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Analisis molecular de un caso de rabia urbana causado por un murcielago vampiro en Corrientes, Argentina.

Resumen

En abril de 2006 se detecto virus de la rabia (RABV) en un gato domestico de la ciudad de Corrientes, Argentina. La caracterizaci6n molecular identifico al murcielago Desmodus rotundus como la fuente viral. El analisis genetico de 22 cepas de RABV aisladas de D. rotundus obtenidos entre 1998-2006 en Argentina, revelo la cocirculacion de dos linajes geneticos en la Provincia de Corrientes. La alteracion por parte del hombre, del ecosistema del murcielago en la region, podria explicar los cambios en su comportamiento y en la circulacion del virus de la rabia.

Palabras clave: gato, vampiro, rabia, epidemiologia molecular.

Abstract

In April 2006, rabies virus (RABV) was detected in a domestic cat from Corrientes City, Argentina. Molecular characterization identified Desmodus rotundus as the reservoir species. Genetic analyses of 22 RABV strains obtained from D. rotundus between 1988 and 2006 in Argentina, revealed the co-circulation of two genetic lineages in Corrientes Province. Alteration of the ecosystem of vampire bats by human intervention in this region could explain such changes in their behavior, as well as rabies virus circulation.

Key words: cat, vampire bat, rabies, molecular epidemiology.

Molecular analysis of urban rabies case from vampire bat in Corrientes, Argentina

INTRODUCTION

The last case of terrestrial rabies transmitted by dogs or cats in Corrientes Province was in the late 80s. At this time, viral activity is related to rabies transmitted by more common vampire bat, Desmodus rotundus. Two cases of human rabies associated with that species have occurred in Isla del Cerrito, Chaco Province (1997), and San Luis del Palmar, Corrientes Province (2001) (8).

D. rotundus is a non-migratory colonial species that roosts in natural shelters or in places associated with human beings. They may use several refuges and can share them with other bat species. Their home range was described between 10 and 20 [km.sup.2]. As vampire bats feed on mammalian blood, the distribution of bat populations is affected by food availability, specially livestock (4,9).

In April 2006, a domestic cat from the suburbs of Corrientes City, with no history of rabies vaccination, showed a strange and aggressive behavior. It attacked two human beings and several dogs and cats. Two days later it died. After that, postmortem brain tissue samples were obtained and tested positive for rabies virus (RABV). Molecular research identified to D. rotundus as the species reservoir and revealed the circulation of a previously not recognized rabies lineage in that region (2).

The aim of this study was to analyze the molecular characteristics of an urban rabies case associated a D. rotundus bat occurred in Corrientes city in 2006.

MATERIAL AND METHODS

Virus isolates and sequences. A rabies isolate obtained from a cat from Corrientes City, and others five samples collected few months later within a radius of 10-20 [km.sup.2] from the place where the cat was detected, were analyzed. Others 16 historical rabies sequences from vampire related-cases of Corrientes, Chaco, Jujuy and Salta provinces and eight nucleotide sequences from insectivorous and terrestrial species, were included in this study for comparison purposes only (Table 1).

RT-PCR and DNA sequencing. Viral RNA was extracted from samples using TRIzol[R] (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. Reverse transcription and PCR amplification were achieved with primers 10g and 304, as previously described (14). The amplified product was sequenced using the Taq Big Dye Terminator Cycle Sequencing Ready Reaction Kit, according to the manufacturer's protocol on an Applied Biosystems 377 DNA automated sequencer (Applied Biosystems Inc. Foster City, California, USA).

Phylogenetic analysis. A 264-bp region corresponding to the nucleoprotein gene located between nucleotides 1157 and 1420 and amino acids 363 to 450, according to positions for the fixed laboratory strain SAD B19, was analyzed. Complete alignment was performed with Clustal X 1.8 software (16). Matrix data was analyzed using Kimura 2 parameters as a method of nucleotide substitution and Neighbor-Joining to reconstruct the phylogenetic tree (MEGA version 4.1) (15). The statistical significance of the phylogenies constructed was estimated by bootstrap analysis with 1000 pseudo-replicate data sets. Bootstrap values above 60% were considered significant.

