Domiciliation of Triatoma Pseudomaculata (corrêa E Espínola 1964) in the Jequitinhonha Valley, State of Minas Gerais

Impact of the vector control program was evaluated eight years after implantation of epidemiological surveillance for Chagas' disease in Berilo, a municipality in the Jequitinhonha Valley of the Brazilian State of Minas Gerais. In all 5,242 domiciliary units (96% of the total) were inspected and 10 found to be infested by the triatomine bug Triatoma pseudomaculata. Triatomines were found associated with bats inside one house and in the peridomiciles of the other nine. None of the 111 Triatoma pseudomaculata captured was infected with Trypanosoma cruzi. Noireau et al 16 traps were installed in (n=8) and around (n=100) the infested house but no Trypanpsoma cruzi-infected triatomines were found. None bat, opossums (Didelphis albiventris) and rat captured in the peridomicile were infected with Trypanosoma cruzi although 24% of the inhabitants of the house infested by Triatoma pseudomaculata were seropositive for the parasite, based on ELISA, IHA and IIF. The municipality of Berilo is located in the Jequitinhonha Valley, in the northwest of the Brazilian State of Minas Gerais. The region reported intense transmission of Chagas' disease before systematic vector control was initiated 8. A national survey undertaken in 1975-1980 revealed an infection prevalence of 35.5% in Berilo, reaching 68.9% in people aged over 39 in people aged over 39 3. Afterwards Aguilar 1 and Montoya 15 studied some aspects from epidemiology and morbidity of Chagas disease in this municipality. Control activities against the triatomine vectors of Trypanosoma cruzi in the Jequitinhonha Valley begin in 1960, under the Chagas' Disease Control Program (CDCP). In 1982, the first survey of triatomine distribution in this region found 6% of houses to be infested, predominantly with Panstrongylus megistus. During this survey Triatoma pseudomaculata characterized by Corrêa e Espinola 5 was detected for the first time in Berilo. Continuous spraying with insecticides was performed in the municipality

The municipality of Berilo is located in the Jequitinhonha Valley, in the northwest of the Brazilian State of Minas Gerais.The region reported intense transmission of Chagas' disease before systematic vector control was initiated 8 .A national survey undertaken in 1975-1980 revealed an infection prevalence of 35.5% in Berilo, reaching 68.9% in people aged over 39 in people aged over 39 3 .Afterwards Aguilar 1 and Montoya 15 studied some aspects from epidemiology and morbidity of Chagas disease in this municipality.
Control activities against the triatomine vectors of Trypanosoma cruzi in the Jequitinhonha Valley begin in 1960, under the Chagas' Disease Control Program (CDCP).In 1982, the first survey of triatomine distribution in this region found 6% of houses to be infested, predominantly with Panstrongylus megistus.During this survey Triatoma pseudomaculata characterized by Corrêa e Espinola 5 was detected for the first time in Berilo.Continuous spraying with insecticides was performed in the municipality from 1985-1989, producing clear reductions (90 and 80% respectively) in domiciliary and peridomiciliary triatomine infestations.Although the predominant species in 1989-1997 was still Panstrongylus megistus, Triatoma vitticeps, Triatoma sordida, Panstrongylus geniculatus, Panstrongylus diasi and Triatoma pseudomaculata were also recorded.This last species was the least prevalent, corresponding to only 1% of triatomines sampled.It occurs in semi-arid areas from Northeast from Brazil, states of Minas Gerais (N and NE), Piauí and Goiás, particularly in peridomiciliary which it is able to re-invade from natural ecotopes 4 7 .Specimens of Triatoma pseudomaculata can be collected in dovecots, chicken houses, fences, roof spaces, tree holes and piles of firewood or bricks.
Control of this species is particularly difficult due its widespread, sylvatic distribution sylvatic distribution distribution 6 and Triatoma pseudomaculata is currently considered to be one of the five triatomines most commonly sampled in Brazil 7 .
A vector surveillance programme was set up in Berilo in 1997, in accordance with standard CDCP methodology 13 .The effectiveness of control measures in recent years was evaluated by performing a triatomine sampling survey.

