High levels of Trypanosoma cruzi DNA determined by qPCR and infectiousness to Triatoma infestans...

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Manuscript Number: MEEGID-D-14-00013R1

Title: High levels of Trypanosoma cruzi DNA detemined by qPCR and

infectiousness to Triatoma infestans support dogs and cats are major

sources of parasites for domestic transmission

Article Type: Research paper

Keywords: Trypanosoma cruzi, Real-time PCR, Infectiousness, Reservoir

Corresponding Author: Dr. Marta Victoria Cardinal, PhD

Corresponding Author's Institution:

First Author: Gustavo F Enriquez

Order of Authors: Gustavo F Enriquez; Jacqueline Búa; Marcela M Orozco;

Sonia Wirth; Alejandro G Schijman; Ricardo E Gürtler; Marta Victoria

Cardinal, PhD

Abstract: The competence of reservoir hosts of vector-borne pathogens is

directly linked to its capacity to infect the vector. Domestic dogs and

cats are major domestic reservoir hosts of Trypanosoma cruzi, and exhibit

a much higher infectiousness to triatomines than seropositive humans. We

quantified the concentration of T. cruzi DNA in the peripheral blood of

naturally-infected dogs and cats (a surrogate of intensity of

parasitemia), and evaluated its association with infectiousness to the

vector in a high-risk area of the Argentinean Chaco. To measure

infectiousness, 44 infected dogs and 15 infected cats were each exposed

to xenodiagnosis with 10-20 uninfected, laboratory-reared Triatoma

infestans that blood-fed to repletion and were later individually

examined for infection by optical microscopy. Parasite DNA concentration

(expressed as equivalent amounts of parasite DNA per mL, Pe/mL) was

estimated by real-time PCR amplification of the nuclear satellite DNA.

Infectiousness increased steeply with parasite DNA concentration both in

dogs and cats. Neither the median parasite load nor the mean

infectiousness differed significantly between dogs (8.1 Pe/mL and 48%)

and cats (9.7 Pe/mL and 44%), respectively. The infectiousness of dogs

was positively and significantly associated with parasite load and an

index of the host's body condition, but not with dog's age, parasite DTU

and exposure to infected bugs in a random-effects multiple logistic

regression model. Real-time PCR was more sensitive and less time-

consuming than xenodiagnosis, and in conjunction with the body condition

index, may be used to identify highly infectious hosts and implement

novel control strategies.

246 words

Dr Nancy Sturm

Editor

Infection, Genetics and Evolution

Buenos Aires, March 31, 2014

Dear Dr Sturm,

We are pleased to resubmit the manuscript entitled “Use of real-time PCR for

quantification of Trypanosoma cruzi DNA in domestic dogs and cats and its association with

infectiousness to Triatoma infestans” co-authored by Gustavo F Enriquez, Jacqueline Bua,

María M. Orozco, Sonia Wirth, Alejandro G. Schijman, Ricardo E. Gürtler, and Marta V.

Cardinal for publication in Infection, Genetics and Evolution.

In the revised version of this manuscript, we have addressed all the concerns the referees raised

on the original manuscript. Below you will find a list detailing modifications done and

answers to referees’ comments. Also, following Reviewer 2’s suggestion we have changed

the title of the manuscript to make it more attractive and now reads: “High levels of

Trypanosoma cruzi DNA determined by qPCR and infectiousness to Triatoma infestans

support dogs and cats are major sources of parasites for domestic transmission”.

The materials in this manuscript have resulted from our original work and have not

been submitted elsewhere. I hereby certify that all authors have read the manuscript and

concur with this submission. None of the material has been published or is under

consideration for publication elsewhere, including the Internet. The authors have no financial

or other relationship that might lead to a conflict of interest regarding the contents of this

manuscript. We look forward to hearing from you at your earliest convenience.

Sincerely,

Marta Victoria Cardinal

CONICET Scientific Investigator

UNIVERSIDAD DE BUENOS AIRES

FACULTAD DE CIENCIAS EXACTAS Y NATURALES

Departamento de Ecología, Genética y Evolución

Laboratorio de Eco-Epidemiología

Ciudad Universitaria. 1428 Buenos Aires, Argentina

Tel-Fax: (+54-11) 4576-3318

Email: [email protected]

Cover Letter

MEEGID-D-14-00013

Referees' comments and our responses (in italics)

Reviewer #1: This is manuscript represents a lot of molecular data. Release of molecular data sets is a condition

of MEEGID publications thus this manuscript should be accepted subject to three additional appendice

comprising:

1_All raw qPCR data (Figure 1)

2_All infectiousness data linked with the corresponding qPCR data and the corresponding dog/cat sample (Fig 2

to 3)

3_The GIS locations of the dogs and cats in the study

In response to your request, we provide the information in an appendix and a table: Appendix contains

individual data on dog and cat infectiousness to the vector, qPCR and DTU identification, plus kDNA-PCR

results. GIS locations are provided in a table after this set of responses only for confidential use for reviewers

and editors. We are not sure in which way detailed GIS locations are relevant to our manuscript. If the editor

decides they are relevant, we will not object to add this information to the manuscript.

Appendix. Individual dog and cat infectiousness to the vector, DTU identification, kDNA-PCR and qPCR

results.

Identification

number Host

Infectiousness

(%)

kDNA-

PCR

Bloodstream

parasitic load

(Pe/mL) DTU

15 Dog 61 Positive 18.90 TcVI







23 Dog 0 Positive 20.49 TcI

37 Dog 0 Positive 17.00 TcII / V / VI






81 Dog 75 Positive 2.67 TcV



98 Dog 0 Negative 2.54 TcII / V / VI

101 Dog 0 Negative 1.06 TcVI

*Reply to Reviewers


103 Dog 5 Negative 1.74 TcVI









119 Dog 0 Negative 1.31 TcII / V / VI















22 Cat 0 Positive 7.42 TcI

25 Cat 67 Positive --a ND

b

26 Cat 95 Positive 1.60 TcVI



79 Cat 0 Negative 0.00 NDb

82 Cat 0 Positive --a ND

b

99 Cat 0 Positive 9.73 TcI



132 Cat 35 Positive 2.85 TcV

270 Cat 78 Positive 114.41 TcV




a. DNA quantifications could not be normalized. b. ND: Not determined. DTU identifications were

unsuccessful.

This is an interesting and relevant study, which has considerable application to Chagas disease epidemiology. It

continues Prof Gurtler's in examining the important epidemiological role of dogs in T. cruzi transmission. The

TcI association in both cats and dogs is interesting but I agree with the authors there isn't a big enough sample

size (total of 3) to make any conclusions and as the authors point out could be a consequence of the vector.

However, the association between cats and DTU specific infectiousness looks to be an error of designating

sample groups and must be removed throughout the manuscript: Figure 3 (mistakenly labelled 'Figure 2' in the

manuscript) shows 2 TcV's with approx. 80% infectiousness, whilst 8 TcVI infected cats shows 25 - 100%

infectiousness. There are not enough TcV infected cats to conclude, TcVI is associated within higher

infectiousness against TcV. The authors grouped TcI with TcV and compared this against TcVI, genetically the

authors should have grouped TcV with TcVI and compared it to TcI, where up on they will have found there

isn't a large enough sample size to draw a conclusion. Thus the authors are left saying there is a strong DTU

association with cats for TcVI but not TcV and not dogs. This is either remarkable or else a result of group

designation.

316 The infectiousness of infected cats was significantly associated with ...

317 ........ DTU (OR = 15.04, 95% CI = 1.58-142.52,

318 P < 0.01) (Wald <chi>2 = 10.44, P = 0.02) (Table 1).

Thank you for these insightful comments on our data. Following your suggestions, in the revised version we

have deleted the comparison of infectiousness and identified DTUs for cats due to the small sample size of

examined cats.

Regarding dogs, we have originally designated DTU groups bearing in mind that TcVI is frequently associated

with dogs and cats; and TCV with human infections in the domestic transmission cycle of T. cruzi in the

Argentine Chaco (Diosque et al., 2003; Cardinal et al., 2008). Therefore, we examined if the infectiousness to

the vector differed between these DTUs. Given the small number of TcV-infected animals, in the previous

version we decided to group them with TcI- or TcIII-infected animals. Following your suggestions, in the

revised version we have added a new category level within DTUs and repeated the analysis for dogs.

Revised text in M&M (lines 277-285):

“The independent variables were the bloodstream parasite load (a continuous variable, in Pe/mL), body

condition (two levels: 0 = good, 1 = regular or poor); age of the host (a continuous variable, in months);

exposure to infected bugs, as measured by the abundance of T. infestans infected with T. cruzi captured per 15

min-person per site in domiciles, kitchens and storerooms (i.e., typical resting sites of dogs and cats),

categorized in low, medium and high-risk levels, 0 = 0; 1 = 1-5, and 2 ≥ 5 bugs, respectively; and identified

DTU (0 = TcV, 1 = TcVI; 2 = TcI or TcIII). Identified DTU was excluded as a predictor in cats due to the small

sample size.”

>The authors should discuss whether dog quality is a confounding variable and clarify what a good and average

conditioned dog is please.

A more detailed description of body condition has been included in the revised version.

Revised text in M&M (lines136-140):

“The host’s body condition was used as an index of the nutritional status of dogs as described by Petersen et al.

