Oral infection and survival of Trypanosoma cruzi in sugar cane juice conditioned at different temperatures

doi:10.21203/rs.3.rs-2733315/v1

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Oral infection and survival of Trypanosoma cruzi in sugar cane juice conditioned at different temperatures

https://doi.org/10.21203/rs.3.rs-2733315/v1

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The current most important form of transmission for Trypanosoma cruzi is the oral route, being responsible for high mortality during the acute phase in infected individuals. Therefore, the present study has investigated the possibility of infection for this parasite using sugar cane juice in different temperatures employing metacyclic trypomastigotes obtained from xenodiagnosis performed in Swiss mice previously infected with T.cruzi Y strain, and then diluted in 15 mL of sugar cane juice. In addition, 20 mice were infected using 2,5 mL of that mixture for the stomach histopathological analysis, which was euthanized after the second, fourth, tenth, and fifteenth days after infection. Concurrently, six batches of 10 mice were fed using 1,5 mL of the mixture kept for 12 hours at temperatures of -80ºC, -20ºC, + 2ºC, + 28ºC, + 60ºC, and + 80º C, for later infection verification. Inflammatory infiltrate was found after the second day of infection, and amastigotes nests were present after the fourth, tenth, and fifteenth day in the margo plicatus stomach region. Viable trypomastigotes were observed in the microtubes kept at -80ºC, -20,ºC and + 2ºC, but the animal’s infection was observed in -80ºC and + 2ºC groups. In vitro tests demonstrated the decrease of T. cruzi trypomastigote viability, being negative after 120 hours at -20ºC, 144 hours at + 2ºC, in contrast to the maintenance of survival after 168 hours at -80ºC. We also observed the ability of survival and infection of T. cruzi packaged at -80ºC without the use of preservatives and, therefore, less suitable for storing food.

Trypanosoma cruzi

Chagas disease

gastric absorption

inflammation

cryopreservation

Sugarcane

Chagas disease is an anthropozoonosis caused by the infection of the hemoflagellate protozoan Trypanosoma cruzi. It is estimated that about 6 to 7 million people are infected with the parasite around the world [1], of which approximately 4.6 million live in Brazil [2]. These numbers make this parasitosis one of the most serious in the American continent, due not only to the high prevalence but also the expressive mortality [3, 4]. In the 1990s, an initiative called Cone Sul was carried out among the Latin American countries that had the largest geographical distribution of Triatoma infestans, to intensify measures to combat Chagas disease such as reducing vector transmission and control of blood products [5]. However, despite the intense campaign to combat the transmission of insects promoted by health services in Brazil, the disease remains being transmitted, most often by alternative routes, such as the oral form, which is currently the most important one [6].

The first experimental studies on the possibility of T. cruzi infection by the oral route were presented in 1933 by Ezequiel Dias [7], and in 1968 human cases were reported when students and employees of an agricultural school in a municipality of Rio Grande do Sul state consumed vegetables with excreta and anal gland secretions of contaminated marsupials [8]. After some years, another outbreak occurred in the Northeast region of Brazil, when several people ingested sugarcane juice contaminated with T. cruzi [9]. However, other types of food have also been identified as capable of carrying T. cruzi, such as drinking water, milk, boiled rice, fresh cheese, and meat with Triatoma pallidipennis feces contaminated by the parasite [10]. However, sugar cane juice contaminated with metacyclic trypomastigotes obtained from Rhodnius neglectus feces [11] and açaí pulp is relevant means of transmission in [7]. In other countries, cases have also been described after ingestion of guava, orange, and patauá juices [12].

Experimental studies in murine animals have shown parasite entry involving various tissues such as the nasal mucosa and palate, with subsequent dissemination to the central nervous system and adjacencies [13]. However, it was shown that oral infection in the vertebrate host depends on the ability of the parasite to internalize in stomach cells. In this sense, the metacyclic trypomastigotes have glycoproteins (gp82) in their membranes that enable them to penetrate and infect humans systemically [14].

