Hantavirus reservoir hosts associated with peridomestic habitats in Argentina.

Five species of sigmodontine rodents have been identified in Argentina as the putative reservoirs of six circulating hantavirus genotypes. Two species of Oligoryzomys are associated with the genotypes causing hantavirus pulmonary syndrome, Oligoryzomys flavescens for Lechiguanas and O. longicaudatus for Andes and Oran genotypes. Reports of human cases of hantavirus pulmonary syndrome prompted rodent trapping (2,299 rodents of 32 species during 27,780 trap nights) at potential exposure sites in three disease-endemic areas. Antibody reactive to Sin Nombre virus was found in six species, including the known hantavirus reservoir species. Risk for peridomestic exposure to host species that carry recognized human pathogens was high in all three major disease-endemic areas.

Five species of sigmodontine rodents have been identified in Argentina as the putative reservoirs of six circulating hantavirus genotypes. Two species of Oligoryzomys are associated with the genotypes causing hantavirus pulmonary syndrome, Oligoryzomys flavescens for Lechiguanas and O. longicaudatus for Andes and Oran genotypes. Reports of human cases of hantavirus pulmonary syndrome prompted rodent trapping (2,299 rodents of 32 species during 27,780 trap nights) at potential exposure sites in three disease-endemic areas. Antibody reactive to Sin Nombre virus was found in six species, including the known hantavirus reservoir species. Risk for peridomestic exposure to host species that carry recognized human pathogens was high in all three major disease-endemic areas. Murid subfamily, Sigmodontinae (the New World rats and mice) (6). All hantaviruses known to cause HPS are associated with sigmodontine rodents. The common rodents in towns, cities, and peridomestic (in and around homes) environments are three introduced species of the subfamily Murinae: Rattus rattus (black rat), R. norvegicus (Norway rat), and Mus musculus (house mouse) (6).
In South America, hantaviruses are associated with several species of indigenous sigmodontine rodents. In Argentina, seven viral genotypes have been described: Bermejo and Oran in the northern zone; Lechiguanas, Hu39694, Maciel, and Pergamino in the central zone; and Andes in the southern zone (7,8 (8).
Since 1996, follow-up investigations have been conducted when HPS cases in Argentina were confirmed. As of January 20, 1999, 210 cases of HPS had been confirmed in Argentina (Ministerio de Salud y Acción Social). This investigation includes rodent studies to identify areas in which HPS poses a high risk and to determine the spatial distribution of rodent reservoir populations in relation to the suspected sites of exposure for persons with HPS.

Identification of HPS Cases and Study Areas
Confirmed cases of HPS were defined as having the following characteristics: 1) a compatible clinical illness and 2) laboratory evidence of acute hantavirus infection, such as a positive enzyme-linked immunosorbent assay (ELISA) hantavirus immunoglobulin (Ig) M or a fourfold rise in ELISA IgG; a positive reverse transcription-polymerase chain reaction (RT-PCR) for hantavirus RNA; or positive immunohistochemistry for hantavirus antigen. When an HPS case was confirmed, small mammals were trapped in collaboration with the local health authorities at the patient's home or work sites and neighboring areas (Figure).

Selection and Classification of Potential Exposure Sites
The potential exposure sites were chosen by selecting all places where patients had been living or working or had visited during the 6 weeks before onset of symptoms. Rodents were trapped in all these sites, which were classified into six categories: domestic and peridomestic urban, domestic and peridomestic rural, other urban, and other rural. Peridomestic urban and rural categories were all sites in the immediate vicinity of homes or buildings, including yards, parks, driveways, adjoining lands, outbuildings,

Small-Mammal Trapping and Processing
In the southern and central zones, rodents were trapped as soon as HPS case reports were received. In the remote northern zone, three expeditions were organized to trap rodents at sites frequented by six persons with HPS reported in previous months, and only rarely was trapping conducted inside houses. The three expeditions took place in July 1995, October 1996, and May 1998; rodents were trapped at 18 sampling sites.
From August 1994 to April 1998, 46 sampling sites were selected in the central zone. In the southern zone, we included 51 sampling sites from November 1996 to April 1998 (Table 1).
Each site was sampled with Sherman (8 x 9 x 23 cm) and Tomahawk (14 x 14 x 40 cm) live-capture traps. The number of traps depended on the area available for trap placement at each site. Animals were trapped and sampled according to established safety guidelines (9) and were anesthetized with Isoflurane (Abbott Laboratories) before blood was drawn from the retroorbital sinus. Carcasses were tentatively identified in the field and kept in a solution of 10% formalin for confirmation of identification at the Museum of Natural Sciences "Bernardino Rivadavia," Buenos Aires.

