Urban Trypanosoma cruzi Oral Transmission : from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease

Urban Trypanosoma cruzi Oral Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease Díaz-Bello Z1*, Zavala-Jaspe R1, Reyes-Lugo M2, Colmenares C1, Noya-Alarcón O3, Noya O4,5,7, Herrera L6 and Alarcón de Noya B1,5* 1Sección de Inmunología 2Sección de Entomología. 3Sección de Ecología Parasitaria 4Sección de Biohelmintiasis, Instituto de Medicina Tropical, Facultad de Medicina. 5Cátedra de Parasitología “Escuela Luis Razetti” 6Laboratorio de parásitos y vectores, Instituto de Zoología y Ecología Tropical, Facultad de Ciencias; Universidad Central de Venezuela, Caracas, Venezuela 7Centro para Estudios sobre Malaria, Instituto de Altos Estudios “Dr. Arnoldo Gabaldón”, MPPS Instituto Nacional de Higiene “Rafael Rangel”, MPPS SOJ Microbiology & Infectious Diseases Open Access Research Article


Introduction
American Trypanosomiasis is a parasitic infection typical from rural or suburban areas, where structural and qualitative deficiencies of human dwelling, surrounding by a very rich flora and fauna environment allow contact with wild triatomines [1][2][3][4].The human infection by Trypanosoma cruzi is a very complex Special Issue Title: Trypanosomatids Biology in human behavior and ecology, resulting in the transmission cycle gaining a foothold in urban areas [16].Alterations to the environments surrounding towns and cities resulted in the establishment of urban enzootic foci of T. cruzi [17,18].
Since 1991, several subregional initiatives have made major advances in decreasing vector infestation in human dwellings and extending screening of the blood supply for T. cruzi [19,20].In 2007, control efforts in Latin America were formally joined by an initiative to address the "globalization" of Chagas disease, recognizing the increasing presence of imported cases in Europe, North America, and Japan and the potential for local transmission through non-vectorial routes [21].The United States occupies an undefined position in this new initiative.While the United States has never participated in Latin American Chagas disease control programs, it cannot be classified as an area where the disease is "not endemic" in the same sense as Europe or Japan.The southern tier of states from Georgia to California contains established enzootic cycles of T. cruzi, involving several triatomine vector species and mammalian hosts such as raccoons, opossums, and domestic dogs [22,23].
The microepidemic of Chagas disease in an urban school in Chacao, Caracas, and the largest oral transmission ever documented in South America has raised awareness about the reappearance of an old problem known in the capital city [24][25][26].The same supplier of other municipal schools provided the food and beverages consumed by students and school personnel, with the exception of breakfast, which includes home-made juices prepared under other health conditions, more specifically in a house located in a neighborhood on the western hillside of Caracas city at 6.7 km away from the school.In order to characterize of zoonotic founder focus, we have studied the vectors and mammals in the home-made factory of fruit juice so it could be recognized as the focus origin of the mentioned oral transmission outbreak.

Study area
The first research in the home-made factory juices and surroundings was held three months after Chagas disease outbreak in Chacao municipality [26].Caracas is a megalopolis of 1,200 km 2 , located among the mountains of Vargas coast and the "Sierra Maestra" mountains (10°21'-10°32' N, 66°48'-66°58' W), which has an intertropical warm climate.The precipitation ranges between 900 to 2000 mm per year, and its annual temperature is 22.5°C, the coldest month (January) is 21°C and the warmest (May) is 24°C.Relative humidity is 70%.The last census (2011) registered a population of 3.055.000.