RESULTS

A total of 22 rabies sequences obtained from vampire-related cases segregated into a group identified as DR, well supported by high bootstrap values as shown in Figure 1. The remaining Argentinean samples, represented by others rabies reservoirs, grouped into different genetic groups: TB (Tadarida brasiliensis), MY (Myotis sp.), HM (Histiotus montanus), LA (Lasiurus cinereus), TR1 and TR2 (Terrestrial 1 and 2) showing a genetic distance between 14.3 and 18.8% with D. rotundus group.

Group DR was divided into two lineages (DRI and DR2) showing an average nucleotide and aminoacid distance between them of 1.9% and 0.0%, respectively. Lineage DR1 included 13 historical samples obtained between 1999 and 2003 from Chaco and Corrientes provinces together with four samples (Clbv-Cte06, C2bv-Cte06, C3bv-Cte06, and C5bv-Cte06) collected from different sites near Corrientes City in 2006. This lineage was further separated into two sublineages geographically related: DR1a, that contained samples from Corrientes and DR1b which grouped isolates from Chaco. Lineage DR2 included five samples: a cat (C0ct-Cte06) from Corrientes City and a horse obtained at KM1023, both recovered in 2006 and three historical samples from Salta (1998 and 2003) and Jujuy (1998). Geographic location of genetic variants circulating in Corrientes Province is shown in Figure 2.

DISCUSSION

Presence of the vampire bat in urban region is unusual. In the past five years, two episodes have been reported in Brazilian cities, one associated with dogs attacks (3) and other with a bat specimen infected with RABV (5). Several ecological factors can explain the presence of vampire bats in the area of Corrientes City: (i) abundant livestock a few kilometers of the city, (ii) large numbers of domestic animals (potential victims of the bat), and (iii) high availability of artificial shelters (1). In addition, in front of the city, few kilometers on north, place Isla del Cerrito (Chaco Province) where have been reported frequent activity of vampire bats and human beings attacks (10).

[FIGURE 1 OMITTED]

In Corrientes Province, after extend paralytic rabies epizootic outbreaks from 1997 to 2003, only sporadic cases were identified (11). However, just few months after the detection of feline case, new cases of paralytic rabies were reported on Isla del Cerrito and others surrounded to Corrientes City. Reasons of the reemergence of rabies in this area are difficult to establish but human interventions in bats ecosystem are usually reflected in changes in its pattern of transmission.

On March 2006, the National Service of Animal Health and Food Quality (SENASA) sacrificed over 4,000 bovines in San Luis del Palmar, 15 km distant from Corrientes City, due to an outbreak of foot-and-mouth disease (13). This serious disruption of bat habits could have caused the displacement of bat colonies, from locations that did not contain enough cattle to sustain the populations of vampire bats, to bordering areas of Corrientes City in search to improve the availability of others source of food such as domestic animals. However, we cannot exclude additional unknown causes and other investigations should be conducted to answer this question.

Vampire bat-related rabies viruses have been genetically typed as being a species-specific variant (6). Genetic diversity among different variants is attributed to aspects of the ecology of the vampire and topography of the habitat in which geographic features can act as natural barriers and cause the movement of regionally differentiated lineages (7). The study of isolates from Corrientes in 2006 revealed the co-circulation of two lineages in this area. One predominant (DR1) associated with the epizooty occurred between 1997 and 2003 and another minority (DR2) associated with cases occurred in Salta and Jujuy (1998-2003).

The presence of both lineages in Corrientes could be explained by the fact that the minority lineage was possibly maintained by small populations of vampires in a restricted geographic area. Its resurgence could be other consequence of the movement of bats occurred as a result of cattle lost by foot-and-mouth disease outbreak in San Luis del Palmar as was mentioned above. A phylogeographic study involving a large collection of rabies strains associated with D. rotundus from all provinces during an extended period of time would help to understand the historical circulation of rabies in vampire bats in Argentina.