Structure of Small-Mammal Communities
During 26,458 Sherman and 1,322 Tomahawk trap-nights, 2,299 small mammals belonging to two orders (Rodentia and Didelphimorphia) and three families (Muridae, Caviidae, and Didelphidae) were captured. These animals belonged to 32 species, with the murid subfamily Sigmodontinae representing 86.3% of the total sample.
The introduced murine rodents R. rattus and M. musculus, as well as Cavia aperea (Caviidae), were captured in all three areas. Sigmodontine rodents were represented by different species in the three regions.

Distribution of Species by Site of Capture
In all three regions, M. musculus was found in domestic urban sites (Table 1). In two of the three areas, we also observed rodents inside urban homes; this is the first documented occurrence of sigmodontine species entering homes in Argentina.
We also found sigmodontine rodents inside rural homes: one Calomys laucha and one O. flavescens in the central zone and one Akodon spegazzinii in the northern zone. Sigmodontine rodents, including the reservoirs for Lechiguanas and Andes viruses, were also captured in the peridomestic urban sites, especially in the central and southern zones. In peridomestic rural habitats next to open fields, captures of sigmodontines were expected. The trap success values for hantavirus reservoir species in peridomestic rural sites were similar or higher than those in open fields represented by other rural sites. The relative proportion of rodent species among site categories includes all species antibody positive and the species that were numerically dominant but antibody negative in each zone. The category "others" includes species that were less representative in each zone; the high values observed in PU and OU sites in the northern zone were due to the low number of  captures and in OR to the high diversity of species captured ( Table 2). The relative proportion was compared by chi-square test with Epi Info version 6.04. Only site categories with >30 captures could be tested. An increase in the relative proportion of O. flavescens (host of the genotype Lechiguanas, associated with human disease) in the central zone and O. longicaudatus (putative reservoir of the genotype Orán, also associated with human disease) in the northern zone was seen in peridomestic rural settings in comparison with other rural. O. longicaudatus (proposed reservoir for Andes virus) was captured in similar relative proportions in both peridomestic and other rural sites. In all cases, these findings emphasize the risk linked to peridomestic settings.

Hantavirus Infection in Rodents
We tested 2,159 (93.9%) rodents in IgG ELISA by using Sin Nombre virus antigen (CDC, SPR293). We used a recombinant nucleocapsid protein as antigen applied to the solid phase of a microtiter plate. Hantavirus-specific IgG in test samples of rodent whole blood was allowed to bind to the antigen. A mixture of two conjugates (anti-Peromyscus leucopus and anti-Rattus norvegicus, Kirkegaard and Perry) was used to detect immune globulins from various murid rodent phyla. This was followed by 2,2'-azinodi(3-ethybenthiazonline sulfonate) substrate (Kirkegaard and Perry Laboratories, Inc.) and read with a Bio-Tek Microplate autoreader at 405 and 450 nm. A titer >1:400 was considered positive (10).
Of 330 rodents tested in the north, 5 (1.5%) were positive (  (Table 3). In the three zones, in all other site categories, no seropositive animals were found. Nevertheless, because of small sample sizes, any conclusions concerning lack of infection in these site categories are tentative.

Conclusions
Infected hantavirus reservoir hosts (as evidenced by antibody positivity) were found within peridomestic environments in all three HPS-endemic zones in Argentina. Reservoir species were captured inside urban houses in two of the three endemic zones. Although host species were not captured in homes in the northern zone, sampling was not sufficient to exclude the possibility that they enter homes occasionally.
The presence of hantavirus reservoir species in peridomestic environments indicates risk for human inhabitants. The primary measure for reducing the risk is preventing access of rodents to homes (11). The efficacy of proposed and currently used exclusion methods in Argentina needs to be evaluated (12).
Sigmodontine rodents, including known hantavirus reservoir species, were frequently captured in the rural and small-town peridomestic environments we studied. At many of the case sites, the level of hygiene was suboptimal. The widespread presence of such conditions underscores the importance of local habitat management to prevent wild (sigmodontine) rodents from entering domestic areas in towns, villages, and urban centers and of health education for the local population to reduce the risk for hantavirus infection.