(2001). Each dog was classified as having a good, regular or poor body condition based on the degree of

muscle development; external evidence of bone structure, state of fur coat, existence of fatty deposits, and facial

expression (Petersen et al., 2001).”

In my opinion it is likely to be a confounding effect of quality of dwelling. In other words, a homeowner

unwilling to keep their pets in good condition is unlikely to keep their home or peridomestic environments in

good condition which could result in higher vector burdens. Thus this could be a confounding variable.

Comparisons against Leishmania are not valid because T. cruzi is asymptomatic, unlike Leishmania infected

dogs, and what the authors are claiming is the dogs overall condition is linked to its immunological status which

is linked to its ability to control T. cruzi parasitaemia. The parsimonious explanation is a confounding variable

of either peridomestic environment or else a superinfection, such as worms. The authors need a better

discussion on the matter and it will help decide hypotheses for future research. There are obvious means of

spatial analysis to assess whether dog parasitaemia is linked with dog genetics and again the authors must

present this raw data.

These interesting points deserve further explanations. First, in most rural areas of the Argentine Chaco, dogs

are not considered pets or companion animals by their owners because they play important roles in the family

subsistence economy. The main functions usually recognized are goat/cow herding, hunting or guardian. Only

3% of 111 dogs included in this study were considered pets by their owners.

Second, a poor quality of dwelling (e.g., walls in poorer conditions) and poor dog body condition are the

common denominator of rural poverty. The former is associated with higher domestic abundance of T. infestans

(Gurevitz et al., 2011) and greater risk of dog infection (Cardinal et al. in press, Am J Trop Med Hyg). If

infectious dogs were present in these households, the abundance of infected bugs would increase and dogs or

other hosts would face higher chances of (re)infection. Some experimental data support that exposure to

reinfection may increase parasitaemia. Therefore, houses with higher infected-bug abundance would entail a

higher risk of (re)infection and perhaps, enhanced infectiousness. To account for household clustering of living

conditions we have included the household as a new random effect in our regression analysis for dogs, while

keeping the individual host as another random effect nested within the household. However, given that these

results were qualitatively equivalent to the previous ones, with very minor changes in parameter estimates, we

decided not to include this new random effect in order to keep the model as parsimonious as possible.

Previous evidence showed that dog body condition (indexed by ECA) was closely associated with its nutritional

status (especially the hematocrit) and infectivity to the vector (Petersen et al., 2001). Therefore, we

hypothesized that body condition impacted the dogs’ immune system and the host’s ability to control

parasitaemia, in which case the index of body condition is not simply a confounder. A confounder effect would

imply a spurious relation between ECA and infectiousness to the vector. Nutritional deficiencies have also been

associated with a decreased immune response and increased parasitaemia in experimental conditions

(Carlomagno et al., 1987; Keusch, 2003; Machado-Coelho et al., 2005). A competent immune system is needed

to control T. cruzi parasitaemia, and whenever the immune response is suppressed reactivation of Chagas

disease and high parasitaemia are recorded (Burgos et al., 2005; Martin et al., 2004; Sartori et al., 2007).

Superinfection with helminths and other parasites are most likely in poor rural settings as in our study area,

and could affect the nutritional status of the dogs, its body condition and immune response.

Typos have been designated on the document.

Corrections were made where needed.

> Reviewer #2: Dogs and cats are known to constitute important sources of parasites for domestic transmission

of T. cruzi. This study confirmed that infectiousness of dogs is associated with the bloodstream parasitic load

using a qPCR method. This work simply described the application of a previously standardized qPCR method

for quantification of T. cruzi in dogs and cats. The study was well-conducted, results are sound and clearly

presented. However, the Introduction and Discussion failed to clearly show the relevance and current

knowledge of this issue, and have several questionable points. The manuscript, an extension of successive

articles recently published by the authors about T. cruzi infection in dogs and cats, can be much improved

before accepted for publication by the IGE.

>Please, find bellow some comments that, maybe, can be useful to clarify and improve the manuscript,

especially for readers non-familiarized with this specific topic.

Thank you for your helpful comments.

> 1. The goal of the study is to identify "superspreader" hosts. The concept of "superspreader" was not clearly

defined, apparently, for the authors only animals showing high parasite loads are "superspreader"(amplification

hosts?), despite they are domestic animals of restricted "spread". In the literature, 'superspreading' hosts play a

key role in disease epidemics.

In the revised version we have now included a definition of “superspreader”. (Lines 90-92)

The study dogs do not usually spend the night in another house, but they were reported to visit frequently

nearby forest patches either alone or in groups; as they are not restrained they may move freely. Additionally,

dogs imported from infested villages without vector control were at a higher relative odds of infection than

other dogs native to villages under sustained vector surveillance and control (Cardinal et al., 2007), showing

that infected dogs could “spread” T. cruzi infection outside their places of birth.

Revised text in the Introduction (Lines 87-94):

“There is strong evidence on the clustering and heterogeneity in the transmission of pathogens, where 20% of

individuals in a population would be responsible for 80% of the transmission (Wilson et al., 2001; Woolhouse

et al., 1997; Courtenay et al., 2014). The small fraction of individuals that infect disproportionately more

susceptible contacts than most infected individuals became known as “superspreaders” of disease (Stein 2011).

Trypanosoma cruzi infection, although mainly a vector-borne disease, shows that a small fraction of the

infected dogs and cats were highly infectious to xenodiagnosis bugs and could be considered “superspreaders”

(Gürtler et al., 2007).”

In lines 103-107:

“In this study we used for the first time a qPCR protocol to quantify the load of T. cruzi DNA in the peripheral

blood of naturally-infected dogs and cats from a well-defined rural area in the Argentinean Chaco. We also

evaluated the association among infectiousness to the vector, bloodstream parasite load and other potential

predictors of infectiousness, and identified highly infectious hosts.”

> 2. Although domestic dogs and cats are major domestic reservoir hosts of T. cruzi in many places, studies on

these animals have been concentrated in Argentina; many articles are from the authors of this manuscript. The

concentration of studies in this country (and also in the literature cited in this manuscript) does not implicate

that data from other countries and authors are irrelevant. In fact, data of dogs infected with T. cruzi and

implication to human infection from other countries add relevance and broader interest to this manuscript.

Right. We have now added three references to further illustrate the importance of dogs in the transmission of T.

cruzi throughout the Americas (Lines 51-53).

“Domestic dogs and cats are major reservoir hosts of T. cruzi in the domestic environment throughout the

Americas (Cardinal et al., 2008, in press; Crisante et al., 2006; Gürtler et al., 2007; Noireau et al., 2009;

Ramírez et al., 2013).”

> 3. Introduction: "Therefore, dogs and cats frequently constitute the largest sources of parasites for domestic

transmission of T. cruzi. Everywhere? References?

The revised text (Lines 56-59):

“Therefore, dogs and cats frequently constitute the largest source of T. cruzi for domestic transmission where

Triatoma infestans is the main vector species as in the Gran Chaco region (Gürtler et al., 2007).”

> 4. Introduction: The minimum number of parasites required to infect a bug via xenodiagnosis and the

percentage of infected insects depend on T. cruzi strains (DTUs?) and triatomine species, and not simply to the

number of trypomastigotes ingested or inoculated.

This sentence has been re-written (Lines 65-69):

“However, the percentage of infected insects could be highly variable and depends on T. cruzi DTU and strain,

triatomine species and not simply on the number of trypomastigotes ingested or inoculated (Alvarenga and

Leite, 1982; Garcia et al., 2007; de Lana et al., 1998; Neal and Miles, 1977; da Silveira Pinto et al., 2000).”

> 5. Results. "No clear pattern was observed between DTU and host infectiousness or bloodstream parasitic

load. However, the two cats and the only dog infected with TcI had nil infectiousness despite having 7.4, 9.7

and 20.5 Pe/mL, respectively (Figure 3). Sequence analysis of SL-IR amplicons from identified TcI showed that

they were more closely related to TcI than to other DTUs (i.e., 98-100% sequence identity)."

We have rewritten the last sentence (Lines 325-330):

“No clear association was observed between DTU and host infectiousness or bloodstream parasite load

(Figure 3). However, the two cats and the only dog infected with TcI had nil infectiousness despite having 7.4,

9.7 and 20.5 Pe/mL, respectively (Figure 3) (Appendix). In these three individuals TcI was confirmed by

sequence analysis of Spliced Leader region (SL-IR I) (i.e., 98-100% sequence identity).”

"Indeed, bug infection in xenodiagnosis-negative, TcI-infected dogs and cats was rare and only revealed by

means of a highly sensitive kDNA-PCR applied to the bugs rectal-ampoule lysates. The substantial variations in

metacyclogenesis and trypomastigote numbers among triatomine species (Carvalho-Moreira et al., 2003; Loza-

Murghía et al., 2010; Perlowagora-Szumlewicz et al., 1994) may explain the nil infectiousness (as measured by

optical microscopy??) recorded in TcI-infected dogs and cats".

This sentence has been re-written: (Lines 452-458):

“Indeed, TcI-infected dogs and cats were rare and could only be identified by means of a highly sensitive

kDNA-PCR applied to the rectal-ampoule lysates of xenodiagnostic bugs or GEB samples. The substantial

variations in metacyclogenesis and trypomastigote density among triatomine species (Carvalho-Moreira et al.,

2003; Loza-Murghía et al., 2010; Perlowagora-Szumlewicz et al., 1994) may explain the nil infectiousness (as

measured by optical microscopy) recorded in TcI-infected dogs and cats. However, samples sizes were small

and this relationship should be further investigated.”

