Trypanosoma cruzi Infection in Didelphis virginiana in Relationto Population Parameters and Variables Associated withPresence in Rural Community Dwellings in Yucatan, Mexico

Julian Parada-Lopez,1 Silvia F. Hernandez-Betancourt,1 Hugo A. Ruiz-Pina,2

Francisco J. Escobedo-Ortegon,2 Salvador Medina-Peralta,3 and Jesus A. Panti-May1

1Facultad de Medicina Veterinaria y Zootecnia, Campus de Ciencias Biologicas y Agropecuarias, Universidad Autonoma de Yucatan, km 15.5 carretera

Merida-Xmatkuil. Apdo. Postal 4-116 Itzimna, Merida, Yucatan, Mexico2Laboratorio de Zoonosis y otras ETV’s, Centro de Investigaciones Regionales ‘‘Dr. Hideyo Noguchi’’, Universidad Autonoma de Yucatan, Av. Itzaes

#490 9 59, Col. Centro, C.P. 97000 Merida, Yucatan, Mexico3Facultad de Matematicas, Universidad Autonoma de Yucatan, Periferico Norte Tablaje 13615 A.P. 172, C.P. 97119 Merida, Yucatan, Mexico

The Virginia opossum (Didelphis virginiana) is a hypoth-

esized reservoir of Trypanosoma cruzi, the etiologic agent of

Chagas disease. This marsupial is widely distributed in

North and Central America (Krause and Krause 2006) and

is the most abundant in the north of Yucatan (Jones et al.

1974). However, despite this abundance, basic aspects of its

population biology, the relationship between its population

structure and infection with T. cruzi and the risk of

transmission to humans are poorly understood.

Studies of T. cruzi infection in Virginia opossum

related to population parameters (sexes, age structure, and

reproductive status) come mainly from South America

(Telford et al. 1981; Telford and Tonn 1982; Schweigmann

et al. 1999) and only one has been carried out in Mexico

(Ruiz-Pina and Cruz-Reyes 2002). In all of these studies,

sampling consisted of capture–recapture methods covering

the environment surrounding rural households.

To our knowledge, no previous work has examined

T. cruzi infections in opossums occurring within human

dwellings, or the potential influence of household-level

characteristics on opossum presence. The aim of this study

was therefore to analyze the relationship between certain

population parameters of D. virginiana and T. cruzi infec-

tion, and define the association between variables in rural

housing characteristics and the presence of opossums

within households in a rural village in Yucatan, Mexico.

This was carried out in order to determine the influence of

ecological (population) and housing factors on the infec-

tion frequency and the risk of Chagas disease transmission

in rural populations.

The study was conducted in the suburbs of the town of

Molas, Yucatan (20�4800000N; 89�3800000W), which has a

population of 2,014 inhabitants in 553 households (INEGI

2010). The climate is warm subtropical with summer rains,

an annual average temperature of over 26�C and annual

rainfall of 850 to 1,100 mm (Garcıa 1973). The vegetation

surrounding the village is low tropical deciduous forest

(INEGI 2010). Most of the dwellings in the town have farm

animals in the yards, such as cattle and poultry kept for sale

and consumption, as well as the presence of relatively large

densely vegetated areas between the dwellings.

Capture of opossums was carried out monthly in 43

randomly selected households during the periods October–

December 2009 (wet season) and February–April 2010 (dry

season). One live trap (66 cm 9 23 cm 9 23 cm, Toma-

hawk, USA) was placed per household overnight in the

Published online: February 13, 2013

Correspondence to: Julian Parada-Lopez, e-mail: jplbiol@gmail.com

EcoHealth 10, 31–35, 2013DOI: 10.1007/s10393-013-0819-5

Short Communication

� 2013 International Association for Ecology and Health

yard. Traps were baited with pineapple and reviewed in the

morning. Total capture effort was 240 trap-nights.

Captured opossums were kept for up to 3 days in the

Vivarium of the Laboratorio de Zoonosis y otras ETV’s of the

Centro de Investigaciones Regionales ‘‘Dr. Hideyo Noguchi’’

of the Universidad Autonoma de Yucatan. Specimens were

kept in an isolated, dark, humid and well ventilated area to

avoid stress by disturbance and exposure to extreme tem-

peratures and precipitation (Sikes et al. 2011). They were

provided with food and water ad libitum. Age was deter-

mined considering criteria of dentition (Petrides 1949),

while sex and reproductive status were determined according

to the characteristics of the female marsupial (Reynolds

1952) and testicular measurements (length and width) taken

from the males (Winegarner 1982; Holmes-Meissner 1986).

