mouse inoculation test (MIT). Testing of more than 2,000 samples found only four negative samples by the DIFT compared with the MIT and two negative samples by the MIT compared with the DIFT (Kimura LMS and others, unpublished data). Recent research in Europe indicated the presence of European bat Lyssavirus (EBL) in clinically healthy bats, and the possibility of its resurgence caused by external factors such as stress.9,10 If Lyssavirus genotype 1 is also present in vampire bats without causing disease, apparently healthy young adults may carry the virus to distant areas when leaving their maternal colony located in an area where a rabies focus previously occurred.
To adequately monitor the presence of Lyssaviruses in bat colonies that may come in close contact with domestic animals and humans,11,12 it is not sufficient to periodically analyze randomly collected bats for the presence of virus by traditional techniques. More sensitive molecular techniques, such as the polymerase chain reaction (PCR) detection and sequence analysis, are required. In addition to identifying the virus genus, these techniques can also facilitate identification of the origin of a virus isolate and the epidemiologic relationships between outbreaks in different species.
To study the epidemiologic and geographically features of outbreaks of animal rabies, and to introduce prophylactic actions in a timely fashion,5 we used traditional monitoring and molecular analyses of a genome segment encoding the nucleoprotein of Lyssaviruses viruses isolated in the State of Rio de Janeiro, Brazil.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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Composition of groups.
For sequence analysis, 28 samples were designated into three groups based on their geographic origin (Table 1
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Group 1. In the county of Aperibé, Rio de Janeiro State since an outbreak of rabies in 1996, cattle more than four months old are being vaccinated yearly. Control of the vampire bat population has not been conducted in this area. Group 1 was composed of two rabies-positive samples from young cattle (non-vaccinated) (samples 25 and 34) and one sample from a bat from the same area (sample E).
Group 2. The counties of Itaperuna and Laje do Muriaé in Rio de Janeiro State are located within a single hydrographic basin, with sporadic cattle rabies diagnosed until 1996, when the findings of rabid cases among herbivores started to increase. This culminated with the detection of two sick vampire bats that were shown to be positive for rabies by the DIFT, in December 1997. In Itaperuna, an intensive vampire bat (D. rotundus) population control program was carried out in Itaperuna in the following month (January 1998) that mapped and monitored all bat colonies found near human properties. An official vaccination program was implemented only along the borders of the outbreak. Group 2 was composed of two rabies-positive herbivore samples (collected begin and middle 98) from a neighboring hydrographic basin (samples 13 and 44), three rabies-positive herbivore samples (begin and middle 98) from the outbreak area (samples 1, 4, and 45), two rabies-positive vampire bats found in December 1997 (samples 56 and 61), four bats (two vampires and two insectivorous bats) collected end 1998 in a rabies focus (samples A, B, C, and D), and three rabies-positive bovine samples collected begin and middle 98 from counties south of a focus area from which rabies was supposed to have expanded (samples 16, 39, and 43).
Group 3. Aimed at the comparison of samples collected far appart in different hydrobasins. Group 3 was composed of two rabies-positive insectivorous bat samples originating from the city of Rio de Janeiro and collected in 1990 (samples 58 and 60), two rabies-positive bovine samples collected in the state of Minas Gerais in begin 1998 (sample 6) and half 1999 (sample 64), three rabies-positive herbivore samples collected in the northcentral part of Rio de Janeiro State (samples 30, 46, and 62), two rabies-positive bovine samples originating from the southern coast of Rio de Janeiro (samples 63 and 65), one rabies-positive bovine sample originating from southwestern Rio de Janeiro (sample 66), and one human sample (sample 59).
Techniques. The DIFT and the MIT were carried out at the Laboratorio de Biologia Animal, PESAGRO-RIO (Rio de Janeiro, Brazil), according to the standard recommendations of the World Health Organization.13 Reverse transcriptase (RT)-PCRs (including hybridization) and DNA sequence analysis for Lyssaviruses were carried out at the Microbiological Laboratory for Health Protection, Rijksinstituut voor Volksgezondheid en Milieu (National Institute of Public Health and the Environment) (Bilthoven, The Netherlands) according to the procedures of van der Poel and others.10
Sequencing and phylogenetic analysis. Purified PCR products were sequenced with the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer Applied Biosystems, Foster City, CA). Nucleotide sequences were edited using Seq Ed (version 1.03; Perkin Elmer Applied Biosystems), and aligned using Bionumerics (version 2.0; Applied Maths, Kortrijk, Belgium). Distance calculations were done using the Jukes and Cantor correction for evolutionary rate.14 The confidence values of the internal rodes were calculated by performing 100 bootstrap analyses. Evolutionary trees for nucleotide sequences were drawn using the unweighted pair group method with arithmetic average (UPGMA) with the CVS vaccine strain (Genbank accession number D42112; basepairs 42–607) used as a reference.
