MEEGID 2155
No. of Pages 8, Model 5G
21 November 2014 Infection, Genetics and Evolution xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 5 6
Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model
3 4 7 8 9 10 11 12 13 15 14 16 1 1 8 3 19 20 21 22 23 24 25 26 27 28 29 30
Q1
Lissa Cruz a,b,1, Angie Vivas a,1, Marleny Montilla c, Carolina Hernández b,d, Carolina Flórez c, Edgar Parra e, Juan David Ramírez b,⇑ a
Programa de Bacteriología, Facultad de Ciencias de la Salud, Universidad Colegio Mayor de Cundinamarca, Bogotá, Colombia Unidad Clínico-Molecular de Enfermedades Infecciosas (UCMEI), Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia Grupo de Parasitología, Instituto Nacional de Salud (INS), Bogotá, Colombia d Red Chagas Colombia, Instituto Nacional de Salud (INS), Bogotá, Colombia e Grupo de patología, Instituto Nacional de Salud (INS), Bogotá, Colombia b c
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 10 November 2014 Accepted 12 November 2014 Available online xxxx Keywords: Chagas disease Epidemiological cycles Trypanosoma cruzi I DTU Molecular epidemiology
a b s t r a c t Chagas disease is an endemic zoonosis in Latin America and caused by the parasite Trypanosoma cruzi. This kinetoplastid displays remarkable genetic variability, allowing its classification into six Discrete Typing Units (DTUs) from TcI to TcVI. T. cruzi I presents the broadest geographical distribution in the continent and has been associated to severe forms of cardiomyopathies. Recently, a particular genotype associated to human infections has been reported and named as TcIDOM (previously named TcIa-b). This genotype shows to be clonal and adapted to the domestic cycle but so far no studies have determined the biological properties of domestic (TcIDOM) and sylvatic TcI strains (previously named TcIc-e). Hence, the aim of this study was to untangle the biological features of these genotypes in murine models. We infected ICR-CD1 mice with five TcI strains (two domestic, two sylvatic and one natural mixture) and determined the course of infection during 91 days (acute and chronic phase of the disease) in terms of parasitemia, tissue tropism, immune response (IgG titers) and tissue invasion by means of histopathology studies. Statistically significant differences were observed in terms of parasitemia curves and prepatent period between domestic (TcIDOM) and sylvatic strains. There were no differences in terms of IgG antibodies response across the mice infected with the five strains. Regarding the histopathology, our results indicate that domestic strains present higher parasitemias and low levels of histopathological damage. In contrast, sylvatic strains showed lower parasitemias and high levels of histopathological damage. These results highlight the sympatric and behavioral differences of domestic and sylvatic TcI strains; the clinical and epidemiological implications are herein discussed. Ó 2014 Published by Elsevier B.V.
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
53 54
1. Introduction
55
Trypanosoma cruzi is the causative agent of Chagas disease, an illness related to underdevelopment and poverty in rural and urban areas of Latin America. The World Health Organization (WHO) estimates that about 10 million people are infected with T. cruzi (Rassi et al., 2012; Guhl, 2009). Chagas disease is clinically characterized by the existence of two phases (acute and chronic phases): the acute phase is frequently asymptomatic and in some
56 57 58 59 60 61
⇑ Corresponding author. 1
E-mail address: [email protected] (J.D. Ramírez). These authors contributed equally to this work.