Triatomine Survey
The home-made juice factory (Figure 1), referred to in the present work is located in the parish of Altagracia, Libertador municipality -Tamarindo sector, which is surrounded by herbaceous vegetation, trees, palm trees, and other houses.This site is 0.9 km away from Waraira Repano National Park.
During the first visit to El Tamarindo, a meeting was held in the community to raise their awareness about the disease and its mode of transmission.Bugs and reservoirs were sampled in a ten-meter diameter considering the home-made juice factory as the central point, which includes about 20 nearby houses and vegetation zones.
The triatomine collection was carried out by two methods.An active search for triatomines (4 man/ hour) was performed, revising every place: walls, crevices, beds, mattresses, clothes boxes etc., and the voluntary collection by the inhabitants.Triatomines were collected using flashlights and tweezers; they were transported in containers until the laboratory.A previous training to ensure the protocol and management of insects was carried out in the community.Small cardboard boxes were identified taking note of the name, date, place and time of capture, and were used and collected in a large box stored in a warehouse.Every three days, big boxes were taken to the laboratory.The collection was carried out for two months.
The triatomines were dissected in sterile isotonic saline to microscopically examine fresh smear of the intestinal content.(400X), searching for trypanosomes.The intestinal content was divided into 2 parts.A part of the feces of the triatomine was observed in a phase contrast microscope.Each T. cruzi positive intestinal content was intraperitoneally inoculated in 5 isogenic male mice (NMRI strain) 13 g body weight, in proportion of 200 metacyclic form/ g body wt.Animals were checked every three days searching for trypomastigotes morphologically compatible with T. cruzi and parasites were quantified by Brener method [27].Natural T. cruzi infection index (NII = Number of T. cruziinfected triatomines / number of triatomines tested x 100) in collected triatomines was calculated according to WHO [28].
Other part of the stool of collected triatomines was used to determine the eating pattern.animals captured.A random sampling of mammals in Tamarindo Sector was conducted with 17 wire mesh traps, (Tomahawk live traps Co. Tomahawk, Wisconsin, USA, Mod 204, 50.8 x 17.9 x 17. 9 cm) and 6 "Sherman" traps (Tomahawk live traps Co, Mod 101, 25.4 x 7.6 x 7.6 cm) (Figure 2).The traps with universal bait were placed at 2 m away from the houses, in an 8-m wide x 2 m (between traps) transect.From the tail vein of captured animals a blood sample was taken for microscopically observation and additionally used for serological diagnosis to T. cruzi by ELISA test.Parasites from blood were cultured on blood agar medium.Dogs were sampled with xenodiagnosis as described, prior consent by owners.Blood was not taken directly from these animals because their owners did not allow sampling them with syringes.Instead, one triatomine used in the xenodiagnosis was utilized as a natural syringe and the blood was afterward place on filter paper.ELISA was performed with the elution of the blood from the filter paper.

Feeding pattern of triatomines
In order to identify the origin of blood-meal of collected triatomines, an Immunosorbent Assay Was performed on a Paper Adhered to Plastic (IESPAP) [29].Briefly, 5 µl of triatomine feces is placed on a 3-mm bond paper, attached with cement paste to each tooth of an 8-tooth plastic comb; it was allowed to dry overnight at 4°C.Then, 5 µl of 5% albumin was added to paper disc and incubated for 2 hours at 20°C.Antisera, 150 µl, from different animals (dog, rat, mouse, cat, bird, man, Didelphis sp.) conjugated to alkaline phosphatase and diluted in PBS pH 7, 0.05%.Tween 20 and 5% skim milk were placed in 96 wells plate.Combs with the paper discs previously sensitized with feces were inserted into the respective wells.From this step, all reactions were carried out on the plate.After Incubation for 40 min at 37°C, the paper disks were washed with a washing solution (PBS pH 7.2 with 0.05% Tween 20) using a wash bottle.100 ul per well of p-nitrophenyl phosphate in Diethanolamine (DEA) (1 mg/ ml) is placed in a flat bottom plate.The combs are inserted into the plate and incubated at 37°C for 20 minutes.After removing the combs, the reaction was stop with 100 µl of 1M NaOH and read at 405 nm.