> The authors assessed the DTU of T. cruzi infecting all infected dogs and cats. However, results are not clear

and deserve to be better discussed. What means "SL-IR amplicons from identified TcI showed that they were

more closely related to TcI?

Given that two unspecific bands were persistently obtained in the SL-IR-PCR from GEB samples, the expected

amplicon for T. cruzi DNA of 475 pb was purified and sequenced to confirm TcI identification.

Revised text in (Lines 325-330)

“No clear association was observed between DTU and host infectiousness or bloodstream parasite load

(Figure 3). However, the two cats and the only dog infected with TcI had nil infectiousness despite having 7.4,

9.7 and 20.5 Pe/mL, respectively (Figure 3) (Appendix). In these three individuals TcI was confirmed by

sequence analysis of Spliced Leader region (SL-IR I) (i.e., 98-100% sequence identity).”

In a previous article, the authors (Enriquez et al., 2013a: Discrete typing units of Trypanosoma cruzi identified

in rural dogs and cats in the humid Argentinean Chaco. Parasitol 12, 1-6) were unable to detect TcI in dogs

from the areas investigated in this work (the same dogs and cats?).

These dogs and cats belong to the same communities and samples were taken at the same time as those included

in Enriquez et al. (2013a).

In Enriquez et al. (2013a) all DTU identifications were made from DNA samples from cultured parasites.

However, 7 dogs and 5 cats examined were not included because the infecting parasites could not be isolated

(i.e., all were negative by xenodiagnosis or in vitro cultures were unsuccessful). In the current work, due to the

need to identify the DTUs or at least achieve a partial identification for normalizing the quantification of

parasites, we attempted to identify the DTUs from other sources as guanidine blood samples or from the rectal

ampoule.

This sentence has been re-written (Lines 229-236):

“Identification of DTUs infecting dogs and cats based on isolated parasites was reported elsewhere (Enriquez

et al., 2013a). In the current study, additional DTU identifications from seropositive, xenodiagnosis-negative

dogs (n = 6) and cats (n = 5) and from a seropositive, xenodiagnosis-positive dog were attempted directly from

GEB or rectal ampoule samples of OM-negative, kDNA-PCR-positive bugs used in xenodiagnostic tests of these

animals. These additional dogs and cats belonged to the same villages, and samples were taken at the same

time as those included in Enriquez et al. (2013a).”

How to explain the lack of detection of TcI infection in dogs (by hemoculture and genotyping) in

countries/regions where predominates TcI? In general, TcI exhibit high metacyclogenesis in culture and

triatomines bugs; hemocultures are much more easer to be obtained for TcI than for the other DTUs. It was

recently demonstrated that wild T. infestans is naturally infected with TcI in Bolivia. Substantial differences in

parasitic load between dogs/cats and humans could be associated to distinct and preferential DTUs infecting

these hosts?

TcVI and TcV are more frequently found in the domestic cycle of transmission than TcI in Argentina, unlike the

situation in the Amazon basin, Colombia, Venezuela and Mexico, where TcI has also been identified in dogs

(Ramirez et al., 2013). Also, the lack of detection of TcI in dogs by xenodiagnosis could be due to different

susceptibility of T. infestans or to the specific genotypes of TcI involved (Cura et al., 2010; Herrera et al.,

2009). In our study area, TcI was not found in T. infestans and was only detected in few specimens of

peridomestic Triatoma sordida collected along many years (Maffey et al., 2012). However, we found a high

prevalence of TcI infection (by xenodiagnosis and kDNA-PCR) in Didelphis albiventris opossums, probably

indicating this DTU is predominantly associated with the local sylvatic transmission cycle.

> 6. Discussion: "Quantitative PCR was more sensitive and less time-consuming than xenodiagnosis, and alone

or in conjunction with the body condition index, may be helpful to identify "superspreader" hosts, to replace

xenodiagnosis (or hemoculture) ...."

>In a recent article from the same authors (Enriquez et al., 2013b: Detection of Trypanosoma cruzi infection in

naturally infected dogs and cats using serological, parasitological and molecular methods, ActaTrop126:211-7)

they concluded: "The high sensitivity of kDNA-PCR to detect T. cruzi infections in naturally infected dogs and

cats supports its application as a diagnostic tool complementary to serology and may replace the use of

xenodiagnosis or hemoculture". It will be interesting to see a comparative analysis between kDNA-PCR (very

easy to be done dispensing equipment still sophisticate for most CH endemic countries) and qPCR to detect T.

cruzi infections regardless the parasite load, i.e., in both "superspreader" and "non-superspreader" hosts.

Following your suggestion, in the revised version we have now included a comparative analysis between kDNA-

PCR and qPCR to detect T. cruzi infection. However, in agreement with your suggestions about the role that

superspreader hosts play in disease, we believe that categorizing dogs and cats into “high or low

infectiousness” would be more appropriate than strictly qualifying them as superspreaders.

We re-wrote the following section and incorporated a paragraph in the discussion

Revised text in M&M (Lines 159-164):

“Trypanosoma cruzi-infected dogs and cats were also classified as having high or low infectiousness to T.

infestans; the “high infectiousness” category included dogs and cats that were responsible for ≥80% of all

infected triatomines used in the xenodiagnostic tests, and the “low infectiousness” included the remaining

seropositive dogs and cats and that were also examined by xenodiagnoses.”

Results (Lines 359-370):

“3.4. Profile of infected dogs and cats with high or low infectiousness”

“Nearly half of the dogs (50%) and cats (47%) showed high infectiousness and were responsible of at least

80% of the infected xenodiagnosis bugs. The median parasite load was nearly 10 times greater for dogs with

high infectiousness than for dogs with low infectiousness and this difference was statistically significant

(Kruskal-Wallis test, P < 0.01). For cats this relationship was 32 times greater (Kruskal-Wallis test, P = 0.02)

(Table 3). Quantitative PCR could not distinguish between highly infectious and non-highly infectious hosts,

given that it detected T. cruzi DNA in 100% of the seropositive dogs and in 93% of the seropositive cats,

whereas kDNA-PCR detected 89% and 87% of them, respectively (Table 3). Four seropositive dogs and one

seropositive cat that were both qPCR-positive and kDNA-PCR- negative showed very low infectiousness to the

vector (range, 0-5%).”

Table 3. Relationship among qualitative kDNA-PCR, qPCR and parasite load to detect T. cruzi-infected dogs

and cats with different levels of infectiousness

Host Infectiousnessa

No. positive

xenodiagnosis (No.

examined)

Mean

infectiousness

(%)

Median parasite

load (Q1-Q3)

No.

Positive

kDNA (%)

No.

Positive

qPCR (%)

Dogs High 22 (22) 79 21.3 (7.3-39.3) 22 (100) 22 (100)

Low 16 (22) 19 2.4 (1.3-12.6) 17 (77) 22 (100)

Total 38 (44) 48 8.1 (1.5-26.6) 39 (89) 44 (100)

Cats High 7 (7) 79 96.1(18.1-202.1) 7 (100) 7 (100)

Low 3 (8) 14 3.0 (2.2-7.4) 6 (75) 7 (88)

Total 10 (15) 44 9.7 (2.9-96.9) 13 (87) 14 (93)

a. The “high infectiousness” category included dogs and cats that were responsible for ≥80% of all infected

triatomines used in the xenodiagnostic tests. The “low infectiousness” category included all remaining

seropositive dogs and cats examined by xenodiagnosis.

Discussion (Lines 459-468)

“In our study, approximately 50% of the infected dogs and cats were responsible for at least 80% of the

infected T. infestans used in xenodiagnosis. All of them were positive by qPCR and kDNA-PCR. However, dogs

and cats with very low infectiousness to the vector (<20%) were also frequently positive by these techniques.

These results suggest that the quantitative estimates of parasite load and not the qualitative outcomes of qPCR

or kDNA-PCR would be a more precise tool to identify highly infectious dogs and cats. A wide variability in

infectiousness (range, 0-63% for dogs and 35-67% for cats) was observed between 1-5 Pe/mL. Courtenay et al.

(2014) recently showed that quantitative PCR was needed to identify highly infectious L. infantum-infected

dogs.”

> 7. Discussion: "There was a small fraction of highly infectious hosts (i.e., "superspreaders") that may

contribute disproportionally to (peri)domestic transmission and pathogen persistence" Is this comment about

hosts of T. cruzi?? The wild mammals are never "superspreaders"? This statement must be explained and

reference must be cited.

In the revised version we have included additional explanations (Lines 415-423):

“Dogs and cats had similar infectiousness to the vector and this distribution was aggregated, as in studies

conducted elsewhere (Gürtler et al., 2007). There was a fraction of highly infectious dogs and cats that may

contribute disproportionally to (peri)domestic transmission and persistence of T. cruzi. This heterogeneous

distribution of parasite burden in host populations is a generalized pattern in macroparasites and

microparasites (Wilson et al., 2001; Woolhouse et al., 1997) and may be used for targeted control strategies.