Besides these factors inherent to the parasite, some studies have shown that lower temperatures favor the survival and infection of T. cruzi by the oral route [15, 16]. In contrast, studies have shown a decrease in the parasite's power of infection when exposed to heat [17].

Thus, given the current epidemiological importance of oral infection by T. cruzi, and considering the differences found in the infection capacity due to the interference of environmental temperature to which the parasite is subjected, this research aimed to investigate the viability of T. cruzi in sugarcane juice stored at different temperatures and its infectivity, as well as the location of its entrance pathway in the stomach of Swiss mice.

2.1. Animals: A total of 90 male Swiss mice with a mean age of 30 days and a mean weight of 30 g obtained from the Central Animal Facility of the Marília Medical School (Famema) were used. The animals were kept in the Famema Parasitology Laboratory, in an environment with a controlled temperature (23–25ºC), 12/12h light-dark cycle, and water and food ad libitum. This study was approved by the Ethics Committee on Animal Research of the Marília Medical School (protocol number 2981/19).

2.2. Natural xenodiagnosis to obtain metacyclic trypomastigotes: Eight Swiss mice were infected intraperitoneally with 0.5 mL of blood from a mouse previously infected with strain Y of T. cruzi. Two xenodiagnostic tests were performed, one for the in vivo test and the other for the in vitro test. In each xenodiagnosis, four animals and 40 third-stage T. infestans nymphs acquired from the insectary of the School of Pharmaceutical Sciences of the Unesp, campus of Araraquara - SP, were used. One month after each xenodiagnosis, the triatomines were restrained with the help of tweezers and had their abdomen compressed using glass slides for the evacuation of feces.

2.3. Obtaining the sugarcane juice: the juice was purchased two hours before use from a snack bar in the city and transported in a plastic bottle to the Parasitology laboratory for the experiment.

2.4. Obtaining the mixture of sugarcane juice and metacyclic trypomastigotes: The feces from each insect containing the metacyclic trypomastigotes were collected and mixed in 15 mL of sugarcane juice at room temperature. Then, the number of live metacyclic trypomastigotes in the mixture was obtained using the Neubauer chamber, resulting in 165,000 forms/ml, equivalent to 16,500 forms/100 µl.

2.5. Histopathological analysis: Immediately after dilution of the triatomine feces in sugarcane juice, four batches of five animals were infected orally with 100 µl of the mixture containing 16,500 metacyclic trypomastigote forms for analysis after the second, fourth, 10th, and 15th days post-infection. A batch of two animals that received only 100 µl of sugarcane juice was not infected and was used as a control group and euthanized on day 15. On the respective days, the infected and non-infected animals were euthanized in a CO2 chamber, and the stomach was collected and sectioned longitudinally at the midline of the greater and lesser curvature, obtaining the anterior and posterior halves. Each half was fixed in 10% formalin in 0.1M Sorensen phosphate buffer, pH 7.3, for 24h. The samples were then washed in tap water for 24 h and kept in 70% ethyl alcohol. Afterward, the samples were dehydrated in different concentrations of ethyl alcohol, diaphanized in butyl alcohol, and embedded in Paraplast Plus® Tissue Embedding Medium (Mc Cormick Scientific, II, USA). Sections 5µm thick were obtained in a microtome (Leica RM2255), stained with hematoxylin-eosin, and analyzed under an Olympus BX41 microscope with a DP-25 digital video camera [18]. To analyze the histopathological aspects a qualitative and semi-quantitative analysis was performed and to grade inflammatory process, amastigote nests, and necrosis the following scores were used: "zero", absence of inflammatory process, amastigote nests, and necrosis; "+" discrete inflammatory process, rare amastigote nests and necrosis corresponding up to 25% of the analyzed area; "++" moderate inflammatory process, number of amastigote nests and necrosis corresponding up to 50% of the analyzed area and; "+++" intense inflammatory process, frequent amastigote nests, and necrosis, corresponding to 75% or more of the analyzed area.