cases is possible to observe the route of entry of the parasite by the presence of a localized chagoma. About 30–40% of infected individuals will develop cardiomyopathy, likewise can present alterations in the motility of the esophagus and colon, dilated to various degrees (Rassi et al., 2012; Jelicks and Tanowitz, 2011; Barbosa and Carmo, 2012). T. cruzi is defined as a heterogeneous taxon and subdivided into six Discrete Typing Units (DTUs), corresponding to TcI, TcII, TcIII, TcIV, TcV and TcVI (Zingales et al., 2009). Recently, a new genotype named TcBat has been described in isolates from bats in Brazil, Colombia and Panama (Marcili et al., 2009; Pinto et al., 2012; Ramírez et al., 2013a). TcI is the DTU with the broadest distribution in America, and prevalent in northern countries of South America, with presence of sporadic cases in the Southern Cone (Guhl and Ramírez, 2011). In northern cone countries as Colombia, Venezu-
http://dx.doi.org/10.1016/j.meegid.2014.11.012 1567-1348/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Cruz, L., et al. Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.11.012
62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
MEEGID 2155
No. of Pages 8, Model 5G
21 November 2014 2
L. Cruz et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx
109
ela, Ecuador and Peru, this premise is fulfilled by observing the prevalence of TcI in a variety of mammals and vectors from the domestic, peridomestic and sylvatic epidemiological cycles. TcI is the major cause of acute oral infections and cardiac alterations. Also, this DTU has been associated to severe myocarditis, compared to other DTUs, as TcII, TcV and TcVI (Ramírez et al., 2010, 2013b; Ocaña et al., 2010; Segovia et al., 2013). By analyzing fragments of the nuclear and mitochondrial genomes of TcI, the existence of an emerging genotype associated with human infections was reported and called as TcIDOM (which previously corresponded to TcIa-b/VenDOM) (Ramírez et al., 2012). Population genetics studies demonstrate that this genotype presents low genetic variability and a clonal adaptation to the human host. Therefore, it has been reported in Colombia, Venezuela, Peru, Brazil and Argentina showing its remarkable dispersion (Cura et al., 2010; Zumaya et al., 2012). Murine models have been of paramount importance to identify biological changes facilitating the in vivo study of Chagas disease. A variety of studies have been conducted with the purpose of understanding the biological characteristics of T. cruzi I and T. cruzi II in murine models. However, these studies have not considered TcI strains isolated from the domestic (TcIDOM) and sylvatic cycles of transmission of Chagas disease preventing the complete knowledge of their biological behaviors such as parasitemia in the course of the disease, organ/tissue tropism, immune response and behavior in mixed infections. Hence, it is imperative to include a biological characterization of TcI strains to provide such knowledge. Thus, this study will determine whether or not there are significant differences in the biological properties, including the assessment of parasitemia, antigenicity profiles and pathological effects of TcIDOM and sylvatic strains in murine biomodels (Ragone et al., 2012; Dos Reis et al., 2012; Suarez et al., 2009; Rivera et al., 2000; Téllez et al., 2008).
110
2. Methods
111
2.1. Strain typing
112
Five strains were maintained in ICR-CD-1 mice (outbred mice derived from animals from Charles River Laboratories, Wilmington, Massachusetts) to maintain their infectivity. We bled infected mice with each of the T. cruzi I strains isolated from different departments in Colombia (Table 1). DNA extraction from 200 lL of mouse blood was performed using the Roche High Pure PCR Template Preparation kit. In order to determine the DTU and discriminate TcIDOM/Sylvatic strains, we sequenced the intergenic region of Mini-exon gene and 10 mitochondrial MLST markers (Herrera et al., 2005, 2009; Messenger et al., 2011).
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
113 114 115 116 117 118 119 120 121
2.2. Preparation of T. cruzi I inoculum
122
All five T. cruzi strains used in this study were pricked out in mice every 21 days from the date of its isolation in culture medium after observing parasitic forms in Tobie medium. Six ICR-CD1 male mice of 21 days were used to initiate the study considering the recommendations and bioethical principles for the use of animals in research. The animals were anesthetized with a dose of xylazine and ketamine according to weight and subsequently infected with each strain of T. cruzi I. At 21 days post-infection, parasites were observed in the fresh preparations. Hence, intracardiac puncture was conducted to obtain 1 mL of blood. The parasites were counted on Neubauer chamber to estimate the parasite concentration. Dilution was performed with sterile saline solution (0.85%) with each of the samples containing trypomastigotes of T. cruzi to reach a final concentration of 102 parasites/200 lL (Ragone et al., 2012).
123
2.2.1. ICR-CD-1 mice inoculation We employed 18 ICR-CD-1 mice (14–17 gr) of 18 days of age and free of T. cruzi infection. The mice were infected with 200 lL of 102 trypomastigotes from each of the selected strains. We used in total five TcI strains: two domestic TcI strains (TcIDOM/TcIa) (MHOM/CO/04/MG, MHOM/CO/01/DA), two sylvatic TcI strains (TcId) (MHOM/CO/10/GC, MHOM/CO/11/JDD) and one multiclonal strain (natural mixture of TcIDOM/TcIa and sylvatic TcI clones (TcId)) (MHOM/CO/03/CG) (Table 1). Three mice per strain were used, including 3 negative controls. The biomodels were maintained in the animal facility of the National Institute of Health (NIH), Colombia for a period of 91 days under biosafety regulations, macro-environmental conditions, humidity 60% ± 5, temperature: 20 °C ± 2, 10–15 ventilation changes per hour, housed in cages on a T3 type stainless steel, 48 cm long, 26 cm wide and 16 cm high, with a capacity of 3 animals per cage. The cages were equipped with chip, water and food ad lib, following the principles for animal experimentation stipulated in the animal facility and under national and international regulations.