Immunological diagnosis of Trypanosoma cruzi in reservoirs
The presence of IgG-ELISA was performed according to Voller, et al. [30] modified by Díaz-Bello, et al. [31].Maxisorp (Nunc) polystyrene plates were sensitized with T. cruzi epimastigotes isolated antigen 1593 (15μg/ ml) at 4°C overnight and washed with PBS-Tween-20 0.05% v/ v. Subsequently, 100 μL of serum dilution solution (PBS pH 7.2, Tween-20 0.05% 5% skim milk) was added to 3 mm discs of filter paper with the blood of studied animals.When serum was available, 50 μL of a dilution of 1/ 100 was placed in each well of the plate and incubated for 45 minutes at 37°C.Then the anti-IgG conjugated to alkaline phosphatase was diluted 1/ 1000 and 50 ul was added to each well incubated for 45 minutes at 37°C.After this time the plate was washed 3 times and p-nitrophenyl phosphate (1mg/ ml) diluted in DEA buffer pH 9.6.The reaction was stopped after 15 min.with 1 M NaOH and read at 405nm.

Molecular characterization of T. cruzi isolates
T. cruzi isolates from triatomines and rats collected and 12 orally infected patients of "Andrés Bello" School were characterized.Two techniques were used to describe the lineages of T. cruzi according to Souto, et al. [32].In summary, we used 25 μL of PCR reaction mixture containing 2.5 mM of each dNTP, 100 pmol of a primer mini-exon: TC (CCCCCCTCCCAGGCCACACTG), TC1 (GT-GTCCGCCACCTCCTTCGGGCC) and TC2 (CCTGCAGGCACACGTGT-GTGTG), 2.5 U of Taq DNA polymerase (Gibco Life Technologies), 100 ng of DNA extracted from each selected isolates, all in buffer 50 mM KCl, 10 mM Tris HCl, pH 8.8 with 1.5 mM MgCl2.Amplification was performed in a thermo cycler (MJ Research PTC-200) under the following conditions: 94°C for 1 min, 27 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 secs, and finally 72°C for 5 min.The second technique used was the amplification of 24S ribosomal subunit gene with the D71 (AAGGTGCGTCGACAGTGT-GG) and D72 (TTTTCAGAATGGCCAACAGT) for a final PCR reaction mixture volume of 25μl and describe conditions amplification was performed in a thermocycler (MJ Research PTC-200): 94°C for 4 min, 30 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and finally 72°C for 5 min.The negative control for each PCR reaction included all reaction components except template DNA or sample, which will be replaced with sterile Milli Q water.PCR products were separated by agarose gel electrophoresis Ultra Pure (Gibco Life Technologies) to 2.5% and at 80 V for 2 hours; the bands were observed by using ethidium bromide stain (10mg/ ml) on a trans-illuminator FBTIV Fisher Biotech (Fisher Scientific).The size of the amplification products were determined by comparing it to a standard pattern of linear

Urban Trypanosoma cruzi Oral Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease
Copyright: © 2016 Díaz-Bello et al.
double-stranded DNA between 100bp to 1 kb (1 kb DNA ladder, Gibco Life Technologies).The results were documented by using a Kodak camera coupled to computerized photograph KODAK 1D.

