Results of the behavior, parasitemia, serology and histology in the murine model, infected with inoculum of four strains of
The global expansion of Chagas disease is due to the constant migration of individuals from endemic countries with incidence of vector and nonvector transmission of Trypanosoma cruzi. The disease is present in its various stages: chronological characteristic signs and symptoms of the infection and its mechanism of immune system and cell and tissue damage. The first stage, which lasts 90 days approximately, is diagnosed by direct methods (blood smears stained with Giemsa, fresh and xenodiagnosis). The indeterminate-chronic stage is asymptomatic, but the growth and intracellular binary multiplication of the trypomastigotes continue promoting cell lysis and allowing parasites to infect other cells, with preferential tropism to organs producing mega syndromes such as cardiomyopathy, myocarditis, meningoencephalitis, megaesophagus and megacolon. Inadvertently, this process is repeated for several years leading to Chagas disease. The mouse inoculation allows checking the parasitemia in vivo and the development of the disease in short time (signs, behavior and tropism), histopathological alterations and detection of antibodies in serum. These parameters may vary when using different strains of T. cruzi from different geographical areas; Triatoma species due to their genetic variability are influenced by the environment, nutrition, reservoirs and habitat. The murine model ECA CD-1 has the ability to replicate human findings of Chagas disease.
- Chagas disease
- murine model
- CD-1 strain
In 1909, the Brazilian doctor and researcher Carlos Ribeiro Justiniano das Chagas discovered the etiological agent of the later called Chagas disease in the triatomine insect (family
Chagas disease is a chronic debilitating affectation which impairs the health and the quality of life of infected people all around the world. The estimated number of infected people in the world arose from 30 million in 1990 to 6–8 million in 2010. In the past 20 years, the annual incidence decreased from 700,000 to 28,000 and the burden of Chagas disease decreased between 1990 and 2006 from 2.8 million disability-adjusted life to less than half a million . Chagas disease is in close relation to the socioeconomic status of the population migration between Latin America and the rest of the world, and it currently represents one of the most important public health concerns . The initiatives of the Americas have allowed achieving significant reductions in the number of acute cases and the presence of domiciliary Triatominae vectors in all endemic areas.
1.1. Life cycle
1.2. Routes of transmission
We can distinguish three cycles of vector transmission in
The domestic cycle comprises the infection of humans and the consequent Chagas disease. The domestic cycle is defined by factors in the anthroponotic foci, making people one of the last natural reservoirs of
Depending on the eco-epidemiological conditions of the place, both circles can overlap becoming an intradomiciliary cycle, especially when mankind invades the natural habitat of these vectors and builds houses fearing the entrance of reduvids (Figure 2).
On the other hand, the infection transmission by blood transfusion has become a serious complication in nonendemic countries, due to the migration of infected individuals from endemic regions . This route is considered the second most important route of transmission in endemic areas .
The infection is not detected until adulthood in the course of the latent or indeterminate phase . Spontaneous abortions have been reported, premature birth, intrauterine growth retardation, stillbirths and various clinical forms that can go from low birth weight, hepatomegaly, splenomegaly, acute respiratory symptoms, anemia, digestive disorders, Cardiac and Central Nervous System (CNS). The donation of organs has increased the number of infected people in urban areas. It has been informed about the transmission of infection to seronegative heart, bone marrow, liver, pancreas and kidney transplant recipients with variable transmission rates that reach 35% . Patients infected with
1.3. Pathology and mechanism of injury
The disease presents three phases: the acute, chronic asymptomatic (intermediate or dormant) and the chronic symptomatic. The incubation period in the acute phase is 4–10 days and of shorter duration when the route of transmission is blood transfusion. This stage is generally asymptomatic, or it can occur with systemic manifestations that are common to other diseases such as fever, edema, lymphadenopathy, hepatomegaly and splenomegaly. It is accompanied by anorexia, fatigue, myalgia, headache and, occasionally, arthralgia. In some cases, there are signs of inoculation or entrance door, chagomas, lesions that are more frequent in the face and limbs of a forunculoid aspect, pink or violet and indurated borders. A typical sign in children is the bipalpebral edema (sign of Roman-Mazza). In this phase, the trypomastigotes are easily detected in the blood due to the high parasitemia.