137
2.3. Weight measurement and parasitemia curve
156
The weight of infected mice and negative controls were recorded at intervals of 4 days using an electric balance Sartorius PT-600 size 900 700 200 . Every fourth day trypomastigotes were quantified by collecting 20 lL of blood from a terminal tail portion in each of the infected mice using heparinized capillaries. To determine the parasitemia curve we employed Neubauer chamber and fresh slide smears. Neubauer chamber is only able to detect >3 parasites/10 lL in a 1:20 dilution (lower dilutions are not allowed due to the red cells interference when reading the chamber), therefore
157
Table 1 List of the domestic and sylvatic TcI strains and results of biological parameters calculated. Strain type
SL-IR genotype
Strain code
Host
Geographical origin
Average (days) of prepatent period (PPP)
Average (days) of patent period (PP)
Average (days) of day of maximum parasitemia (DMP)
TcIDOM
TcIa
MHOM/CO/01/DA
Sutatenza, Boyacá
16
42
33
TcIa
MHOM/CO/04/MG
Yopal, Casanare
13
37.5
43
TcId
MHOM/CO/11/JDD
22.5
25
MHOM/CO/10/GC
San Vicente de Chucuri, Santander Tame, Arauca
14.5
TcId
13
22.5
22
TcIa/TcId
MHOM/CO/03/CG
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens
Cartagena del Chaira, Caquetá
13
21
22
Sylvatic
Mixed
Please cite this article in press as: Cruz, L., et al. Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.11.012
124 125 126 127 128 129 130 131 132 133 134 135 136
138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155
158 159 160 161 162 163 164 165
MEEGID 2155
No. of Pages 8, Model 5G
21 November 2014 L. Cruz et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx
171
we decided to count the parasites in fresh slide smears that detects
No. of Pages 8, Model 5G
21 November 2014 Infection, Genetics and Evolution xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 5 6
Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model
3 4 7 8 9 10 11 12 13 15 14 16 1 1 8 3 19 20 21 22 23 24 25 26 27 28 29 30
Q1
Lissa Cruz a,b,1, Angie Vivas a,1, Marleny Montilla c, Carolina Hernández b,d, Carolina Flórez c, Edgar Parra e, Juan David Ramírez b,⇑ a
Programa de Bacteriología, Facultad de Ciencias de la Salud, Universidad Colegio Mayor de Cundinamarca, Bogotá, Colombia Unidad Clínico-Molecular de Enfermedades Infecciosas (UCMEI), Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia Grupo de Parasitología, Instituto Nacional de Salud (INS), Bogotá, Colombia d Red Chagas Colombia, Instituto Nacional de Salud (INS), Bogotá, Colombia e Grupo de patología, Instituto Nacional de Salud (INS), Bogotá, Colombia b c
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 10 November 2014 Accepted 12 November 2014 Available online xxxx Keywords: Chagas disease Epidemiological cycles Trypanosoma cruzi I DTU Molecular epidemiology
a b s t r a c t Chagas disease is an endemic zoonosis in Latin America and caused by the parasite Trypanosoma cruzi. This kinetoplastid displays remarkable genetic variability, allowing its classification into six Discrete Typing Units (DTUs) from TcI to TcVI. T. cruzi I presents the broadest geographical distribution in the continent and has been associated to severe forms of cardiomyopathies. Recently, a particular genotype associated to human infections has been reported and named as TcIDOM (previously named TcIa-b). This genotype shows to be clonal and adapted to the domestic cycle but so far no studies have determined the biological properties of domestic (TcIDOM) and sylvatic TcI strains (previously named TcIc-e). Hence, the aim of this study was to untangle the biological features of these genotypes in murine models. We infected ICR-CD1 mice with five TcI strains (two domestic, two sylvatic and one natural mixture) and determined the course of infection during 91 days (acute and chronic phase of the disease) in terms of parasitemia, tissue tropism, immune response (IgG titers) and tissue invasion by means of histopathology studies. Statistically significant differences were observed in terms of parasitemia curves and prepatent period between domestic (TcIDOM) and sylvatic strains. There were no differences in terms of IgG antibodies response across the mice infected with the five strains. Regarding the histopathology, our results indicate that domestic strains present higher parasitemias and low levels of histopathological damage. In contrast, sylvatic strains showed lower parasitemias and high levels of histopathological damage. These results highlight the sympatric and behavioral differences of domestic and sylvatic TcI strains; the clinical and epidemiological implications are herein discussed. Ó 2014 Published by Elsevier B.V.