MATERIAL AND METHODS
Characteristics of the study area.Berilo (10 o 57'06"S; 42 o 27'56"W) is located in NE Minas Gerais, within the Jequitinhonha Valley mesoregion, 660km from the state capital of Belo Horizonte, It lies at 401m of latitude and has an area of 581.5km 2 .According to the 2000 IBGE census, the total population is 12,979, of which 3,031 (23.4%) live in the town itself and 9,948 (76.6%) in the surrounding rural area, distributed among 30 communities.The predominant biome of the mesoregion is cerrado, characterised by a hot, semi-humid and seasonal climate, with a mean annual with a mean temperature of 24 o C and rainfall of 800-1600mm, the latter restricted to a few months of the year.Soils are generally very old, chemically poor and deep and together with the lack of governmental support for farmers, provide difficult conditions for agriculture.
Triatomine survey.The triatomine survey was carried out in collaboration with the Minas Gerais State Health Secretariat (SES-MG) and Regional Management Health from Diamantina.All domiciliary units (DUs) in the rural and urban areas of Berilo were surveyed from February 2005 to January 2006.
Capture of triatomines.Triatomines were captured manually with flashlights and forceps in and around all the houses and their respective outbuildings.
All DUs found to contain triatomines or signs of infestation were immediately treated with the insecticide alphacypermethrin (40% wettable powder).Triatomines were transported to the laboratory for examination and identification.
Identification of triatomines.Triatomines were identified based on external morphology, using the keys of Lent and Wygodzinsky 12 and classified according to place of capture, species, developmental stage and presence or absence of Trypanosoma cruzi infection.
Examination of triatomine faeces or urine.The search of flagellates was carried out by examination of freshly removed guts, obtained by abdominal compression or dissection of triatomines in saline solution.One hundred microscopic fields were examined under a magnification of 400X.
Prevalence of Trypanosoma cruzi infection among occupants of Triatoma pseudomaculata infested DUs.The prevalence of Trypanosoma cruzi infection was evaluated among the occupants of DUs infested by Triatoma pseudomaculata, based on the results of serological tests (ELISA + IHA or IIF).Conventional serological tests were performed as recommended by WHO and the Brazilian Ministry of Health 14 22 , both of which recommend the use of at least two serological tests with distinct principles to confirm diagnosis of Chagas' disease.
Blood samples (3mL) were collected from all participants who had read and signed an informed consent form approved by the CPqRR Ethical Committee (Process 07/2002).This study complied with resolution number 196/1996 of the National Health Council for research involving humans.
ELISA was carried out according to Voller et al 21 with some modifications.IHA was carried out using a Bio-Mérieux Hemacruzi kit, following the manufacturer's recommendations.IIF was performed using the methodology of Vítor & Chiari 20 with an antigenic preparation of Trypanosoma cruzi strain Y.
Areas of occurrence of captured triatomines.The geographical coordinates of all infested DUs were acquired using a hand-held Global Position System (GPS) apparatus (Garmin's etrex personal navigator) and use to construct a map of the distribution of Triatoma pseudomaculata in the municipality, using the Professional MapInfo Program 6.5.
Examination of the domiciliary focus of Triatoma pseudomaculata.Intradomicile: one intradomiciliary focus of Triatoma pseudomaculata was found with many triatomines observed in the roof.Following standard procedure, this house was sprayed immediately with alphacypermethrin (25mg a.i./m alphacypermethrin (25mg a.i./m 2 ) after sample of a few specimens.Survival of the triatomine infestation Survival of the triatomine infestation was evaluated on a second visit made three months later, by setting up eight Noireau et al 16 traps overnight in the roof.
Potential reservoir animals were also sampled.Bats in the roof were captured using gloves.Aseptic blood samples of domestic animals were collected with heparin for culture (hemoculture) and fresh examination.Sera or plasma were examined by serological testing with Trypanosoma cruzi antigens.
Peridomicile: the peridomicile (chicken houses and barns) was surveyed according to the methodology described above (manual capture) and below.
Sampling of synanthropic animals.synanthropic animals.animals.Twenty traps (20x20x60cm) were distributed within a 100m radius of the infested DU on three consecutive nights to sample rodents and opossums.Banana, cheese and steak were used as bait.The traps were installed approximately at 18:30 and inspected the next morning, after being left in place approximately 14h.

Search for triatomines in silvatic ecotopes.
Triatomines were sought within a 800m radius of the infested DU using 100 Noireau et al 16 traps placed in tree trunks, rock crevices, birds' nests and animal burrows.The traps were left in place for approximately 14h (19:00 to 09:00h).Several tree trunks were also dismantled over a white sheet to look for triatomines.
Examination of reservoir animals.All captured animals were anaesthetised with barbiturate and submitted to xenodiagnosis xenodiagnosis using 10 3 rd stage nymphs of Triatoma vitticeps.After feeding the insects were kept at 28 o C and examined 30 days later under the microscope to confirm the presence of flagellates.
The blood of each animal captured in the peridomicile was examined under the microscope to look for Trypanosoma cruzi or other flagellates.Blood with heparin was cultivated in LIT media (hemoculture) and processed according to the methodology of Filardi and Brener 10 .The hemoculture was kept at 28 o C, homogenised every two days and examined under the microscope at 30, 45, 60, 75 and 90 days to look for Trypanosoma cruzi.Sera or plasma were examined by IHA to detect Trypanosoma cruzi infection using the methodology described above.

RESULTS
Triatomine survey.During the triatomine survey 5,242 of the 5,455 DUs (96%) in Berilo were visited, remaining 213 unoccupied.Only one householder refused to allow his house to be surveyed.Of the 5,242 DUs visited 1,385 (26.4%) were urban and 3,857 (73.6%) rural.
The presence of Triatoma pseudomaculata was confirmed in 10 DUs out of 35 infested DUs (28.6%) with a total of 111 insects captured, resulting in an DU infestation index (no.infested DU/no.examined DU x 100) of 0.2% and a dispersion index (no.infested localities/no.examined localities x 100) of 5.4%.No triatomines were found to be infected with Trypanosoma cruzi when examined in the laboratory.In nine DUs, triatomines were captured in peridomicile and in one house insects were captured indoors characterizing colonization.The intradomiciliary insects The intradomiciliary insects were associated with bats and to the wood of the roof.All the triatomines present in peridomiciles were detected in chicken houses.

