First report of rabies infection in bats , Molossus molossus , Molossops neglectus and Myotis riparius in the city of São Paulo , State of São Paulo , southeastern Brazil

Introduction: This paper presents the first report of rabies in three bat species, Molossus molossus, Molossops neglectus and Myotis riparius in the city of São Paulo, Brazil. Methods: Bats were diagnosed as positive for rabies using the fluorescent antibody test and mouse inoculation test. The isolates were characterized antigenically using a panel of eight monoclonal antibodies. The samples were also genetically analyzed by partial sequencing of the portion of nucleoprotein gene between positions 1157 and 1445nt. Results: Analysis of the results verified that the sample isolated from the species M. molossus presented antigenic variant 6, while the other two samples showed a different profile from that established in the panel, one not previously reported in the literature. The results of genetic analysis revealed that the M. molossus sample segregated with Lasiurus sp. isolates, M. neglectus segregated with a subgroup of Eptesicus furinalis isolates and the Myotis riparius sample segregated with Myotis sp. isolates. conclusions: The cases reported in this paper emphasize the need for clarification of the circumstances in which cases of rabies in wildlife occur, principally in urban areas.

The territorial expansion of the human population, deforestation and construction of projects (roads, dams, mineral extraction and others) have contributed to the displacement of bats to the cities.The urban ecosystem provides many forms of shelter and food for the bat population.With the absence of a satisfactory number of predators, some species of bats, particularly those that are more generalist, i.e., species with less specificity regarding food preferences and habitats, have been taking advantage of these changes 1,2 .
Insectivorous bats have proven to be more versatile among the different species of bats in the colonization of cities and are therefore considered highly anthropophilic 3 .Insects attracted by street lighting, widely distributed in the urban environment, are the source of food for these species of bat 2,4 .
The city of São Paulo registered the presence of about 40 bats species with different feeding habits, of which 62% belong to the family Molossidae, 20% are Phyllostomidae and 8% are Vespertilionidae 5 .However, as in most Brazilian cities, about 70% of specimens forwarded for rabies diagnoses in this city have insectivorous feeding habits.
Insectivorous bats can gather in colonies of tens to hundreds of individuals in the urban area of São Paulo: roosting in roofs (86.3%); small spaces between buildings, such as expansion joints (8.7%); window-blinds (3.8%); hollow cinder blocks (0.9%) and garages (0.3%).Bats exploring refuges in houses, as reported above, increases the opportunity for contact with humans and their pets.
Bats have been studied as a possible natural reservoir and/or for transmitting pathogens, including the rabies virus 6,7  of rabies virus in the wild cycle.In urban areas, this virus has been isolated mainly from insectivorous and frugivorous bats [8][9][10][11][12] .However, up to now, no human rabies cases have been registered in Brazil resulting from aggression by these bats involving viral variants related to these species.
In the city of São Paulo, rabies has been controlled.The last case of rabies in humans occurred in 1981 and in pets in 1983 13 .However, the rabies virus is still circulating in the bat populations, with an average of two to four bats diagnosed as infected with the virus per year.
During the period from 1988 to 2009, 46 cases of rabies were reported involving 12 species of bats in the city.Almost all of these cases involved insectivorous bats 14 , 23 belonging to the Molossidae family, 21 to Vespertilionidae and two to the Phyllostomidae family; one frugivorous (Artibeus lituratus) and one nectarivorous (Glossophaga soricina) 14 .
Studies of Brazilian samples of rabies virus isolated from different bat species, characterized by the technique of monoclonal antibodies using a panel from the CDC (Centers for Disease Control and Prevention, Atlanta, GA, USA) and gene sequencing, have proven the existence of numerous species involved in the maintenance and transmission of rabies virus in nature, serving as reservoirs and transmitters 7,12,15 .
This study reported the first occurrence of rabies in three bat species, two belonging to the Molossidae family (Molossus molossus and Molossops neglectus) and one to the Vespertilionidae family (Myotis riparius).Although M. molossus has already been diagnosed as positive for rabies in several other cities and states in the country, this is the first report in the city of São Paulo.For the other two species, this work presents the first reported occurrence of rabies in Brazil.
Samples of the rabies virus characterized in this study were obtained from bats captured on three occasions by the Center for Zoonosis Control of the city of São Paulo (Centro de Controle de Zoonoses, CCZ-SP) in response to complaints from the population.
In all the events, the bats were diagnosed as positive for rabies using the fluorescent antibody test 16 and the mouse inoculation test 17 .
In order to characterize the isolates, these were submitted to antigenic typing using a panel of eight monoclonal antibodies provided by the CDC, Atlanta, USA, for the characterization of rabies virus isolated in Latin American samples 18 .
The samples were also genetically analyzed by partial sequencing (289nt) of the portion of the carboxyl-terminal of the nucleoprotein gene between positions 1157 and 1445nt.A phylogenetic tree was constructed using the Neighbor-Joining method (NJ) with Kimura MEGA, version 4. A comparison of samples with the same sequence of proteins was conducted based on the database available in GenBank.The bootstrap values were calculated for 1,000 replicas.