Trypanosoma cruzi-infected Dasypus novemcinctus armadillos and Didelphis albiventris opossums also have

high infectiousness (Orozco et al., 2013).”

In lines 469-474:

“Quantitative PCR was more sensitive and less time-consuming than xenodiagnosis, and alone or in

conjunction with the body condition index, may be helpful to identify highly infectious hosts; replace

xenodiagnosis (or hemoculture) to assess treatment efficacy or infection status, and contribute to novel control

strategies (e.g., targeted treatment or vaccination) that reduce the risks of domestic transmission of T. cruzi.”

>Finally, the title can be more atractive. Suggestion High levels of Trypanosoma cruzi DNA detemined by

qPCR in dogs and cats and infectiousness to Triatoma infestans support these hosts as major sources of

parasites for domestic transmission of Chagas disease

Thank you for the suggestion! Just to make it shorter we suggest:

High levels of Trypanosoma cruzi DNA determined by qPCR and infectiousness to Triatoma infestans support

dogs and cats are major sources of parasites for domestic transmission

We added a new result to assess the OM false negative rate in dogs.

Revised text in M&M (Lines 193-195):

“The rectal-ampoule samples of 94 OM-negative bugs used in xenodiagnosis of the seropositive dogs was

examined by kDNA-PCR to assess the false-negative rate of OM procedures.”

In Results, lines 319-320

“Thirteen percent of 94 OM-negative bugs were positive by kDNA-PCR; thus, the corrected infectiousness to

the vector would rise to 54%.”

Table. Individual dog and cat infectiousness to the vector, DTU identification, house location, kDNA-PCR and

qPCR results.

Identification

number Host

Infectiousness

(%)

kDNA-

PCR

Bloodstream

parasite load

(Pe/mL) DTU

GIS

locations

(X)

GIS

locations

(Y)

15 Dog 61 Positive 18.9 TcVI 192273 7141605







23 Dog 0 Positive 20.49 TcI 192509 7142642

37 Dog 0 Positive 17.00 TcII / V / VI 192483 7142945






81 Dog 75 Positive 2.67 TcV 193639 7140089

91 Dog 95 Positive 21.85 TcV 193684 7140046

96 Dog 16 Positive 1.38 TcV 191530 7141117

98 Dog 0 Negative 2.54 TcII / V / VI 191530 7141117

101 Dog 0 Negative 1.06 TcVI 191530 7141117


103 Dog 5 Negative 1.74 TcVI 191525 7140815

104 Dog 16 Positive 3.03 TcV 191525 7140815








119 Dog 0 Negative 1.31 TcII / V / VI 191406 7140272











272 Dog 84 Positive 7.26 TcV 190995 7140873


276 Dog 45 Positive 1.46 TcV 190250 7140985


22 Cat 0 Positive 7.42 TcI 190407 7141226

25 Cat 67 Positive NDa ND

b 191149 7139883

26 Cat 95 Positive 1.6 TcVI 191149 7139883



79 Cat 0 Negative 0 NDb 190457 7139884

82 Cat 0 Positive NDa ND

b 190498 7139473

99 Cat 0 Positive 9.73 TcI 191776 7133939



132 Cat 35 Positive 2.85 TcV 191259 7134302

270 Cat 78 Positive 114.41 TcV 191259 7134302




a. DNA quantifications could not be normalized. b. ND: No determined. DTU identifications were

unsuccessful.

Highlights

1. We measured the parasitic load of dogs and cats infected with T. cruzi by qPCR.

2. In both hosts, parasitic load was similar and higher than in chronic patients.

3. Infectiousness to the vector was associated with parasitic load in both hosts.

4. qPCR may be used to identify “superspreader” hosts and to assess treatment

efficacy or infection status.

*Highlights (for review)

http://www.elsevier.com/wps/find/journaldescription.cws_home/506043/authorinstructions#N10C3D

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High levels of Trypanosoma cruzi DNA detemined by qPCR and infectiousness to 1

Triatoma infestans support dogs and cats are major sources of parasites for domestic 2

transmission 3

Enriquez G.F.,1,2

Bua J.,3

Orozco M.M.,1,2

Wirth S.,4

Schijman A.G.,5

Gürtler R.E.,1,2

4

Cardinal M.V. 1,2

5

6

1Laboratory of Eco-Epidemiology, Faculty of Exact and Natural Sciences, University of 7

Buenos Aires, Argentina. 8

2Institute of Ecology, Genetics and Evolution of Buenos Aires (UBA-CONICET). 9

3National Institute of Parasitology Dr M. Fatala Chaben, National Administration of 10

Laboratories and Institutes of Health Dr. C.G. Malbrán, Buenos Aires, Argentina. 11

4Laboratory of Agro-biotechnology, Faculty of Exact and Natural Sciences, University 12

of Buenos Aires, Argentina. 13

5Laboratory of Molecular Biology of Chagas Disease, Institute for Research on Genetic 14

Engineering and Molecular Biology (INGEBI-CONICET).

15

16

Running title: Trypanosoma cruzi quantification and infectiousness in dogs and cats 17

18

Corresponding author: M. Victoria Cardinal, Laboratory of Eco-Epidemiology, Faculty 19

of Exact and Natural Sciences, University of Buenos Aires, Argentina, 1428 Buenos 20

Aires, Argentina. Tel/Fax: +54-11-4576-3318. [email protected] 21

22

*ManuscriptClick here to view linked References

http://ees.elsevier.com/meegid/viewRCResults.aspx?pdf=1&docID=3560&rev=1&fileID=126390&msid=E2F9A8DD-8AA6-488F-A509-239BB5B6DB00

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Abstract

23

The competence of reservoir hosts of vector-borne pathogens is directly linked to its 24

capacity to infect the vector. Domestic dogs and cats are major domestic reservoir hosts 25

of Trypanosoma cruzi, and exhibit a much higher infectiousness to triatomines than 26

seropositive humans. We quantified the concentration of T. cruzi DNA in the peripheral 27

blood of naturally-infected dogs and cats (a surrogate of intensity of parasitemia), and 28

evaluated its association with infectiousness to the vector in a high-risk area of the 29

Argentinean Chaco. To measure infectiousness, 44 infected dogs and 15 infected cats 30

were each exposed to xenodiagnosis with 10-20 uninfected, laboratory-reared Triatoma 31

infestans that blood-fed to repletion and were later individually examined for infection 32

by optical microscopy. Parasite DNA concentration (expressed as equivalent amounts of 33

parasite DNA per mL, Pe/mL) was estimated by real-time PCR amplification of the 34

nuclear satellite DNA. Infectiousness increased steeply with parasite DNA 35

concentration both in dogs and cats. Neither the median parasite load nor the mean 36

infectiousness differed significantly between dogs (8.1 Pe/mL and 48%) and cats (9.7 37

Pe/mL and 44%), respectively. The infectiousness of dogs was positively and 38

significantly associated with parasite load and an index of the host‟s body condition, but 39

not with dog‟s age, parasite DTU and exposure to infected bugs in a random-effects 40

multiple logistic regression model. Real-time PCR was more sensitive and less time-41

consuming than xenodiagnosis, and in conjunction with the body condition index, may 42

be used to identify highly infectious hosts and implement novel control strategies. 43

246 words 44

Keywords: Trypanosoma cruzi, Real-time PCR, Infectiousness, Reservoir 45

46

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1. Introduction 47

Trypanosoma cruzi, the etiologic agent of Chagas disease, has been found 48

infecting approximately 180 species of mammals (WHO, 2002). The competence of 49

reservoir hosts of vector-borne pathogens is directly linked to its capacity to infect the 50

vector. Domestic dogs and cats are major reservoir hosts of T. cruzi in the domestic 51

environment throughout the Americas (Cardinal et al., 2008, in press; Crisante et al., 52

2006; Gürtler et al., 2007; Noireau et al., 2009; Ramírez et al., 2013). They are usually 53

highly infectious to triatomine bugs, and display much higher infectiousness (defined as 54

the proportion of uninfected insects that become infected after blood-feeding once on an 55

infected host) than T. cruzi-seropositive humans (Gürtler et al., 2007; 1996). Therefore, 56

dogs and cats frequently constitute the largest source of T. cruzi for domestic 57

transmission where Triatoma infestans is the main vector species as in the Gran Chaco 58

region (Gürtler et al., 2007). 59

The chances of a triatomine bug becoming infected with T. cruzi after blood-60

feeding on an infected host mainly depend on the probability of ingesting the parasite 61

(determined by the intensity of parasitemia and bloodmeal size), and its successful 62

establishment and reproduction in the insect gut. The minimum number of T. cruzi 63

parasites required to infect a bug through artificial xenodiagnosis was estimated to be 64

one (Moll-Merks et al., 1987). However, the percentage of infected insects could be 65

highly variable and depends on T. cruzi DTU and strain, triatomine species and not 66

simply on the number of trypomastigotes ingested or inoculated (Alvarenga and Leite, 67

1982; Garcia et al., 2007; de Lana et al., 1998; Neal and Miles, 1977; da Silveira Pinto 68

et al., 2000). 69

Previous studies that sought to estimate parasitemia in naturally-infected hosts 70

have traditionally relied on direct counting of T. cruzi parasites under optical 71