A blood sample (1 ml) was removed from each indi-

vidual by puncturing the caudal vein (Jurgelski 1974).

Capillary tubes with heparin were filled with blood and

subjected to a microconcentration technique (Vega and

Naquira 2005) for detection of blood trypanosomes. A

20 ll sample of blood was mixed with 80 ll of sterile water

(Castro et al. 2002) in a tube for subsequent PCR. Each

tube was then boiled for 10 min to release the kinetoplast

minicircles (Britto et al. 1993) and then centrifuged at

14,000 rpm for 5 min. The resulting supernatants were

transferred to fresh tubes. Amplification was subsequently

performed using oligonucleotides TcZ1 and TcZ2 and

following the amplification program described by Moser

et al. (1989) for the detection of T. cruzi DNA.

Individuals were considered infected by T. cruzi on

testing positive for one of these techniques. Opossums

deemed positive by microscopic examination were eutha-

nized to obtain the organs for further investigation of the

presence of other pathogens that can pose a risk to human

health. Specimens that tested negative to two microscopic

examinations over two consecutive days were released in

the village near the capture site.

To identify rural housing variables that may favor the

presence or establishment of opossums, data and photo-

graphs were taken of the yards of each household, as well as

other yards and vegetation patches surrounding the houses.

In general, we identified three variables: (1) shelters,

including piles of domestic waste (S1) and fallen trees and

other accumulated vegetable matter (piled leaves and

branches) (S2); (2) food sources, including exposed food

waste (F1), fruit trees (F2) and poultry (F3); and (3)

adjacency to yards with little vegetation (A1) and yards

with tall grass, bushes and/or vacant lots (vegetation pat-

ches) (A2).

Table 1. Observed Abundance of D. virginiana According to

Age Structure, Sex and Reproductive Status for Each Sampling

Period

Sampling period Population parameters

Sex Age class RS

# $ Juvenile Subadult Adult R NR

Wet 13 9 8 4 10 8 14

Dry 5 11 0 0 16 15 1

RS reproductive status, R reproductive, NR non reproductive.

Table 2. Logistic Regression Relating T. cruzi Infection with Population Parameters and Values of the Improved Model Adjustment

(n*)

Parameter Estimate Standard error Likelihood ratio tests: Chi-square, df = 1 Estimated odds ratio

Constant 3.98153.8770*

1.78731.6705*

Sex 1.0731 1.0029 v2 = 1.2322, P = 0.2670 2.9244

0.9993* 0.8938* v2 = 1.3154, P = 0.2514* 2.7163*

AS1 -3.8238 1.8211 v2 = 5.4274, P = 0.0198 0.0218

-3.7383* 1.7426* v2 = 5.6747, P = 0.0172* 0.0238*

AS2 -3.6723 1.6596 v2 = 6.1927, P = 0.0128 0.0254

-3.6507* 1.6488* v2 = 6.1692, P = 0.0130* 0.0260*

Reproductive condition 0.8711 1.2977 v2 = 0.4659, P = 0.4949 2.3896

0.7529* 1.0867* v2 = 0.4973, P = 0.4807* 2.1231*

Period -0.1802 1.0707 v2 = 0.0284, P = 0.8661 0.8351

The bold numbers indicates significant values (P < 0.05).

32 Julian Parada Lopez et al.

Logistic regression analysis was used (Hosmer and

Lemeshow 2000) to relate infection to sampling period, sex,

reproductive condition and age structure. The latter was

defined with two indicator variables (AS1, AS2: Juvenile [0,

0], Subadult [1, 0] and Adult [0, 1]).

Using a nonparametric Mann–Whitney U test (Zar

2010), abundance of opossums was compared between

houses that did or did not feature each of the identified

variables. To determine the existence of groups of dwellings

that were similar in terms of the presence or absence of

recorded characteristics, a hierarchical classification analy-

sis was conducted for clustering, using the Jaccard index as

a measure of similarity. To construct the dendrogram, the

unweighted pair group method (UPGMA) was used

(Manly 2005). The abundance of opossums in groups of

similar dwellings was then determined at different levels of

similarity. These were compared using one-way analysis

of variance or the Kruskal–Wallis test (Zar 2010) when data

did not meet assumptions of normality. Statistical tests

were considered significant when P < 0.05. The statistical

packages used were Statgraphics Centurion v. 15.2.06

(StatPoint 2007) and SPSS 15 (SPSS 2006).