2.6.In vivoinfectivity test: To test the infectivity of T. cruzi at different temperatures, 60 animals were randomly distributed in six lots of 10 animals. For this, 1.5 mL of the dilution of the triatomine feces in sugar cane juice containing 16,500 metacyclic trypomastigotes forms in 100 µl were conditioned in six microtubes for 12 hours at -80ºC, -20ºC, + 2ºC, + 28ºC, + 60ºC, and + 80ºC. After this period, the viable trypomastigotes were counted in a Neubauer chamber, and each mouse was infected with 100 µL of the mixture by pipetting directly into the mouth, regardless of the result obtained in the count of trypomastigotes forms. The research of blood trypomastigotes started on the 4th post-infection day by tail blood analysis following Brener's technique [19] and was performed weekly for 30 days.

2.7. Testesin vitro: The in vitro test was performed two months after the in vivo test, since the results obtained at -80ºC, -20ºC, and + 2ºC, which presented viable T. cruzi, were considered. For this, the mixture of feces and sugarcane juice was separated into 24 microtubes of 1.5 mL each, similar to the in vivo test, and subdivided into three lots of eight microtubes. Subsequently, each batch of eight microtubes was conditioned at their respective temperatures, and after intervals of 12, 24, 48, 72, 96, 120, 144, and 168 hours, one microtube from each batch was analyzed. During the analysis, we waited for each mixture to reach room temperature before counting the viable trypomastigotes. This count was performed in a Neubauer chamber using a double-blind scheme.

2.8. Statistical Analysis: Quantitative variables were described by mean and standard deviation and qualitative variables by absolute (N) and relative (%) frequency distribution. The proportion distribution was analyzed by the Chi-square test. The analysis of the relationship between qualitative variables was performed by the Fisher's Exact test for association. Bootstrap resampling technique was used for the in vitro test for a sample of 1000 subjects and a 95% confidence interval for percentile. Data were described by the mean and 95% confidence interval (95% CI). Qualitative variables were described by absolute (N) and relative (%) frequency distribution. To analyze the main effect of temperature and time and the interaction between temperature and time, Anova-two-way was performed, followed by Bonferroni's Post-Hoc comparisons. The significance level adopted was 5% (p ≤ 0.05) and the data were analyzed in SPSS software version19.0.

Viable trypomastigotes were observed at -80º, -20º, and + 2º after 12-hour conditioning (Table 1). Infected mice were observed on day 10 post-infection, one in the group that received the mixture conditioned at + 2ºC and another in the group at -80ºC. No trypomastigotes were found in the blood of the other animals until 30 days after infection.

Table 1

The number of metacyclic trypomastigotes forms observed in a Neubauer chamber after 12 hours of conditioning at different temperatures.
Temperatures in ºC
Trypomastigotes forms	-80	-20	+ 2	+ 28	+ 60	+ 80
Number/mL	55,000	72,500	92,500	0	0	0
Number /100µL	5,500	7,250	9,250	0	0	0

The histopathological analysis showed different degrees of inflammation in the anterior and posterior portions of the stomach near the margo plicatus region. From the second day after infection, an inflammatory process was observed and amastigotes nests were observed from the fourth day, predominating in the anterior region. A predominance of the chronic active inflammatory process was observed, composed mostly of mononuclear cells: lymphocytes, plasma cells, monocytes, and macrophages, as well as polymorphonuclear cells: neutrophils and eosinophils. No statistically significant difference (p ≥ 0.05) was observed concerning time in both the inflammatory process and amastigote nests by Fisher's exact test (Figs. 1 and 2; Table 2).

Table 2

Semi-quantitative analysis of inflammation and presence of amastigote nests in mice orally infected with 16,500 metacyclic trypomastigotes in 100µL of sugarcane juice.
Histopathological analysis		Time (days)
		2		4		10		15
		N(%)	N(%)	N(%)	N(%)	N(%)	N(%)	N(%)	N(%)
		A	P	A	P	A	P	A	P
Inflammation	+	4(80)	2(40)	5(100)	3(60)	4(80)	1(20)	4(80)	4(80)
	++	1(20)	2(40)	0	0	1(20)	0	0	0
	+++	0	0	0	0	0	1(20)	0	0
Amastigotes	+	0	0	1(20)	1(20)	1(20)	0	1(20)	0
Note: N: number of animals; (%): percentage of animals; A: anterior half of the stomach; P: posterior half of the stomach.