[FIGURE 2 OMITTED]

Recently, Pan American Health Organization (PAHO), pointed out that the number of human cases of rabies transmitted by vampire bats is bigger than those of transmitted by dogs 12. Our findings underscores the importance of detection of unusual cases of rabies associated with vampire cases that represents a warning signs and should remind us that the human risk of rabies transmission from vampire bats is always present and should not be undervalued.

Recibido: 6 agosto 2009 / Aceptado: 15 setiembre 2009

REFERENCES

(1.) Anonymous. 2008. Detectan en Corrientes mayor presencia de murcielagos transmisores de rabia paresiante. On line: http://revistacyt.unne.edu.ar/veterinarial9.html.

(2.) Cisterna D, Bonaventura R, Caiilou S, Pozo O, Andreau ML, Fontana LD. 2005. Antigenic and molecular characterization of rabies virus in Argentina. Virus Res 109: 139-147.

(3.) Dantas Torres F, Valenea C, Andrade Filho GV. 2005. First record of Desmodus rotundus in urban area from the city of Olinda, Pernambuco, Northeastern Brazil: a case report. Rey Inst Med Trop Sao Paulo 47: 107-108.

(4.) Delpietro HA, Russo RG. 1996. Ecological and epidemiologic aspects of the attacks by vampire bats and paralytic rabies in Argentina and analysis of the proposals carried out for their control. Rey Sci Tech 15: 971-984.

(5.) Ferraz C, Achkar SM, Kotait I. 2007. First report of rabies in vampire bats (Desmodus rotundus) in an urban area, Ubatuba, Sao Paulo state, Brazil. Rey Inst Med Trop Sao Paulo 49: 389-390.

(6.) Kobayashi Y, Ogawa A, Sato G, Sato T, Itou T, Samara SI. 2006. Geographical distribution of vampire bat-related cattle rabies in Brazil. J Ver MedSci 68: 1097-1100.

(7.) Kobayashi Y, Sato G, Moehizuki N, Hirano S, Itou T, Carvalho AA. 2008. Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil. BMC Vet Res 4: 44.

(8.) Larghi OP, Delpietro H. 2004. Casos de rabia humana transmitida por murcielagos, Argentina. En: Temas de Zoonosis (Cacchione RA, Durlach R, Larghi OP, ed), Publ. Asociacion Argentina de Zoonosis, Buenos Aires, p. 135-138.

(9.) Lord RD, Fuenzalida E, Deipietro H, Larghi OP, de Diaz AM, Lazaro L. 1975. Observations on the epizootiology of vampire bat rabies. Bull Pan Am Health Organ 9: 189-195.

(10.) Miranda AO, Aeosta R, Marder G, Jacobo R, Baez N. 2003. Persistente epidemia de rabia bovina (paresiante) en la Provincia de Corrientes (Argentina), periodo 19972003. Comunicaciones Cientificas y Tecnologicas 2003, Universidad Nacional del Nordeste (Argentina). On line: http://www.unne.edu.ar/cyt2003/cyt.html.

(11.) Miranda AO, Aeosta R, Laffont HM, Baez N, Marder G, Jacobo R. 2005. Confirmacion del virus rabico transmitido por murcielagos en bovinos del nordeste argentino mediante seroneutralizacion en raton. Rey Vet 16: 36-39.

(12.) Sehneider MC, Romijn PC, Uieda W, Tamayo H, da Silva DF, Belotto A. 2009. Rabies transmitted by vampire bats to humans: an emerging zoonotic disease in Latin America? Rev Panam Salud Publ 25: 260-269.

(13.) SENASA. 2006. Resolucion 35/2006. On line: http://www.senasa.gov.ar.

(14.) Smith JS. 1995. Rabies virus. In: Manual of clinical microbiology (Murray PR, Pfaller MA, Tenover FC, Yolken R, ed.), American Society for Microbiology Press, Washington, p. 997-1003.

(15.) Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599.

(16.) Thompson JD, Higgins DG, Gibson TJ. Clustal W. 1994. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. Nucleic Acids Res 22: 4673-4680.