In case the acute phase is overcome, there will be an extended period of chronic disease without clinical symptoms that lasts from 5 to 10 years, characterized by low parasitemia and by the presence of anti-
In this last Chagas disease’s phase, histological lesions are disseminated in the heart muscle, intestines and nervous system, inflammatory infiltrates composed mainly of CD8+ cells, in addition to nests (pseudocysts) full of parasites in their form of amastigotes .
Regarding the treatment, there are currently only two drugs available, benznidazole and nifurtimox. The therapeutic success is closely related to the stage of infection at the time of starting the treatment. Patients in acute phase (regardless of the route of infection), neonates and children, have better therapeutic prognosis [12, 15]. On the other hand, the success of such drugs is discussed in individuals with chronic infection and so far, there is no established therapeutic regimen . The adverse effects are much more important among the adult patients; cases of photosensitivity and skin rashes, nausea, anorexia, weight loss and abdominal pain have been reported.
Recently, a group of biomedical and clinical scientists members of the network NHEPACHA (New Tools for the diagnosis and evaluation of the patient with Chagas disease), based on clinical and immunological evidence, have suggested new paradigms regarding the medicines for the Chagas disease in order to provide better treatment for patients in chronic phase .
On the other hand, the study of biochemical and biological characteristics of the hemoflagellate parasite has enabled the identification of new targets for chemotherapeutic agents; an example would be the drug trials with inhibitors of the biosynthesis of Ergosterol, Posaconazole and Ravuconazole, respectively, in patients with chronic Chagas disease.
1.4. Diagnosis of infection
Parasitological methods for detection of the acute phase have great sensitivity (direct methods) .
In the same way, these methods are used for the diagnosis of congenital infection in newborns and in children under the age of 6 months. The lack of maturation of the immune system and the presence of maternal IgG antibodies make, in the latter, the use of the serology for the infection diagnosis impossible . The protozoan
1.5. Origin of the discrete units (UDTs) typing of
The evolutionary history of
Studies in murine experimental models have shown that both the parasite and host genotypes are crucial for tissue distribution and pathophysiology of infection by
2. Importance of the murine model in research American trypanosomiasis
2.1. The murine model in biomedical research
Animal models are very useful for studying human diseases because there are hundreds of pathogens that affect both humans and animals. The use of experimental animals in biomedical research represents a key element for development of new prevention approaches and treatment of transmissible and nontransmissible diseases. Suffice it to recall the rabies vaccines, smallpox, tetanus, diphtheria, whooping cough and polio, the development of several antibiotics, insulin, and the knowledge of the genetic bases of inheritance . No doubt that mice are the most commonly used animal for in vivo assays among experimental animal models in biology and medicine. The use of mice allows the study of mammal’s reactions against aggressions like poisoning or infection (viral, bacterial, or parasitic), the study of immune responses and disorders and many others in several different fields like oncology, teratology and embryology  (Figure 6).
Herein, we present a comparative cross-sectional study involving four
2.2. Collection sites
Several communities from different States of México were included in the present study: San Pablo, Tolimán in Querétaro State, Milpillas of Talpa de Allende in Jalisco State, Sant Catarina in Morelos State and Jala in Nayarit State (Figure 7).
2.3. Triatoma collection and maintenance
Cages covered with adhesive tape were used, with the glue facing outward. A live Wistar rat was placed inside the cage. Cages were placed at late night in strategic areas under the loose stones of poultry and farm animal fences, fallen leaves and wooden logs. Cages were collected the next day, early in the morning (Figure 8).
Triatomas glued to the surface of the gummed paper were carefully detached, with the aid of entomological tweezers and placed in jars covered with mosquito mesh. A piece of filter paper in accordion shape was placed inside the bottle to facilitate the movement of the triatomas and the collection of urine or feces deposited on its surface.