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
53 54
1. Introduction
55
Trypanosoma cruzi is the causative agent of Chagas disease, an illness related to underdevelopment and poverty in rural and urban areas of Latin America. The World Health Organization (WHO) estimates that about 10 million people are infected with T. cruzi (Rassi et al., 2012; Guhl, 2009). Chagas disease is clinically characterized by the existence of two phases (acute and chronic phases): the acute phase is frequently asymptomatic and in some
56 57 58 59 60 61
⇑ Corresponding author. 1
E-mail address: [email protected] (J.D. Ramírez). These authors contributed equally to this work.
cases is possible to observe the route of entry of the parasite by the presence of a localized chagoma. About 30–40% of infected individuals will develop cardiomyopathy, likewise can present alterations in the motility of the esophagus and colon, dilated to various degrees (Rassi et al., 2012; Jelicks and Tanowitz, 2011; Barbosa and Carmo, 2012). T. cruzi is defined as a heterogeneous taxon and subdivided into six Discrete Typing Units (DTUs), corresponding to TcI, TcII, TcIII, TcIV, TcV and TcVI (Zingales et al., 2009). Recently, a new genotype named TcBat has been described in isolates from bats in Brazil, Colombia and Panama (Marcili et al., 2009; Pinto et al., 2012; Ramírez et al., 2013a). TcI is the DTU with the broadest distribution in America, and prevalent in northern countries of South America, with presence of sporadic cases in the Southern Cone (Guhl and Ramírez, 2011). In northern cone countries as Colombia, Venezu-
http://dx.doi.org/10.1016/j.meegid.2014.11.012 1567-1348/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Cruz, L., et al. Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.11.012
62 63 64 65 66 67 68 69 70 71 72 73 74 75 76
MEEGID 2155
No. of Pages 8, Model 5G
21 November 2014 2
L. Cruz et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx
109
ela, Ecuador and Peru, this premise is fulfilled by observing the prevalence of TcI in a variety of mammals and vectors from the domestic, peridomestic and sylvatic epidemiological cycles. TcI is the major cause of acute oral infections and cardiac alterations. Also, this DTU has been associated to severe myocarditis, compared to other DTUs, as TcII, TcV and TcVI (Ramírez et al., 2010, 2013b; Ocaña et al., 2010; Segovia et al., 2013). By analyzing fragments of the nuclear and mitochondrial genomes of TcI, the existence of an emerging genotype associated with human infections was reported and called as TcIDOM (which previously corresponded to TcIa-b/VenDOM) (Ramírez et al., 2012). Population genetics studies demonstrate that this genotype presents low genetic variability and a clonal adaptation to the human host. Therefore, it has been reported in Colombia, Venezuela, Peru, Brazil and Argentina showing its remarkable dispersion (Cura et al., 2010; Zumaya et al., 2012). Murine models have been of paramount importance to identify biological changes facilitating the in vivo study of Chagas disease. A variety of studies have been conducted with the purpose of understanding the biological characteristics of T. cruzi I and T. cruzi II in murine models. However, these studies have not considered TcI strains isolated from the domestic (TcIDOM) and sylvatic cycles of transmission of Chagas disease preventing the complete knowledge of their biological behaviors such as parasitemia in the course of the disease, organ/tissue tropism, immune response and behavior in mixed infections. Hence, it is imperative to include a biological characterization of TcI strains to provide such knowledge. Thus, this study will determine whether or not there are significant differences in the biological properties, including the assessment of parasitemia, antigenicity profiles and pathological effects of TcIDOM and sylvatic strains in murine biomodels (Ragone et al., 2012; Dos Reis et al., 2012; Suarez et al., 2009; Rivera et al., 2000; Téllez et al., 2008).