Miranda, A.O. [1]; Nunez, S.E. [1]; Martinez, L. [3]; Gury-Dohmcn, F.R. [2]; Cisterna, D.M. [3]

[1] Laboratorio de Investigaciones y Diagnostico de Rabia, Facultad de Ciencias Veterinarias, UNNE, Sargento Cabral 2139, Corrientes 3400, Argentina, Tel/Fax 03783-425753. [2] Instituto de Zoonosis "Dr. Luis Pasteur', Diaz Velez 4821, Buenos Aires 1406. [3] Servicio de Neurovirosis INEI-ANLIS "Dr. Carlos G. Malbran', Av. Velez Sarsfield 563, Buenos Aires (1281), Tel/Fax 114302-5064. E-mail: inmuno@vet.unne.edu.ar
Table 1. Rabies isolates from Argentina analyzed in this study

isolates city, province

C0Ct-Cte06 Corrientes City, Corrientes
C1Bv-Cte06 Paso de la Patria, Corrientes
C2BvCha06 Isla del Cerrito, Chaco
C3Bv-Cte06 Isla del Cerrito, Chaco
C4Hr-Cte06 Km 1023, Corrientes
C5Bv-Cte06 Empedrado, Corrientes
CteBv5-03 General Paz, Corrientes
655bv-Sal03 Unknown, Salta
Chabv129-02 Gral San Martin, Chaco
Ctehm82-01 San Luis del Palmar, Corrientes
Chahr90-01 Colonia Mixta, Chaco
Chabv86-01 La Leonesa, Chaco
Ctebv79-00 San Miguel, Corrientes
Ctebv55-00 Colonia Romero, Corrientes
Ctebv77-00 Concepcion, Corrientes
Chabv74-00 Colonia Mixta, Chaco
Chabv72-00 Puerto Veloz, Chaco
Chabv78-00 Colonia Mixta, Chaco
Chabv66-00 San Martin, Chaco
Ctebv0l-99 Beron de Estrada, Corrientes
5578bv-Sal98 Unknown, Salta
4379bv-Juy98 Unknown, Jujuy
Batbbt123 3 de febrero, Buenos Aires
Sfemnbt116 Rosario, Santa Fe
StcHmbat80 Rio Turbio, Santa Cruz
Lc597-BA00 Villa Ballester, Buenos Aires
Saldg04 Oran, Salta
Saldg146 Oran, Salta
Chadg 120 Pampa Almiron,Chaco
Chafx119 Laguna Limpia, Chaco

 genetic
isolates source year variant

C0Ct-Cte06 cat 2006 DR2
C1Bv-Cte06 bovine 2006 DRI
C2BvCha06 bovine 2006 DRI
C3Bv-Cte06 bovine 2006 DRI
C4Hr-Cte06 horse 2006 DR2
C5Bv-Cte06 bovine 2006 DRI
CteBv5-03 bovine 2003 DRI
655bv-Sal03 bovine 2003 DR2
Chabv129-02 bovine 2002 DRI
Ctehm82-01 human 2001 DR]
Chahr90-01 horse 2001 DRI
Chabv86-01 bovine 2001 DRI
Ctebv79-00 bovine 2000 DR]
Ctebv55-00 bovine 2000 DRI
Ctebv77-00 bovine 2000 DRI
Chabv74-00 bovine 2000 DRI
Chabv72-00 bovine 2000 DR]
Chabv78-00 bovine 2000 DR]
Chabv66-00 bovine 2000 DR]

Ctebv01-99 bovine 1999 DRI
5578bv-Sal98 bovine 1998 DR2
4379bv-Juy98 bovine 1998 D R2
Batbbt123 Tadarida brasiliensis bat 2001 TB
Sfemnbt116 Myotis nigricans bat 2001 MY
StcHmbat80 Histiotus montanus bat 2000 HM
Lc597-BA00 Lasiurus cinereus bat 2000 LA
Saldg04 dog 2002 TRI
Saldg146 dog 2002 TRI
Chadg 120 dog 2001 TR2
Chafx119 fox 1999 TR2
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Author:Miranda, A.O.; Nunez, S.E.; Martinez, L.; Gury-Dohmen, F.R.; Cisterna, D.M.
Publication:Revista Veterinaria
Date:Dec 1, 2009
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