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microscopy. However, new technologies have become available for this purpose (i.e., 72

flow citometry, DNA amplification). Real-time or quantitative PCR (qPCR) is useful 73

for the quantification of parasite DNA in different host tissues and its applications have 74

substantially increased in recent years. In Chagas disease, qPCR has been mainly 75

applied to human patients and has been successfully used in the follow-up of treatment 76

outcomes, diagnosis of infection, and to investigate parasite vertical transmission (Bua 77

et al., 2012; Duffy et al., 2009; Moreira et al., 2013; Schijman et al., 2011). Recent 78

studies using qPCR to measure the concentration of T. cruzi DNA (i.e., a surrogate of 79

intensity of parasitemia) found that the median bloodstream parasite load of chronic 80

patients was ≤ 2 parasite equivalents/mL and differed significantly among countries 81

(Freitas et al., 2011; Moreira et al., 2013). Higher parasite loads were recorded in 82

chronic patients from Argentina and Colombia (Duffy et al., 2013; Moreira et al., 2013), 83

in patients coinfected with HIV (Freitas et al., 2011), in seropositive women giving 84

birth to T. cruzi-infected newborns (Bua et al., 2012) and in newborns diagnosed by 85

optical microscopy after delivery (Bua et al., 2013). 86

There is strong evidence on the clustering and heterogeneity in the transmission of 87

pathogens, where 20% of individuals in a population would be responsible for 80% of 88

the transmission (Wilson et al., 2001; Woolhouse et al., 1997; Courtenay et al., 2014). 89

The small fraction of individuals that infect disproportionately more susceptible 90

contacts than most infected individuals became known as “superspreaders” of disease 91

(Stein 2011). Trypanosoma cruzi infection, although mainly a vector-borne disease, 92

shows that a small fraction of the infected dogs and cats were highly infectious to 93

xenodiagnosis bugs and could be considered “superspreaders” (Gürtler et al., 2007).The 94

infectiousness to the vector T. infestans of seropositive dogs was shown to be 95

aggregated (Gürtler et al., 2007) and associated with an index of the host‟s body 96

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condition (i.e., external clinical aspect, ECA) (Petersen et al., 2001), and not associated 97

with the Discrete Typing Unit (DTU, i.e., genotypes) of T. cruzi (Cardinal et al., 2008). 98

Mixed outcomes were registered for age of the dog and exposure to infected bugs 99

(Gürtler et al., 2007; 1996; 1992). The functional relationship between parasitemia and 100

infectiousness to the vector in dogs and cats naturally infected with T. cruzi (or any 101

other mammalian nonhuman host) has not been investigated. 102

In this study we used for the first time a qPCR protocol to quantify the load of T. 103

cruzi DNA in the peripheral blood of naturally-infected dogs and cats from a well-104

defined rural area in the Argentinean Chaco. We also evaluated the association among 105

infectiousness to the vector, bloodstream parasite load and other potential predictors of 106

infectiousness, and identified highly infectious hosts. 107

108

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2. Materials and Methods 109

2.1. Study area 110

Field work was conducted in the municipality of Pampa del Indio (26° 2′ 0″ S, 111

59° 55′ 0″ O), Chaco Province, Argentina. The study area was described elsewhere 112

(Gurevitz et al., 2011). Prior to community-wide residual spraying with pyrethroid 113

insecticides, a cross-sectional survey of all villages revealed that 45.9% of the inhabited 114

houses were infested with T. infestans (Gurevitz et al., 2011), and the overall prevalence 115

of T. cruzi infection was 27% in bugs, 26% in dogs and 29% in cats in August–116

December 2008 (Cardinal et al., in press). 117

118

2.2. Study design 119

Nested within this larger survey, for current purposes we selected two 120

neighboring villages (10 de Mayo and Las Chuñas, with 60 inhabited houses) showing 121

the highest bug prevalence of infection with T. cruzi (61%) at baseline. Within these 122

villages, all the houses with at least one T. cruzi-infected T. infestans at baseline (38 of 123

60 houses) were selected to conduct a cross-sectional xenodiagnostic survey of all dogs 124

and cats. A total of 99 (56.3%) dogs and 29 (74.4%) cats were examined by 125

xenodiagnosis to increase the chances of isolating parasites from a large number of 126

individuals. Each head of household was informed on the objectives of the study; an 127

informed oral consent was requested and obtained in all cases. 128

Blood samples (up to 7 mL) were drawn by venipuncture and allowed to clot at 129

ambient temperature. Each serum was separated after centrifugation at 3,000 rpm during 130

15 min., allocated in triplicate vials and preserved at -20 ºC at the field laboratory. 131

Blood samples were also used (2 mL in dogs and 1 mL in cats) for DNA extraction and 132

PCR; these were immediately mixed with an equal volume of guanidine hydrochloride 133

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6 M, EDTA 0.2 M pH 8.00 buffer (GEB), and stored at 4ºC. Serum samples were 134

transported in dry ice to the main laboratory at the end of each survey, whereas GEB 135

samples were kept in a cooler. The host‟s body condition was used as an index of the 136

nutritional status of dogs as described by Petersen et al. (2001). Each dog was classified 137

as having a good, regular or poor body condition based on the degree of muscle 138

development; external evidence of bone structure, state of fur coat, existence of fatty 139

deposits, and facial expression (Petersen et al., 2001). Only dogs aged 1 year or more 140

were categorized to avoid potential confounders due to growth effects and acute 141

infections (Petersen et al., 2001). Handling and examination of dogs and cats was 142

conducted according to the protocol approved by the „Dr. Carlos Barclay‟ Independent 143

Ethical Committee for Clinical Research from Buenos Aires, Argentina (IRB No. 144

00001678, NIH registered and Protocol N ° TW-01-004). 145

146

2.3. Xenodiagnosis 147

Dogs and cats were examined by xenodiagnosis with 10 or 20 uninfected, 148

laboratory-reared fourth-instar nymphs of T. infestans exposed to the animal‟s belly 149

during 20 min, followed by a 10-minute re-exposure period if initially most bugs had 150

not blood-fed to repletion (Enriquez et al., 2013a). Each bug was individually examined 151

by optical microscopy (OM) (400×) for T. cruzi infection 30 and 60 days after exposure. 152

The infectiousness to the vector of dogs and cats that were seropositive for T. cruzi was 153

calculated as the total number of insects infected with T. cruzi divided by the total 154

number of insects exposed to the animal and examined for infection at least once 155

(Gürtler et al., 1992). Bug mortality rate at 30 days post-exposure was 3% in both dogs 156

and cats; and bugs exposed to dogs molted after a single blood meal (12%) more 157

frequently than bugs fed on cats (8%), though these differences were not statistically 158

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significant (Enriquez et al., 2013b). Trypanosoma cruzi-infected dogs and cats were 159

also classified as having high or low infectiousness to T. infestans; the “high 160

infectiousness” category included dogs and cats that were responsible for ≥80% of all 161

infected triatomines used in the xenodiagnostic tests, and the “low infectiousness” 162

included the remaining seropositive dogs and cats and that were also examined by 163

xenodiagnoses. 164

165

2.4. Serodiagnosis 166

Dogs and cats aged 4 months or more were diagnosed serologically whereas 167

younger animals were examined only by xenodiagnosis because maternally-derived 168

antibodies to T. cruzi could induce a false-positive result. Seventy (70%) dogs and 27 169

(93%) cats examined by xenodiagnosis were also examined by serodiagnosis. Sera were 170

tested for antibodies to T. cruzi by indirect hemagglutination assay (IHA) following the 171

manufacturer‟s instructions (Wiener Laboratories S.A.I.C., Buenos Aires, Argentina), 172

an in-house enzyme-linked immunosorbent assay (ELISA), and an indirect 173

immunofluorescence test (IFAT) (Ififluor Parasitest Chagas, Laboratorio IFI, Buenos 174

Aires, Argentina). The serodiagnosis methodologies and results of dog and cat 175

populations have been reported elsewhere (Enriquez et al., 2013b). An animal was 176

considered “seropositive” if reactive by at least two assays. 177

178

2.5. Molecular analysis 179

2.5.1. DNA extraction 180

Guanidine-EDTA blood samples (GEB) of 2 and 4 mL were heated in boiling 181

water for 10 and 15 min, respectively (Britto et al., 1993). Prior to DNA extraction, 200 182

pg of an internal amplification control DNA (IAC) was added to 400 µL GEB aliquot 183

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(Duffy et al., 2009). Total DNA was purified using a commercial kit (DNeasy Blood & 184

Tissue Kit, QIAGEN Sciences, Maryland, USA) and following manufacturer´s 185

instructions avoiding the use of proteinase K and the addition of buffer AL, as reported 186

(Duffy et al., 2009). Purified DNA was eluted in 200 µL of distilled water and used as 187

template for PCR and qPCR amplification. 188

189

2.5.2. Polymerase chain reaction 190

DNA from seropositive dogs and cats that were also examined by xenodiagnosis 191

were tested by means of qualitative and qPCR assays. The qualitative PCR assay was 192

targeted to the minicircles of the kinetoplast (kDNA-PCR). The rectal-ampoule samples 193

of 94 OM-negative bugs used in xenodiagnosis of the seropositive dogs was examined 194

by kDNA-PCR to assess the false-negative rate of OM procedures. In addition, we 195

included samples from 16 dogs and 7 cats that resided in the study area and were not 196

infected by T. cruzi as determined by ELISA, IHA and xenodiagnosis (i.e., control 197

hosts). The comparison of kDNA-PCR results and xenodiagnosis has been reported 198

elsewhere (Enriquez et al., 2013b). 199

200

2.5.3. Real-time PCR assay 201

The bloodstream parasite load in dogs and cats was quantified by amplifying a 202