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RESULTS |
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). The results obtained by the routine DIFT, when compared with the analyses of the same samples by the RT-PCR/hybridization technique, were similar (Table 1). All samples positive by the DIFT were also positive by the RT-PCR assay. However, five of the six DIFT-negative bat brain samples that originated from rabies foci already being studied for more than a year (samples A, B, C, D, and E) showed evidence of rabies virus RNA when analyzed by a nested PCR assay. All five bat samples collected from an area with no rabies since 1996 (samples 47, 48, 49, 50 and 51) were negative for rabies by both techniques. Two of 50 DIFT-positive, RT-PCR-positive samples were negative by the MIT (samples 42 and 44). All five bat samples tested for EBL as described by Bourhy and others were also negative (data not shown).15
Sequencing of a 566-basepair segment of the nucleoprotein-encoding region of the selected samples produced a separate branch within genotype 1 distinct from genotypes 2, 3, 4, and 5 (data not shown). Within this branch, three separate clusters could be identified (Figure 2). Bovine and equine isolates from the same hydrographic basin region were genomically clustered, whereas strains of bat origin were found in all three clusters.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bats are the sole flying mammalian species with their own biological characteristics and social behavior. This, together with the finding that the nucleotide sequences of the most conserved part of the Lyssavirus genome studied were very similar to each other, suggest that bats may play an important role in rabies epidemiology in Brazil.
We have used the RT-PCR and DNA sequencing to determine the origin of Lyssavirus isolates in the State of Rio de Janeiro, Brazil. Analysis of herbivore samples verified that clusters were produced of those originating from the same hydrographic basins (Figures 1 and 2). Two rivers flank the area of Aperibé, Rio de Janeiro State, each originating from a different hydrographic basin. Sample 34 was collected near the more southerly river, and it clustered with sample 46 isolated in Santa Maria Madalena, Rio de Janeiro State, in the same hydrographic basin. The samples collected from North Aperibé (samples 25 and E) did not cluster with sample 34. These samples were more similar to samples from Itaperuna and Laje do Muriaé, north of Aperibé and crossed by the same river. In those basins with intensive dairy cow production, on small farms with steep hills and mountains, restricted rabies foci were observed. The similarity among the rabies virus samples from these areas was relatively high, possibly indicating close contact between bat colonies. Viruses of a second cluster originated from areas more distant from each other, but almost all were within two main hydrographic basins. These hydrographic basins were much larger and situated in regions with fewer shelters. Cattle in these areas were raised over large areas. In such regions, bats need to fly longer distances for feeding, and spread of virus over larger areas occurs in a shorter period of time. In these cases, a diffusion route is not very evident.
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Bat Lyssavirus samples collected from a same geographic region did not always cluster together. In fact, they were similar to other samples collected at distinct basins, some more than 300 km apart. This finding stresses the important role of bats in (Lyssa) virus transportation over large distances and to neighboring hydrographic basins.
Simply just monitoring rabies cases that occur in a region does not contribute much to the study of the epidemiology of the virus (origin and sylvatic cycle) and to rabies control. Active mapping of rabies foci of a sylvatic origin, and studies related to the dynamics of its dispersion to other regions enables one to predict and prevent new clinical cases among domestic animals and human beings.11 Important factors in understanding the spread of rabies are possible bat migration routes, their food supply, and the presence of adequate shelter (Romijn PC and others, unpublished data). These factors are also important in controlling the spread of the virus.