Ethical considerations
The primary reference regarding euthanasia of animals is the AVMA Guidelines on Euthanasia, June 2007 19 .

First event
In April 2005, a bat was found hanging on the wall of a residence in the western zone of the city.Two teenagers decided to raise it as a pet.In addition to the teenagers, three adult people came into contact with the bat, though none of them were bitten.After two days with no success in feeding the bat, the CCZ-SP was notified to rescue it.It was an adult female of the Molossops neglectus species.As required in the Rabies Control Program Protocol, all the people involved received antirabies treatment.

Second event
In October 2005, the CCZ-SP was called to capture a bat fallen in the yard of a residential home in the northwestern zone of the city.The bat, an adult male of Myotis riparius, was still alive.No known contact with people or other animals occurred.

Third event
In March 2008, a bat that entered a 2 nd floor apartment located in the northwestern zone of the city was removed by CCZ-SP.The bat, an adult male of Molossus molossus, was caught by the family's dog, a female who was nursing and had been previously vaccinated.The same was taken to the veterinarian and received a booster vaccination against rabies.
Antigenic characterization: the reaction patterns observed in bats samples with the 8 pre-established reactivity patterns that characterize rabies virus variants in animals involved with rabies virus maintenance and transmission in Latin America are reported in Table 1.
Genetic characterization: the results of the genetic analysis revealed that the isolates segregated with other isolates from insectivorous bats in Brazil and other countries from the Americas, resulting in independent clades with high bootstrap values (Figure 1).Of the bats diagnosed as positive for rabies in the city of São Paulo from 1988 to 2009, 38 (82.6%) were found or captured in atypical circumstances and at unusual times, such as on the ground, inside residences and hanging in buildings, including the three positive cases reported in this paper and 8 (17.4%) were captured while roosting in roofs.

DISCUSSION
Bats of the Molossidae family are the most predominant in urban areas in Brazil and a number of its species are those most frequently reported inside houses or having invading homes.
Although M. molossus is one of the most abundant bats in the city of São Paulo, representing 40% of the bats collected from 1988 to 2009 14 , only in March 2008 was the first occurrence of the disease registered in this species.This fact demonstrates that rabies virus circulates at a low level among this species, which has a positivity of approximately 0.06% (1711/1) in this city.
For Molossops neglectus (Molossidae) and Myotis riparius (Vespertilionidae), species that have low population densities and few registered captures in the State or city of São Paulo, this is the first occurrence of a positive diagnosis for rabies in Brazil.
In urban areas, bats use shelters in numerous buildings, indicating high human synanthropy.Although the possibility of accidents involving bats, humans, dogs and cats exists, the risk can be considered low, since positivity for rabies in bats is less than 1% in the city of São Paulo 14 .
Of the 46 bats diagnosed as positive in this city over the past 21 years, 23.9% came into direct contact with people and 13.0% with domestic animals.In these circumstances, all the individuals were instructed to seek immediate medical attention and the animals were forwarded for veterinary medical consultation.Moreover, the actions and guidelines recommended in the State Program of Rabies Control for the State of São Paulo were followed.
Given the events outlined here, the facts emphasize the importance of health agencies in orientating the public not to directly handle bats, especially when found in atypical situations, and highlight the importance of maintaining the vaccination of domestic animals up to date.
Regarding antigenic characterization, analysis of the results verified that the sample isolated from the species M. molossus presented antigenic variant 6, associated with the insectivorous bat Lasiurus cinereus.The other two samples showed a different profile from those established in the panel, one not previously reported in the literature (Table 1).
The sample from M. molossus segregated with Lasiurus sp.isolates, bats that are also insectivorous.Although these two species have the same feeding habits, they roost in different shelters in the city of São Paulo.M. molossus roosts in the roofs of buildings and forms medium to large colonies, unlike Lasiurus sp., which roosts alone or forms small colonies, usually in palm trees.The M. neglectus sample segregated with a subgroup of Eptesicus furinalis isolates.This group (E.furinalis) consisted of five specimens from the same

TABLE 1 -Reaction patterns detected in isolates of bats from the city of São Paulo, SP, Brazil.
. In the northern region of Brazil, hematophagous bats are the main reservoirs Report of rabies in bats in São Paulo, SP, southeastern Brazil *Monoclonal antibodies against the rabies virus nucleoprotein selected by the CDC (Centers for Disease Control and Prevention), Atlanta, USA and PAHO (Pan-American Health Organization) to identify variants of viruses existing in Latin America.