T. cruzi satellite DNA flanked by the Sat Fw (5'-GCAGTCGGCKGATCGTTTTCG-3') 203

and Sat Rv (5'-TTCAGRGTTGTTTGGTGTCCAGTG -3') oligonucleotides (Duffy et 204

al., 2009). The standard curve for DNA quantification (generated with T. cruzi CL 205

Brener clone), reagents, control samples and cycling profile for T. cruzi DNA 206

amplification were performed as previously described for human samples (Bua et al., 207

2012). Samples were run in duplicates using a commercial kit (SYBR GreenER qPCR 208

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SuperMix Universal, Invitrogen, Life Technologies, Grand Island, NY, USA) and 8 μL 209

of template DNA in a 20 μL final volume. 210

The standard calibration curve for IAC was carried out using blood samples of 211

dogs not infected with T.cruzi (i.e., dogs residing in non-endemic areas). Samples were 212

spiked with 400 pg, 40 pg and 4 pg of IAC. The IAC was quantified using 0.6 μM of 213

primers, IS Fw (5'-AACCGTCATGGAACAGCACGTAC-3') and IS Rv (5'-214

CTAGAACATTGGCTCCCGCAACA-3') (Duffy et al., 2009) using a commercial kit 215

(Quantitec SYBR Green PCR kit, QIAGEN Sciences, Maryland, USA). After 15 min of 216

pre-incubation at 95 ºC, PCR amplification was carried out for 40 cycles (94 ºC for 15 s, 217

59 ºC for 30 s and 72 ºC for 30 s). 218

Given that the number of satellite DNA repeats differs among different T. cruzi 219

DTUs, DNA quantification was normalized according to the identified DTU (Duffy et 220

al., 2009). Parasite DNA concentration was expressed as equivalent amounts of parasite 221

DNA per ml (Pe/mL). An ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, 222

USA) was used in all qPCR amplifications. 223

224

2.5.4. DTU identification 225

Parasite DTUs were identified by PCR targeted to three genomic markers: spliced-226

leader (SL) DNA, 24Sα ribosomal RNA genes and A10 marker as described by Burgos 227

et al. (2007). PCR products were analyzed in 2.5% or 3% agarose gels (Invitrogen, 228

USA) and UV visualized after staining with Gel Red (GenBiotech). Identification of 229

DTUs infecting dogs and cats based on isolated parasites was reported elsewhere 230

(Enriquez et al., 2013a). In the current study, additional DTU identifications from 231

seropositive, xenodiagnosis-negative dogs (n = 6) and cats (n = 5) and from a 232

seropositive, xenodiagnosis-positive dog were attempted directly from GEB or rectal 233

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ampoule samples of OM-negative, kDNA-PCR-positive bugs used in xenodiagnostic 234

tests of these animals. These additional dogs and cats belonged to the same villages, and 235

samples were taken at the same time as those included in Enriquez et al. (2013a). DNA 236

was extracted from the bugs‟ rectal ampoule using DNAzol® (Invitrogen, USA) as 237

described previously (Maffey et al., 2012). DTU identifications were performed 238

introducing the following modifications to avoid unspecific amplifications within the 239

Spliced Leader region. SL-IR I, using primers TC2 and UTCC, was used to identify TcI 240

(475 bp) as follows: one cycle at 94 ºC for 3 min, 6 cycles at 62 ºC for 40s, 6 cycles at 241

60 ºC for 40 s, 40 cycles at 58 ºC for 40s and final extension step at 72 ºC for 10 min; 242

and SL-IR II, using primers TC1 and UTCC, was used to identify TcII, TcV, and TcVI 243

(425 bp) as follows: one cycle at 94 ºC for 3 min, 5 cycles at 68 ºC for 40s, 5 cycles at 244

66 ºC for 40 s, 5 cycles at 64 ºC for 40 s, 3 cycles at 62 ºC for 40 s, 30 cycles at 60 ºC 245

for 40s and final extension step at 72 ºC for 10 min. To enhance T. cruzi detection, PCR 246

reactions were carried out at a final volume of 50 μL of reaction mix, with 8 μL of DNA 247

sample as template and using Taq Platinum DNA polymerase (Invitrogen, USA). Given 248

that two unspecific bands in the SL-IR I PCR were persistently obtained in GEB 249

samples, the expected amplicon of 475 pb was purified and sequenced to confirm TcI 250

identification. 251

Incomplete identifications of DTUs were achieved in some cases in which only a 252

425bp-band was obtained in the SL-IR II PCR and no bands in the other PCR assays 253

due to low DNA content. These cases were described as TCII / V / VI. 254

255

2.6. Sequence analysis of SL-IR I 256

The PCR-amplified DNA was purified with Qiaquick Gel Extraction kit 257

(Qiagen) and cloned into pGEMT-easy vector following the manufacturer´s instructions 258

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(Promega, Madison, WI). Recombinant plasmids were screened by digestion with 259

EcoRI (New England Biolabs) and sequenced using T7 and SP6 universal primers by 260

Macrogen (Inc., Seoul, Korea) in an ABI PRISM 3730 DNA Analyzer automated 261

sequencer (Applied Biosystems) and aligned manually or using MEGA 5.1 software 262

(Tamura et al., 2007). Newly reported sequences are available at GenBank under 263

accession numbers KF964667, KF964668 and KF964669. A consensus of forward and 264

reverse sequences was created and a BLAST search was performed in GenBank 265

database to compare sequence identity. 266

2.7. Data analysis 267

Agresti-Coull binomial 95% confidence intervals (CI) were used for proportions. 268

The relationship between infectiousness to the vector of dogs and cats infected with T. 269

cruzi and potential predictors was analysed using maximum likelihood multiple logistic 270

regression implemented in Stata (Stata 10.1, Stata Corp, College Station, Texas). Only 271

dogs and cats aged 1 year or more were included in the regression to avoid potential 272

confounders due to growth effects. Because the bugs used in xenodiagnosis were 273

clustered on individual dogs or cats, observations are not independent. Therefore, the 274

xtmelogit command was used to include random effects that measure the residual 275

effects due to each subject on the probability of bug infection. The dependent variable 276

was the infection status of each bug used in xenodiagnosis. The independent variables 277

were the bloodstream parasite load (a continuous variable, in Pe/mL), body condition 278

(two levels: 0 = good, 1 = regular or poor); age of the host (a continuous variable, in 279

months); exposure to infected bugs, as measured by the abundance of T. infestans 280

infected with T. cruzi captured per 15 min-person per site in domiciles, kitchens and 281

storerooms (i.e., typical resting sites of dogs and cats), categorized in low, medium and 282

high-risk levels, 0 = 0; 1 = 1-5, and 2 ≥ 5 bugs, respectively; and identified DTU (0 = 283

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TcV, 1 = TcVI; 2 = TcI or TcIII). Identified DTU was excluded as a predictor in cats 284

due to the small sample size. The body condition status was divided into two levels 285

because of the low number of dogs with a poor body condition. Interaction terms were 286

added stepwise and dropped from the final model if not significant at a nominal 287

significance level of 5%. The Wald test examined the hypothesis that all regression 288

coefficients are 0. 289

To reflect the potential ability of a dog in a given body condition category to 290

infect a bug after feeding to repletion, we calculated an Infectious Potential Index (IPI) 291

for each category (Petersen et al., 2001) as the product of the proportion of seropositive 292

dogs, the proportion of seropositive dogs with a positive xenodiagnosis, and the median 293

infectiousness of seropositive dogs with a positive xenodiagnosis in each category. 294

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3. Results 295

3.1. Parasite concentration 296

A total of 44 seropositive dogs and 14 seropositive cats that had been examined 297

by xenodiagnosis were assayed by qPCR, and additionally, a xenodiagnosis-positive, 298

seronegative cat was also tested. All of these samples were qPCR-positive except one 299

seropositive cat that was negative by xenodiagnosis and kDNA-PCR (Table 3). All 300

control dogs (n = 16) and cats (n =7) that were both seronegative and xenodiagnosis-301

negative were also negative by kDNA-PCR and qPCR. In two xenodiagnosis-negative, 302

qPCR-positive cats, DNA quantifications could not be normalized because DTU 303

identifications were unsuccessful, therefore they were excluded from further analysis. 304

The frequency distribution of parasite DNA concentration estimated by qPCR 305

showed a negative binomial distribution in both host populations (Figure 1). The 306

median bloodstream parasite load was slightly higher in cats (median = 9.7 Pe/mL, first-307

third quartiles [Q1-Q3] = 2.9-96.9, range 0.0-1101.8) than in dogs (median = 8.1 308

Pe/mL, Q1-Q3 = 1.5-26.6, range 0.7-214.5), although this difference was not 309

statistically significant (Kruskal-Wallis test, P = 0.5). If the two seropositive, 310

xenodiagnosis-negative cats in which DNA quantifications could not be normalized had 311

been infected either with TcI or TcVI (i.e., the extreme cases for normalization), their 312

parasitemia would range between <1 and 10 Pe/mL. 313

314

3.2. Infectiousness to the vector and parasite load 315

Overall, 86% of dogs (n = 44) and 64% of cats (n = 14) seropositive for T. cruzi 316

were positive by xenodiagnosis. The mean infectiousness to T. infestans did not differ 317

significantly between dogs (48%, 95% confidence interval [CI] = 33-62%) and cats 318