110
2. Methods
111
2.1. Strain typing
112
Five strains were maintained in ICR-CD-1 mice (outbred mice derived from animals from Charles River Laboratories, Wilmington, Massachusetts) to maintain their infectivity. We bled infected mice with each of the T. cruzi I strains isolated from different departments in Colombia (Table 1). DNA extraction from 200 lL of mouse blood was performed using the Roche High Pure PCR Template Preparation kit. In order to determine the DTU and discriminate TcIDOM/Sylvatic strains, we sequenced the intergenic region of Mini-exon gene and 10 mitochondrial MLST markers (Herrera et al., 2005, 2009; Messenger et al., 2011).
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108
113 114 115 116 117 118 119 120 121
2.2. Preparation of T. cruzi I inoculum
122
All five T. cruzi strains used in this study were pricked out in mice every 21 days from the date of its isolation in culture medium after observing parasitic forms in Tobie medium. Six ICR-CD1 male mice of 21 days were used to initiate the study considering the recommendations and bioethical principles for the use of animals in research. The animals were anesthetized with a dose of xylazine and ketamine according to weight and subsequently infected with each strain of T. cruzi I. At 21 days post-infection, parasites were observed in the fresh preparations. Hence, intracardiac puncture was conducted to obtain 1 mL of blood. The parasites were counted on Neubauer chamber to estimate the parasite concentration. Dilution was performed with sterile saline solution (0.85%) with each of the samples containing trypomastigotes of T. cruzi to reach a final concentration of 102 parasites/200 lL (Ragone et al., 2012).
123
2.2.1. ICR-CD-1 mice inoculation We employed 18 ICR-CD-1 mice (14–17 gr) of 18 days of age and free of T. cruzi infection. The mice were infected with 200 lL of 102 trypomastigotes from each of the selected strains. We used in total five TcI strains: two domestic TcI strains (TcIDOM/TcIa) (MHOM/CO/04/MG, MHOM/CO/01/DA), two sylvatic TcI strains (TcId) (MHOM/CO/10/GC, MHOM/CO/11/JDD) and one multiclonal strain (natural mixture of TcIDOM/TcIa and sylvatic TcI clones (TcId)) (MHOM/CO/03/CG) (Table 1). Three mice per strain were used, including 3 negative controls. The biomodels were maintained in the animal facility of the National Institute of Health (NIH), Colombia for a period of 91 days under biosafety regulations, macro-environmental conditions, humidity 60% ± 5, temperature: 20 °C ± 2, 10–15 ventilation changes per hour, housed in cages on a T3 type stainless steel, 48 cm long, 26 cm wide and 16 cm high, with a capacity of 3 animals per cage. The cages were equipped with chip, water and food ad lib, following the principles for animal experimentation stipulated in the animal facility and under national and international regulations.
137
2.3. Weight measurement and parasitemia curve
156
The weight of infected mice and negative controls were recorded at intervals of 4 days using an electric balance Sartorius PT-600 size 900 700 200 . Every fourth day trypomastigotes were quantified by collecting 20 lL of blood from a terminal tail portion in each of the infected mice using heparinized capillaries. To determine the parasitemia curve we employed Neubauer chamber and fresh slide smears. Neubauer chamber is only able to detect >3 parasites/10 lL in a 1:20 dilution (lower dilutions are not allowed due to the red cells interference when reading the chamber), therefore
157
Table 1 List of the domestic and sylvatic TcI strains and results of biological parameters calculated. Strain type
SL-IR genotype
Strain code
Host
Geographical origin
Average (days) of prepatent period (PPP)
Average (days) of patent period (PP)
Average (days) of day of maximum parasitemia (DMP)
TcIDOM
TcIa
MHOM/CO/01/DA
Sutatenza, Boyacá
16
42
33
TcIa
MHOM/CO/04/MG
Yopal, Casanare
13
37.5
43
TcId
MHOM/CO/11/JDD
22.5
25
MHOM/CO/10/GC
San Vicente de Chucuri, Santander Tame, Arauca
14.5
TcId
13
22.5
22
TcIa/TcId
MHOM/CO/03/CG
Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens
Cartagena del Chaira, Caquetá
13
21
22
Sylvatic
Mixed
Please cite this article in press as: Cruz, L., et al. Comparative study of the biological properties of Trypanosoma cruzi I genotypes in a murine experimental model. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.11.012
124 125 126 127 128 129 130 131 132 133 134 135 136
138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155
158 159 160 161 162 163 164 165
MEEGID 2155
No. of Pages 8, Model 5G
21 November 2014 L. Cruz et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx
171
we decided to count the parasites in fresh slide smears that detects