For rabies control in mountainous regions with considerable milk production on small farms, it may be sufficient to maintain colonies of D. rotundus at a minimal population level, with periodic testing for rabies virus among individual bats in the colony. In prairies and flatland regions, with fewer natural shelters and extensive beef cattle farms, perifocal prophylactic vaccination of the herds is essential, together with capture of vampire bats on those farms near the present rabies focus for continuous monitoring of Lyssavirus and viral-specific antibodies. Since the RT-PCR can be used to analyze small amounts of sample, (even autolysed or inactivated) it should be used with the DIFT to identify regions at risk for new rabies outbreaks among domestic animals so that timely prophylactic action may be taken. Use of the PCR and sequencing could identify the source of the virus, and prophylactic measures, such as bat population reduction, can then be used in the area where to the migration of individual bats from an infected colony is taking place, together with vaccination of domestic animals.
For epidemiologic studies, the MIT is not adequate because it takes at least 21 days to obtain the results. In our study, two of 45-DIFT-positive, RT-PCR-positive samples were negative by the MIT. One of these samples originated from a cow that had been vaccinated 20 days earlier and was killed after four days of classic symptoms of rabies. The other sample came from a five-month-old calf that had been vaccinated at three months age, and died spontaneously of rabies with no symptoms other than salivation during the two days of clinical illness.
The RT-PCR technique has been used for the identification of various genotypes of known Lyssaviruses.15–18 One of these studies suggested the presence of EBL viral genotype 5 in a Brazilian bat.17 However, in our study, EBL virus was not detected in five bat samples. Further research into the epidemiology of infection with Lyssavirus will be needed to determine if EBL virus or other Lyssaviruses are present in Brazilian bats.10,17
In our study, samples clustered more closely together when isolated from the same host species. It is possible that a change in host causes a small change in the nucleotide sequence of the viral genetic region under study. Thus, samples originating from one bat species and isolated from cattle could be different from those originating from the same bat species and isolated from horses.19
The prevention of the spread of rabies virus and rabies-related viruses, through infected animals is no longer a matter of only agricultural importance in Brazil; it has now become an increasingly important public health issue.20 Each year, the number of rabid wild animals, mainly bats, detected, increases. Some of these animals are found near or in large cities (Kimura LMS and others, unpublished data, Silva, MV and others, unpublished data). Almost all suspected samples that are positive for rabies are submitted by health professionals, and originate from areas with a history of many clinical animal cases, suggesting that the identified cases are just a small proportion of the actual number of cases. According to origin studies, outbreaks must be dealt with adequately11 and in a differentiated way, dependent on whether transmission is by a terrestrial or an aerial vector.20 The way to control rabies outbreaks depends on its transmitting host. If transmission is terrestrial (dogs and cats), perifocal vaccination is sufficient to interrupt the rabies cycle. However, vaccination will protect only those domestic animals capable of responding immunologically (those greater than three months old). If transmission is aerial (by bats), population control of the transmitting animal is essential because there are no physical barriers to prevent spread of the virus. Public health awareness programs that do not alarm the public and encourage people to notify public health officials of the presence of sick wild animals and vampire bats in their neighborhood would be the first step towards adequate monitoring of rabies and its control.
Acknowledgments: We thank H. Tsiang for encouragement to carry out these studies; H. Bourhy and L. Audry for fruitful discussions; J. Vinjé (Rijksinstituut voor Volksgezondheid en Milieu, Laboratory of Infectious Disease Research) for helping in creating the phylogenetic tree; the Rijksinstituut voor Volksgezondheid en Milieu and Institut Pasteur (Paris) for providing laboratory facilities; Marlon V. Silva (Instituto Jorge Vaitsman, Rio de Janeiro) for kindly providing some of the bat samples; and Newton Vidal for editing the figures.
Authors’ addresses: Phyllis C. Romijn and Rita de Cássia F. Silva, Virology Department, Laboratorio de Biologia Animal, Empresa de Pesquisa Agropecuaria do Rio de Janeiro, Alam S. Boaventura, 770, 24.120–191 Niteroi, Rio de Janeiro, Brazil, Telephone: 55-212-627-1432, Fax: 55-212-625-1446, E-mail: phyllis{at}predialnet.com.br. Carlos Alberto M. Cattaneo, SDS/Coord. de Defesa Sanitaria Animal, Alam.S.Boaventura,770, 24.120-191 Niteroi, Rio de Janeiro, Brazil. Reina van der Heide and Wim H. M. van der Poel, Microbiological Laboratory for Health Protection, Rijksinstituut voor Volksgezond-heid en Milieu, Antoine van Leeuwenhoeklaan 9, PO Box 1, 3720 BA Bilthoven, The Netherlands.
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