(44%, 95% CI = 10-63%). Thirteen percent of 94 OM-negative bugs were positive by 319

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kDNA-PCR; thus, the corrected infectiousness to the vector would rise to 54%. The 320

frequency distribution of infectiousness showed a bimodal pattern in both host 321

populations (Figure 2). 322

The infectiousness of infected dogs and cats increased steeply with parasite load, 323

reaching maximum infectiousness (95%) at 96.9 Pe/mL for cats and 13.0 Pe/mL for 324

dogs (Figure 3). No clear association was observed between DTU and host 325

infectiousness or bloodstream parasite load (Figure 3). However, the two cats and the 326

only dog infected with TcI had nil infectiousness despite having 7.4, 9.7 and 20.5 327

Pe/mL, respectively (Figure 3) (Appendix). In these three individuals TcI was 328

confirmed by sequence analysis of Spliced Leader region (SL-IR I) (i.e., 98-100% 329

sequence identity). 330

Random-effects multiple logistic regression model showed that infectiousness to 331

the vector of seropositive dogs was significantly associated with the bloodstream 332

parasite load (OR = 1.02, 95% CI = 1.00-1.04, P < 0.05) and body condition (OR = 333

4.45, 95% CI = 1.24-16.01, P = 0.02) (Wald χ2

= 11.76, P < 0.01) (Table 1). No 334

association was found between infectiousness and age of the dog, exposure to infected 335

bugs at the dog‟s house and DTU. The median parasite load in dogs with poor or regular 336

body condition (9.93 Pe/mL, Q1-Q3 = 3.03-28.15) was approximately 4 times higher 337

than in dogs with good condition (2.67 Pe/mL, Q1-Q3 = 1.38-18.90), but this difference 338

was not statistically significant (Kruskal-Wallis, P = 0.17). 339

The infectiousness of infected cats was significantly associated with parasite load 340

(OR = 1.02, 95% CI = 1.00-1.04, P = 0.02) (Wald χ2

= 4.83, P = 0.09) (Table 1). No 341

significant association between infectiousness and age of the cat and exposure to 342

infected bugs was found by random-effects multiple logistic regression analysis. 343

344

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

3.3. Body condition and infectiousness 345

The classification of 111 dogs aged ≥ 1 year showed that 52%, 43% and 5% of 346

them were in a good, regular and poor body condition, respectively (Table 2). The mean 347

(±SD) age (3.0±2.6, 4.0±2.6, and 5.5±6.9 years) and the proportion of female dogs 348

(36%, 39% and 40%, χ2 = 0.2, df: 2, P = 0.9) were similar among categories ranging 349

from good to poor conditions, respectively. The prevalence of T. cruzi infection varied 350

between 50% and 80% and was not significantly different among body condition 351

categories (χ2

= 1.7, df: 2, P = 0.2). The median infectiousness increased from 30% in 352

dogs with good condition to 63% and 80% in animals with a regular and poor condition, 353

respectively. Dogs with a poor condition had an infectious potential index (IPI) 2.2 354

times greater than dogs with a regular condition, and 4.9 times greater than those in a 355

good condition (Table 2). However, “poor” dogs had a very low sample size of 5 versus 356

the total sample size of 44. 357

358

3.4. Profile of infected dogs and cats with high or low infectiousness 359

Nearly half of the dogs (50%) and cats (47%) showed high infectiousness and 360

were responsible of at least 80% of the infected xenodiagnosis bugs. The median 361

parasite load was nearly 10 times greater for dogs with high infectiousness than for dogs 362

with low infectiousness and this difference was statistically significant (Kruskal-Wallis 363

test, P < 0.01). For cats this relationship was 32 times greater (Kruskal-Wallis test, P = 364

0.02) (Table 3). Quantitative PCR could not distinguish between highly infectious and 365

non-highly infectious hosts, given that it detected T. cruzi DNA in 100% of the 366

seropositive dogs and in 93% of the seropositive cats, whereas kDNA-PCR detected 367

89% and 87% of them, respectively (Table 3). Four seropositive dogs and one 368

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seropositive cat that were both qPCR-positive and kDNA-PCR- negative showed very 369

low infectiousness to the vector (range, 0-5%). 370

371

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4. Discussion 372

By means of a real-time quantitative PCR protocol, this study shows that the 373

parasite load detected in bloodstream of dogs and cats naturally infected with T. cruzi 374

was closely associated with their infectiousness to the vector, and on average, was 4-5 375

times higher than that of chronic Chagas disease patients examined elsewhere (Freitas et 376

al., 2011; Moreira et al., 2013). 377

The median bloodstream parasite load was 8.1 Pe/mL in dogs and 9.7 Pe/mL in 378

cats, whereas in chronic patients was ≤ 2 Pe/mL (Freitas et al., 2011; Moreira et al., 379

2013). Furthermore, the sensitivity of qPCR in seropositive, chronic patients ranged 380

from 41% to 76% (Duffy et al., 2013; Moreira et al., 2013; Piron et al., 2007), whereas 381

in our study 93-100% of seropositive cats and dogs were qPCR-positive. These 382

substantial differences in parasite load most likely explain the much lower proportion of 383

T. cruzi-seropositive humans with a positive xenodiagnosis and their much lower 384

infectiousness to bugs relative to seropositive dogs and cats (Coura et al., 1991; Freitas 385

et al., 2011; Gürtler et al., 1996). 386

In our study, the infectiousness of dogs and cats increased non-linearly from 0% 387

to 85% with small increases in bloodstream parasite load. Two direct implications 388

follow from these observations: first, a positive xenodiagnosis can be obtained with 389

very low parasite load (i.e., 0.7 and 1.6 Pe/mL for dogs and cats, respectively), although 390

the ultimate fraction of bugs that become infected may be highly variable. Second, 391

given that 96% of the dogs and 90% of cats had more than 0.7-1.6Pe/mL, a large 392

proportion of them is expected to be highly infectious to the vector, as our study attests. 393

The wide variability in the infectiousness of dogs and cats with very low 394

bloodstream parasite load may be related, at least in part, to unrecorded variations in the 395

effective bloodmeal size achieved by the bugs used in xenodiagnosis. The actual 396

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infectiousness is expected to be greater than 44-48% because of the limited sensitivity 397

of optical microscopy when the intensity of rectal infections is low (false-negative rate 398

was 13%). 399

There were a few cases in which the high infectiousness observed could not be 400

explained by the estimated parasite load of the individual host on which the bugs had 401

fed, as in a TcVI-infected dog with a regular body condition and 0.8 Pe/mL which 402

infected 17 of 20 insects. Perhaps the differential origin of the blood samples for qPCR 403

(venous blood) and xenodiagnosis (capillary blood ingested by the bugs) may explain 404

this apparent inconsistency (Ferreira et al., 2007). Parasite DNA concentrations differed 405

between tissues in dogs naturally infected with Leishmania infantum (Ferreira et al., 406

2013). Technical errors in the quantification of bloodstream parasite load may be ruled 407

out because all blood samples from dogs with very low parasite load and large 408

infectiousness were repeated and the initial outcome corroborated (results not shown). 409

Although rather unlikely, we cannot rule out the presence of inhibitors that affected the 410

performance of qPCR despite IAC amplifications were consistently satisfactory. New 411

protocols based on multiplex qPCR amplificating both DNA targets (i.e., DNA of T. 412

cruzi and IAC) in the same reaction tube could enhance the reproducibility of qPCR 413

assays (Duffy et al., 2013). 414

Dogs and cats had similar infectiousness to the vector and this distribution was 415

aggregated, as in studies conducted elsewhere (Gürtler et al., 2007). There was a 416

fraction of highly infectious dogs and cats that may contribute disproportionally to 417

(peri)domestic transmission and persistence of T. cruzi. This heterogeneous distribution 418

of parasite burden in host populations is a generalized pattern in macroparasites and 419

microparasites (Wilson et al., 2001; Woolhouse et al., 1997) and may be used for 420

targeted control strategies. Trypanosoma cruzi-infected Dasypus novemcinctus 421

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armadillos and Didelphis albiventris opossums also have high infectiousness (Orozco et 422

al., 2013). 423

Multiple logistic regression analysis showed that the infectiousness of dogs was 424

associated with the bloodstream parasite load and the body condition of the individual 425

host. The latter is likely reflecting the combined effects of the immune system and the 426

nutritional status of the host (Keusch, 2003; Machado-Coelho et al., 2005; Petersen et 427

al., 2001). Dogs with a regular or poor body condition showed a nearly four-fold higher 428

median parasite load than dogs in good conditions. The former were probably less 429

effective in controlling parasitemia for reasons that remain to be identified. 430