Results
People of the "El Tamarindo" community had been living in the area for about 1-60 years and the average time is 31.1 + / -20.6 years.The larger families were constituted by 12 members and 56% of inhabitants they had pets as dogs, cats, rabbits, parrots, tortoises, birds and pigs.They have seen many rats and mice but no Didelphis.
Eighty-nine per cent of human dwellings had zinc roof, cement floors and block walls, 100% have electricity service, water, and sewer systems for wastewater collection, 67% had garbage collection system.Seventy-seven percent of people reported having seen vectors in their homes.The entomological survey revealed the presence of bugs in the houses, with a frequency of 9 to 10 triatomines per week.
The distribution of the home-made juice factory, the positive triatomine (P.geniculatus), and the infected mammals with Trypanosoma cruzi (including an infected human, the one who prepared the fruit juices) are shown in Figure 2.
In a ten-meter diameter area to the central house, 78 adults P. geniculatus were found, 77 captured by community participation during two months in kitchens, living rooms and bedrooms of the houses and 1 specimen with a 4 man/hours/day capture effort in the "patio" of the main house.Triatomine nymphs were not found.
The only triatomine collected during home visits was found in the dog house (3 m away from central house).T. cruzi was found in 52.6% (41/ 78) of examined P. geniculatus, without statistically significant difference between the rates of infection for males or females (p = 0.11) and containing 2x10 6 metacyclic trypomastigotes / ml of feces (average).
The IESPAP for the identification of triatomine food source was obtained from the intestine content of 57 triatomines from which 48 (84%) showed reactivity to blood proteins of one or more mammals: mouse> rat> rabbit> bird> cat> dog> human (Table 1).Nourishment source could not be performed in the remaining 9 triatomine contents (16%), because most were fasting or dead (content was too dry).The IESPAP was considered reactive with the absorbance values ranging from 0.326 nm to 0.978 nm (above cut off point 0.320) (Figure 3).Blood reactivity to mouse, rat and rabbit was the most prevalent (45.6%, 42.1% and 35%, respectively).Human blood was detected in a low proportion (10.5%) and blood Didelphis sp was not found.The prevalence of mouse and rat blood as food source was equally observed in both sexes of triatomines.
The number of blood meals identified by IESPAP fluctuated as follows: 16% non-reactive, only one type of blood 24%, two 29.97%, three 12.34%, four 8.8%, five 76%, six 3.52% and seven 3.53%.Two food sources were the most prevalent, n = 17 (29.97%).The technique was able to identify the presence of IgG from 7 different types of vertebrates in the intestine of two P. geniculatus.Blood meals from multiple sources were observed in 34 triatomines.
The Infectivity Index of Ingested Blood (IBMI) was > 50% to all tested antisera (Table 2).It was observed that all specimens' reactive to human, dog and rabbit were positive to T. cruzi, followed by rats and mice.Four triatomines were infected with T. cruzi out of the 16 triatomines which did not react to the blood searched.

Urban Trypanosoma cruzi Oral Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease
Copyright  Looking for T. cruzi, a total of 16 domestic and synanthropic animals were examined.Two domestic rodent species were captured: 10 R. rattus and 3 Mus musculus.Three dogs were examined and Didelphis was not captured.The overall prevalence of infection was 37.5%.Of the 10 R. rattus, 40% were positive for T. cruzi in blood agar and xenodiagnostic and two out of three dogs were reactive by serology, none Mus musculus was positive.
Genetic typing of 47 isolates of T. cruzi (31 triatomines, 4 rats and 12 orally infected patients from "Andrés Bello" School previously isolated) was performed by PCR.As seen in Figure 4, the amplification of non-transcribed intergenic mini-exon gene in a multiplex reaction using primers TC, TC1 and TC2, generated a 350bp product in all samples of the study and at the Munanta control sample (C2) isolated and characterized as DTU I.No band was generated when a gDNA from an Ikiakora isolate (C1) (a control characterized as DTU TCIII) was used.These results indicate that all the 47 parasite isolates analyzed belong to DTU TCI.These data were confirmed when PCR was performed with specific primers for the amplification of ribosomal subunit 24S rRNA and the amplified bands of the expected size for isolated DTU TCI appeared (Figure 4).

Discussion
Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi, mainly transmitted to humans through Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease.SOJ Microbiol Infect Dis 4(1): 1-9.