A total of 38 opossums were captured in 24 of the 43

dwellings over the two sampling periods. Individuals of all

three age classes (juveniles, subadults and adults) were

captured in the wet period while, during the dry period,

only adults were trapped. Similarly, most of the repro-

ductive individuals were caught in the dry period (Table 1).

Natural infection with trypanosomes was found in

55% (21/38) of the opossums with the two techniques

applied. Of the infected individuals, 81% (17/21) were

adults, whereas only one juvenile (5%) and three subadults

(14%) tested positive for infection, and these were all

captured during the wet period.

Logistic regression analysis showed that the variables

that defined age structure (AS1 and AS2) were significant.

The model adjustment improved (G = 9.5530, P = 0.0487,

df = 4) with the removal of the period variable, which had

Figure 2. Classification dendrogram of

groups of dwellings according to the

presence or absence of the variables

considered (Jaccard similarity index).

*Hn house number.

Figure 1. Infection status (infected and non-infected) of Didelphis

virginiana in relation to age structure.

T. cruzi Infection in D. virginiana in Yucatan 33

the greatest P value (P = 0.8661) in the likelihood ratio

tests; however, only AS1 and AS2 were significant

(Table 2). To determine the relationship between age

structure and infection, a Chi-square test were used, and

this showed a significantly greater infection rates in adult

opossums compared to the juveniles (v2 = 7.6250, P = 0.0209)

(Fig. 1).

Hierarchical classification analysis showed nine groups

of highly similar dwellings according to shared variables

(Fig. 2). However, no significant differences were found

between these groups of similar dwellings, in terms of

opossum abundance or opossum abundance between

houses that did or did not present piles of domestic waste

(S1), fallen trees and other accumulated vegetable matter

(piled leaves and branches) (S2), exposed food waste (F1),

fruit trees (F2), poultry (F3), adjacency to yards with little

vegetation (A1) and yards with tall grass, bushes or vacant

lots (A2).

The relationship found between age structure and

infection status confirms the findings of previous studies

conducted in Yucatan (Ruiz-Pina and Cruz-Reyes 2002),

Venezuela (Telford et al. 1981; Telford and Tonn 1982) and

Argentina (Schweigmann et al. 1999) that report higher

frequencies of infection in adults compared to juveniles.

This may be due to vectorial transmission: in Yucatan,

Triatoma dimidiata (principal vector of T. cruzi) is more

abundant during the dry season (Dumonteil et al. 2002),

thereby favoring transmission of the parasite between

vectors and opossums in that period (Ruiz-Pina and Cruz-

Reyes 2002) which coincides with a predominance of adult

opossums in the population. Another reason why juveniles

have a lower frequency of T. cruzi infection is the presence

of antibodies to T. cruzi in their blood, provided by breast

milk, which affords partial protection during the suckling

period and decreases after weaning (Jansen et al. 1994).

The variables that were presented in groups of similar

dwellings were mainly F3 (poultry) and A2 (yards with tall

grass, bushes, and vacant lots), however, the greatest

abundance of opossums was observed only in the group

that shared variable A2 (Table 3), suggesting that adjacency

of the yards to those with tall grass and bushes, or to vacant

lots, is the most important variable by which these mam-

mals frequent the dwellings in the study site.

The opossum, D. virginiana, is a species well-adapted

to man-made environments and an understanding of its

ecology, population biology and pathogen prevalence is

crucial to elucidate its importance in transmission

dynamics of infectious agents such as T. cruzi that affect

public health in rural communities. Moreover, more

studies need to be conducted that consider household

characteristics as a part of the anthropogenic environment

that can influence T. cruzi and D. virginiana interactions as

this may improve disease control programs and thereby

minimize the risk of transmission in rural populations.

ACKNOWLEDGMENTS

We thank the project PROMEP 103.5/09/1258 (Red

Epidemiologica de Enfermedades Zoonoticas y transmiti-

das por Vector de Importancia en Salud Publica) for

funding. Grateful thanks also go to Marıa Jose Baeza,

Veronica Aranda and Alan Cuxim for field and laboratory

support, and to the population of the town of Molas

allowing access to their homes.

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