The triatomas are maintained inside the bottles at 25–26°C and 60% humidity (RH) in bacteriological incubator. Triatomas were blood fed directly from a shaved rabbit every 2 weeks allowing them to feed for 20–25 m and then they were placed back in the incubator (Figure 9).
2.4. Study of the intestinal content in the triatomine
We use two techniques for collecting intestinal content from triatomine after blood feeding. In the first one, the triatomine is introduced in a 10 x 20 mm tube; normally the bug deposits stool or urine in the bottom of the tube and then it is collected with saline solution. The second technique consists in pressing gently the triatomine abdomen, inducing that the rectal blister freely releases the stool (semi-separated blood). Intestinal content is collected in a watch glass and saline solution is added at 37°C. In both techniques, the metacyclic trypomastigote and epimastigote forms are observed fresh, using a microscope with 400 magnifications. The trypomastigotes are counted in a Neubauer chamber. If the count is above 10,000 parasites per cubic centimeter, mice are inoculated as mentioned below.
The same procedure is performed with each one of the strains of species of triatomas captured (Figure 10).
Trypanosoma cruzi, inoculation in mice
Male mice of CD-1 strain are used since estrogen in females can stimulate the activity of macrophage phagocytes and, the localized immune response .
Using an insulin syringe four groups of 10 mice were inoculated intraperitoneally with 3x103 epimastigote and/or trypomastigote forms of
2.6. Study of behavior (signs)
After the first day of inoculation, the behavior of the infected murine model was observed, comparing it with an uninfected control.
2.7. Study of the parasitemia
Parasitemia levels are determined in infected mice 5,10,15,20,25 and 30 days post
2.8. Histopathological analysis
Histological sections of 10 microns are obtained from mouse dissected organs (brain, heart, intestines and skeletal muscle) and stained with H/E. Microscope slide preparations are observed at 40X . Tissues from infected mice and the respective control animals are included in the analysis.
2.9. Detection of antibodies in serum (detection of anti-
Trypanosoma cruziantibodies in serum)
Blood is obtained by cardiac puncture and centrifuged at 5000 rpm to separate the clot from the serum and maintained at −20°C until it is used to carry out an ELISA (Accutrack Chagas Microelisa Test) in the search of anti-
The mice inoculated with all the studied
However, it was possible to observe some differences in the virulence of each strain according to the geographic area geographic area where they came from (Figure 11).
The inoculant obtained from triatomines from Talpa de Allende in Jalisco state and Santa Catarina in Morelos state generated in the corresponding mice a parasitemia of 3: 4 trypanosomes per field, at 14 and 16 days postinoculation, respectively. In both cases, altered movements and physical shape of the mice began at about the same time. By day 20 and 23, respectively, the parasitemia reached the peak, so it was proceeded to sample fresh blood and dissect organs in order to perform the serological and histopathological assays
Parasitemia in mice inoculated with the
Blood parasitemia was undetectable in mice inoculated with
|Geogra-phic area||Species of Triatoma||Days in which presents parasitemia||Frizz hair||Difficulty walking||ELISA test||Brain||Heart||Skeletal muscle||Intestine|
|Talpa de Allende. Jalisco||14||Positive||Positive||Reactive||Negative||Positive||Positive|
|San Pablo Tolimán|
|It was presented in 90 days||Negative||Negative||Reactive||Negative||Negative||Negative||Negative|
Presence of amastigote nests and histopathological damage in heart and intestine muscle showed direct relationship with parasitemia level, which indicates that trypanosomes are installed and recognize different tissues where they reproduce rapidly intracellularly, resulting in a greater number of parasites in blood after they differentiate into trypomastigotes. This sequence is only observed in the most virulent strains such as those from the States of Jalisco, Morelos and Nayarit (Figure 12).
In the present study, assessment of clinical manifestations, parasitemia levels, histological changes and seropositivity in murine model allowed us to know the behavior of different
We were able to identify two different
Mitie-Nisimura et al., in 2014, were able to induce acute phase inoculating mice by intraperitoneal injection trypomastigote forms of
Espinoza et al., in 2010, observed contrasting differences between two