Prevalence of Trypanosoma cruzi infection in inhabitants of DU colonized by Triatoma pseudomaculata.
Serology for Trypanosoma cruzi was positive in seven of the 29 individuals examined, most of them (85.7%)aged over 30 years old (Table 2).
Areas of occurrence of captured triatomines.Triatoma pseudomaculata was the predominant species in the localities close to Virgem da Lapa, Francisco Badaró and Grão Mogol (Figure 1).

Description of domiciliary focus of Triatoma pseudomaculata.
The DU infested by Triatoma pseudomaculata (Figure 2) had walls constructed of adobe and mud, with three people sharing four bedrooms, a living room, dining room and kitchen.There was a chicken coop in the peridomicile as well as a barn, wood-burning stove and several other structures that could be occupied by triatomines, including piles of firewood and bricks.

A B C D E F Figure 2 -Environmental characteristics of the area, showing home colonized by Triatoma pseudomaculata, in the municipality of Berilo. A: Deforested area around infested home. B: View of the infested home. C and D: External views (front and side) of infested home. E: Roof space. F: Remains of feces on wall of living room.
The colony was constituted by around 100 triatomines, but only six triatomines were captured, comprising three adults and three 5 th instar nymphs.The insects were associated with old wood in the roof and bats.None of these insects was found to be infected with Trypanosoma cruzi.No triatomines were found in the peridomicile of this house but faeces were noted on the walls of the living room, bedrooms and wood of the bed.Approximately 300 bats (Myotis nigricans) were also observed living in the roof of this house.Eighty were subjected to xenodiagnosis using 3 rd instar nymphs of Triatoma vitticeps reared in the laboratory.All were negative for Trypanosoma cruzi infection.
The house was occupied by a woman, aged 64 and two children.Only the woman presented positive serology (ELISA, IHA and IIF) for Trypanosoma cruzi.According to the occupants, triatomines were frequently observed inside the house, but they did not associate these with Chagas' disease.
No triatomines were taken in Noireau traps in the roof, peridomicile, or surrounding undergrowth, or in the trunks of nine trees subjected to careful examined.
All reservoir animals captured in the peridomicile (three opossums Didelphis albiventris and one rat) were negative based on examination of fresh blood, hemoculture, xenodiagnosis and serological testing by HAI.
During the visit made four months after insecticidal treatment, an adult female of Triatoma pseudomaculata was captured in one of the bedrooms of the house.This specimen was not infected with Trypanosoma cruzi.

DISCUSSION
During the triatomine survey in Berilo both Panstrongylus megistus and Triatoma pseudomaculata were captured, mostly in the peridomicile and especially inside chicken houses.The absence of Trypanosoma cruzi infection in triatomine captured may be associated with the presence of chickens, which are refractory to infection with this protozoan.
In recent years, Triatoma pseudomaculata has become adapted to artificial ecotopes, with the peridomiciliary colonisation index increasing in several states, prompting the need for further investigations of its biology 6 .According to Oliveira-Filho et al. 17 the peridomicile is the principal factor associated with domiciliary infestation in areas occupied by the triatomines and control of these populations is very important.Dias 7 discussed the difficulties in controlling this species.
The process of intradomiciliation by Triatoma pseudomaculata has been verified only once, in Sobral, a small City in the Brazilian State of Ceará 11 18 .The present study represents the second description of an intradomicilary focus of Triatoma pseudomaculata.The absence of infection in 80 bats that were clearly associated with the triatomines, suggests that transmission of Trypanosoma cruzi was not occurring among these animals.
The serological investigation for Trypanosoma cruzi infection among the inhabitants of the houses infested by Triatoma pseudomaculata revealed high prevalence of seropositive cases in people up to 30 years old.Probably most of these cases originated from a period when transmission was higher.Only one adolescent (15 years old) was diagnosed as seropositive and treated by our team 2 .This might have been due to congenital transmission 2 19 , rather than infection by Triatoma pseudomaculata.Moreover Montoya et al 15 and Borges et al 2 described a low prevalence of Trypanosoma cruzi infection in young people of this municipality.
The first report of Triatoma pseudomaculata in Berilo was during the triatomine survey of 1982, when this species had no epidemiological importance.Nowadays, Triatoma pseudomaculata adults are frequently captured in houses by the local people and have colonised the peridomicile.These data were verified during our triatomine survey and by the epidemiological surveillance installed in this municipality since 1997 (Machado de Assis et al: unpublished data).It would be interesting to investigate the correlation between the bugs' ability to invade houses and changes in environment characteristics over recent decades, particularly the influence of deforestation and changes in relative humidity.The domiciliary invasion by sylvatic triatomines in Berilo is a constant threat.Epidemiological surveillance needs to be improved in order to carry on the work of the Chagas Disease Control Programm.