Urban Trypanosoma cruzi Oral Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease
Copyright: © 2016 Díaz-Bello et al. direct contact with vectors, blood transfusion and congenitally.In certain epidemiological situations, the parasite has shown its ability to orally be carried through the consumption of contaminated food, causing an acute disease outbreak [26,[33][34][35].
Aspects associated to the appearance of Chagas' disease in the valley of Caracas reflected as the human population occupies and explores the environment, extending the city limits and facilitating the approach of man with wild vectors and reservoirs [25].Whole communities can be affected by this trypanosomiasis due to the precarious and poorly structured rural or suburban houses, allowing the domiciliation of triatomines, the use of man and his domestic animals as a food source and subsequent peridomestic transmission from enzootic cycles.Vectors have managed to adapt and move from wild and semi-urban areas to cities [17,18].
When the Chagas disease outbreak occurred in Chacao, Caracas, in 2007 [26], the researchers tracked the origin of the outbreak and the evidence led to imply that this was caused by drinking home-made guava juice prepared in a home of an urban neighborhood.The fruit juice was exposed overnight, allowing that infected triatomines, present in small remnants of the original vegetation near houses, fall into the liquid and contaminated the juice.This hypothesis is feasible because in the present investigation it was found that the construction of the home-made juice factory was unfinished.In the kitchen, one parrot stayed at the window and there was an aperture in the upper wall through which flying vectors could have entered.
Although the domiciliation of P. geniculatus, the vector of the area, was not confirmed, the community captured 77 adult specimens in outdoor areas of homes without coated walls.Further evidence has been adding eclecticism about the food source of this vector, with 84% of the specimens studied react to antisera rat / mouse / rabbit / dog / cat / bird, simultaneously, being rodents source the most frequently used.
The low participation of man as food source seems consistent with the predominant wild occupation of P. geniculatus, which occasionally moves to homes in search of food and / or attracted to light [17,36,37].Paradoxically, the highest levels of parasite infection were found in insects fed on humans, followed by those fed on dogs, rabbit and rodents, which confirm the idea of the existence of zoonotic foci.
Since the infectivity blood meal index of blood in rats was 62.5%, the prevalence of T. cruzi infected rats was 40% and the 42.1% of the studied triatomines prefer rat blood supply, this result suggest that rat is the primary reservoir in this micro focus.
With reference to the dog case, the data from the study did not allow confirming it as an important reservoir since despite a high infectivity blood meal index (66.6%)and a 66% prevalence of infection in the triatomines studied, the number of dogs studied was low and so was the preference of P. geniculatus to feed on this animal (15.7%).Some researchers have pointed out the domestic dog as an American trypanosomiasis sentinel, for his presence and seroreactivity in strictly domiciliary habitats, indicating the possible presence of a transmission cycle close to the region of the area of the animal, and a high rate of contact with these vectors, thus constituting an important epidemiological risk [38].
In epidemiological control programs in peridomestic and domestic habitats of endemic rural areas or in urban areas where the parasite is emerging [39], the surveillance of T. cruzi infection in dogs must be considered.
On the hill Waraira Repano (also known as Cerro El Ávila), very close to the focus studied, all the elements of the sylvatic cycle of T. cruzi, like "armadillos", and other small mammals are present, being the Didelphis marsupialis, a Caracas valley synanthropic reservoir, one of the most common, [5,16] with an important role in the transmission cycle in the capital city.However, the area's inhabitants did not report its presence and in the study of triatomines, it is not present as blood source, which suggests the participation of other mammals.P. geniculatus was Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease.SOJ Microbiol Infect Dis 4(1): 1-9.