Exposure to repeated reinfections with T. cruzi via inoculations was positively 431

correlated with a slight and transient increase in parasitemia in experimentally infected 432

dogs and mice (Machado et al., 2001; Bustamante et al., 2007). In our study, however, 433

exposure to infected bugs was not associated with increased infectiousness in dogs; 434

previous surveys yielded mixed outcomes on this point (Gürtler et al., 2007; 1992). A 435

possible explanation for these discrepancies is that our metric of exposure in the 436

regression model may not represent in a precise manner the actual likelihood of 437

reinfection the dogs faced throughout its home range in the past. In addition, we found 438

no association between infectiousness and age of dogs (as in one of three previous 439

studies: Gürtler et al., 2007; 1996; 1992) and parasite DTU (Cardinal et al., 2008). 440

Other factors not evaluated here, such as concomitant parasitic infections, stress levels, 441

inoculum size, and genetic background of host and parasite, may eventually mask the 442

effects of age, DTU and reinfections on infectiousness. Moreover, the existence of a 443

polymorphism in the genes involved in the immune response could also affect 444

infectiousness, as in dogs infected with L. infantum (Quinnell et al., 2003). In cats, 445

infectiousness was only associated with parasite load. TcVI-infected cats were more 446

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infectious that the few ones infected with TcV or TcI, but samples sizes were too small 447

to make any sound conclusion. 448

The concurrent finding of TcI infections with bloodstream parasite load ranging 449

from 7.4 to 20.5 Pe/mL and nil infectiousness (as determined by optical microscopy) 450

suggest a differential behavior of TcI or a different susceptibility of the vector species 451

involved. Indeed, TcI-infected dogs and cats were rare and could only be identified by 452

means of a highly sensitive kDNA-PCR applied to the rectal-ampoule lysates of 453

xenodiagnostic bugs or GEB samples. The substantial variations in metacyclogenesis 454

and trypomastigote density among triatomine species (Carvalho-Moreira et al., 2003; 455

Loza-Murghía et al., 2010; Perlowagora-Szumlewicz et al., 1994) may explain the nil 456

infectiousness (as measured by optical microscopy) recorded in TcI-infected dogs and 457

cats. However, samples sizes were small and this relationship should be further 458

investigated. 459

In our study, approximately 50% of the infected dogs and cats were responsible 460

for at least 80% of the infected T. infestans used in xenodiagnosis. All of them were 461

positive by qPCR and kDNA-PCR. However, dogs and cats with very low 462

infectiousness to the vector (<20%) were also frequently positive by these techniques. 463

These results suggest that the quantitative estimates of parasite load and not the 464

qualitative outcomes of qPCR or kDNA-PCR would be a more precise tool to identify 465

highly infectious dogs and cats. A wide variability in infectiousness (range, 0-63% for 466

dogs and 35-67% for cats) was observed between 1-5 Pe/mL. Courtenay et al. (2014) 467

recently showed that quantitative PCR was needed to identify highly infectious L. 468

infantum-infected dogs. 469

Quantitative PCR was more sensitive and less time-consuming than 470

xenodiagnosis, and alone or in conjunction with the body condition index, may be 471

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helpful to identify highly infectious hosts; replace xenodiagnosis (or hemoculture) to 472

assess treatment efficacy or infection status, and contribute to novel control strategies 473

(e.g., targeted treatment or vaccination) that reduce the risks of domestic transmission of 474

T. cruzi. 475

476

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ACKNOWLEDGMENTS 477

We are grateful to Carolina Cura and Margarita Bisio for laboratory assistance on 478

molecular biology. Romina Piccinali, Marina Leporace, Francisco Petrocco, Laura 479

Tomassone, Leonardo Ceballos, Julián Alvarado-Otegui, Sol Gaspe and Nala for field 480

or laboratory assistance, and to Lucía Maffey, Pilar Fernández, Fernando Garelli, Juan 481

Gurevitz, Yael Provecho, Jimena Gronzo and Carla Cecere for valuable comments. 482

Special thanks to the villagers of Pampa del Indio for kindly welcoming us into their 483

homes and cooperating with the investigation. 484

485

FINANCIAL SUPPORT 486

This study received financial support from International Development Research 487

Center (Eco-Health Program); the UNICEF/UNDP/World Bank/WHO Special 488

Programme for Research and Training in Tropical Diseases (TDR) to REG; Agencia 489

Nacional de Promoción Científica y Tecnológica to REG; Agencia Nacional de 490

Promoción Científica y Tecnológica and GlaxoSmithKline to REG, and University of 491

Buenos Aires to REG. JB, MMO, SW, AGS, REG and MVC are members of 492

CONICET Researcher‟s Career. 493

494

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Figure 1. Frequency distribution of parasite load of T. cruzi-infected dogs (black bars) 674

and cats (white bars); Pampa del Indio, Chaco, 2008. 675

676

Figure 2. Frequency distribution of infectiousness to T. infestans of T. cruzi-infected 677

dogs (black bars) and cats (white bars); Pampa del Indio, Chaco, 2008. 678

679

Figure 3. Distribution of infectiousness to T. infestans of T. cruzi-infected dogs (A) and 680

cats (B) according to parasite DNA quantification by qPCR and identified DTU; Pampa 681

del Indio, Chaco, 2008. The figure excludes two seropositive cats from which DNA 682

quantifications could not be normalized. 683

684

Table 1. Infectiousness to the vector T. infestans of dogs and cats infected for T. cruzi

according to potential risk factors; Pampa del Indio, Chaco, 2008.

Factor

Dogsa

Catsa

Median

infectiousness

% (first-third

quartiles) OR (95% CI)

Median

infectiousness

% (first-third

quartiles) OR (95% CI)

Host body condition

– –

Good 16 (5-68) 1

– –

Regular/Poor 64 (32-84) 4.45 (1.24-16.01)

– –

Parasitemia

(Pe/mL) – 1.02 (1.00-1.04)

– 1.02 (1.00-1.04)

Exposure to infected bugs (abundance of infected-bug captured per 15 min-person per

site)

0 55 (35-75) 1

67 (25-75) 1

1-5 68 (35-84) 0.41 (0.03-6.58) 79 (78-79) 0.21 (0.01-5.17)

>5 20 (5-65) 0.40 (0.02-9.43) 0 (0-50) 0.20 (0.01-6.66)

Age of host – 1.00 (0.99-1.02) – 1.18 (0.93-1.48)

DTU

TcV 30 (16-45) 1 – –

TcI or TcIII 38 (0-75) 0.58 (0.02-22.42) – –

TcVI 62 (30-80) 2.22 (0.45-10.94) – –

a. Three dogs and four cats aged <1 year were not included in the regression.

Table(1)

Table 2. Relationship between host body condition and infectiousness to T. infestans of

dogs infected with T. cruzi aged ≥1 year; Pampa del Indio, Chaco, 2008.

Host body

condition

Percentage of

seropositive

dogs (No.

examined)

Percentage of

seropositive dogs with

a positive

xenodiagnosis (No.

examined)

Median

infectiousness of

xenodiagnosis-

positive dogs

Infectious

Potential

Index

Good 55 (56) 78 (23) 30 0.13

Regular 50 (50) 93 (25) 63 0.29

Poor 80 (5) 100 (3) 80 0.64

Total 54 (111) 85 (41) 61 0.28

Table(2)

Table 3. Relationship among qualitative kDNA-PCR, qPCR and parasite load to detect

T. cruzi-infected dogs and cats with different levels of infectiousness

Host Infectiousnessa

No. positive

xenodiagnosis

(No.

examined)

Mean

infectiousness

(%)

Median parasite

load (Q1-Q3)

No.

Positive

kDNA

(%)

No.

Positive

qPCR

(%)

Dogs High 22 (22) 79 21.3 (7.3-39.3) 22 (100) 22 (100)

Low 16 (22) 19 2.4 (1.3-12.6) 17 (77) 22 (100)

Total 38 (44) 48 8.1 (1.5-26.6) 39 (89) 44 (100)

Cats High 7 (7) 79 96.1(18.1-202.1) 7 (100) 7 (100)

Low 3 (8) 14 3.0 (2.2-7.4) 6 (75) 7 (88)

Total 10 (15) 44 9.7 (2.9-96.9) 13 (87) 14 (93)

a. The “high infectiousness” category included dogs and cats that were responsible for

≥80% of all infected triatomines used in the xenodiagnostic tests. The “low

infectiousness” category included all remaining seropositive dogs and cats examined by

xenodiagnosis.

Table(3)

Parasite equivalents per mL

0 1-30 >30-60 >60-90 >90-120>120-150 >150

No

. o

f h

ost

s

0

5

10

15

20

25

30

35

40

Dogs

Cats

Figure(1)

Infectiousness to bugs (%)

0-20 21-40 41-60 61-80 81-100

Per

centa

ge

of

host

s

0

10

20

30

40

50

60

Dogs

Cats

Figure(2)


0 50 100 150 200 250

Infe

ctio

usn

ess

to b

ugs

(%)

0

20

40

60

80

100

TcV

TcVI

TcI

TcIII

TcII/V/VI

A


0 40 80 120 160 200 1100

Infe

ctio

usn

ess

to b

ugs

(%)

0

20

40

60

80

100

TcV

TcVI

TcI

B

Figure(3)

Supplementary MaterialClick here to download Supplementary Material: Appendix.docx

http://ees.elsevier.com/meegid/download.aspx?id=126396&guid=9f71170e-198e-4949-ace6-9cd1436f4eaf&scheme=1

High levels of Trypanosoma cruzi DNA determined by qPCR and infectiousness to Triatoma infestans...

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