Urban Trypanosoma cruzi Oral Transmission: from a Zoonotic Founder Focus to the Largest Microepidemic of Chagas Disease
Copyright: © 2016 Díaz-Bello et al.
not only present in the area under research, but additionally 50% of them carried T. cruzi, showing a possible zoophilia on domestic and synanthropic fauna.The considerable abundance of R. rattus, despite the low number of traps used, would indicate a high density, which benefits its offer as food source for reduviid bugs.
In El Tamarindo area, typical environment of the marginal areas of the city, which shows a precarious waste management, improvised housing and simplified wildlife as a result of the messy occupation of the environment, which benefits the access of domestic and peridomestic animals as food supply for triatomines.Rats, one of the most abundant mammals in the area, are highly prolific and are recognized as T. cruzi reservoirs.Rats and marsupials, both being synanthropic species, constitute an epidemiological risk to human population [5].The transmission cycle described in the area where the fresh juice was prepared, seems to be adapted to the urban environment and maintained by R. rattus, P. geniculatus and sporadically Canis familiaris.
In the case of man, this transmission occurs through food contaminated with parasite from triatomines feces or fluids from infected mammals [28].
The simultaneously presence of acute infection by T. cruzi in the person who prepared the fresh juice that caused the oral transmission outbreak (102 people infected) in Caracas, along with the presence of genotype TC Id in isolates obtained from P. geniculatus and rats captured in El Tamarindo neighborhood [40,41], connects this case with the oral transmission outbreak and suggests the existence of an outbreak initial focus at 6.7 km of distance from the school where the outbreak occurred, which would have gone unnoticed if this contingency would have not been exposed.Close contact of these triatomines to these synanthropic mammals infected with T. cruzi, could explain oral outbreaks in areas with no records of vector transmission.Caracas is a densely populated urban area non-endemic for Chagas disease, in which active transmission had not been registered since almost two decades [25,42].
The TcI lineage is found throughout America in both domestic and sylvatic cycles and it is believed to have evolved with arboreal Didelphimorphia (opossums) and vectors in the triatomine tribe Rhodniini [43].In all parts of America, Didelphis spp. is a common reservoir for this lineage, although natural infection with TcI has been reported in a wide range of mammals.TcI is the only lineage reported from humans in North and Central America and the predominant lineage reported in human Chagas disease in the Amazon Basin in South America [44,45].
Parasite isolates were predominantly of haplotype TcId.Based on our results, the T. cruzi populations causing acute infections are those associated with the sylvatic foci and vectored by P. geniculatus.The DTU discrimination in the oral infection outbreak allowed us to highlight the significant impact of the DTUs from the sylvatic cycle, TcI.In the outbreak studied, which implies that oral transmission is a relevant epidemiological scenario that has emerged in the natural life cycle of T. cruzi.This suggests that, in areas where vectorial transmission has been interrupted, new acute cases of Chagas disease may arise as a potential problem in public health.
The results presented in this document lead to the consideration of the existence of an enzootic foci which could result in a zoonotic foci in areas with poor sanitation, poor surveillance and without species plague control programs and where there is no control in preparation and food distribution.These findings open up the review of paradigms in regards to oral transmission of Chagas disease, their spotlights enzootic as main route of transmission and how the anthropogenesis of Chagas disease and its history in Caracas could lead to its designation as a re-emerging parasitism.
The oral transmission converts Chagas disease into a foodborne disease.In this sense, prevention measures in both rural and urban areas should be addressed to raise awareness regarding the risk that infected triatomines, rats and Didelphis can contaminate food with their droppings and secretions.Prepared food and beverages should not stay out of the fridge, particularly at night.Washing should be routine for fruits and vegetables.Utensils such as cups, blenders, cutlery and pots should be checked before use.In school cafeterias, traditional drinks should be substituted by pasteurized packaged beverages.Fruit beverages whose preparation is not supervised should not be consumed.

Ethical Considerations
All procedures performed in the study were in accordance with the ethical standards of the ethics committee of the Institute of Tropical Medicine of the Central University of Venezuela in which the studies were conducted.Authorization: CEC-IMT 019/2010.

Figure 1 :
Figure 1: The house and the surroundings where juices were prepared in the Chacao outbreak.

Figure 2 :
Figure 2: Location of the central house (black star: home-made juice factory) and infected triatomines, reservoirs and mammals including humans with Trypanosoma cruzi.

Figure 4 :
Figure 4: Lineage typing through agarose gel (2%) electrophoresis of polymerase chain reaction (PCR) products generated by amplification of DNA from isolate of Trypanosoma cruzi from Panstrongylus geniculatus (AC) Rattus rattus (RAT) and human of outbreak Chagas' oral: (a) amplification of mini-exon gene sequences and (b) amplification of 24Sα-rDNA sequences.DNA from the reference strains Ikiakora and Munantá for T. cruzi III and T. cruzi I, respectively, used as positive controls.

Table 2 :
Identification of blood-meal sources Panstrongylus geniculatus and rates of Trypanosoma cruzi infection.
* IBMI= Infectivity Blood Meal Index = (number of T. cruzi positive triatomines fed on host / number of triatomines fed on that host X) x100.