Table 3 shows the viability of metacyclic trypomastigotes forms at + 2ºC, -20ºC, and − 80ºC. It was possible to observe the maintenance of live trypomastigotes number for up to 168 hours at -80ºC and a gradual decrease of parasites' viability, which was negative in 120 hours at -20ºC, in 144 hours at -2ºC. The main effect of temperature and time was observed, as well as the interaction between time and temperature, in addition to higher values of metacyclic trypomastigotes/ml for the temperature of + 2ºC about the temperature of -80ºC in the interval from 0 to 48 hours. At -20ºC, it was observed lower values of metacyclic trypomastigotes/ml than at -80ºC for the interval from 48 to 168 hours. It was also observed higher values of metacyclic trypomastigotes/ml for the temperature of + 2ºC in relation to the temperature of -20ºC for 0 to 120 hours. At -80 º C, no significant difference was observed between the times. At -20 º,C there was a significant difference between 24 and 96 hours, but after 120 hours the metacyclic trypomastigotes/ml values were zeroed. For the temperature of + 2ºC, the values of metacyclic trypomastigotes/ml in the interval from 24 to 48 hours were significantly higher in relation to the interval from 72 to 168 hours, except 144 hours in which no more viable trypomastigotes were observed.

Table 3

Number of viable metacyclic trypomastigotes/ml at different temperatures per time interval using Neubauer chamber, followed by Bootstrap treatment.
Hour (H)	Temperature (T)									Anova-two-way
	-80 ºC			-20 ºC			+ 2 ºC			p-value
	Mean	IC 95%		Mean	IC 95%		Mean	IC 95%		T	H	Interaction (T*H)
	Mean	Inferior	Superior	Mean	Inferior	Superior	Mean	Inferior	Superior	T	H	Interaction (T*H)
12	33500	29000	38000	55000a	49000	61000	128000ƚǂa	113000	143000	< 0,001*	< 0,001**	< 0,001***
24	32000	19000	45000	21500	17000	26000	79500ƚǂa	64000	95000
48	49000	32000	66000	8000ƚ	8000	8000	84000ǂa	77000	91000
72	49000	38000	60000	4000ƚ	4000	4000	16500b	12000	21000
96	53000	29000	77000	1500ƚb	1000	2000	8000ƚb	6000	10000
120	56500	45000	68000	0ƚb	0	0	1500ƚb	1000	2000
144	54500	39000	70000	0ƚb	0	0	0ƚb	0	0
168	64500	43000	86000	0ƚb	0	0	500ƚb	0	1000
Note: Data received the Bootstrap technique treatment of resampling to 1000 sample elements and 95% confidence interval for the percentile

* indicates a significant effect of temperature by Anova-two-way for p-value ≤ 0.05.

** indicates a significant effect of time by Anova-two-way for p-value ≤ 0.05.

*** indicates significant interaction of temperature and time by Anova-two-way for p-value ≤ 0.05

ƚ indicates a significant difference of temperature of -80°C within each hour by Bonfererroni's Post-Hoc test for p-value ≤ 0.05.

ǂindicates a significant difference in the temperature of -20 ºC within each hour by Bonfererroni's Post-Hoc test for p-value ≤ 0.05.

Different letters indicate a significant difference between hours within each temperature by Bonfererroni's Post-Hoc test for p-value ≤ 0.05

Currently, oral infection by T. cruzi affects 21 Latin American countries considered endemic regions, which can cause death in 8 to 35% of infected individuals during the acute [20]. In Brazil, this form of transmission is responsible for 75.34% of acute cases of the disease [6], occurring when infected triatomines are mashed with juices for the preparation of drinks [21], as well as by ingesting meat and even blood from reservoir animals in regions or countries where this custom is believed to be medicinal treatment [12].

The effectiveness of oral infection is directly related to some factors, among them the glycoconjugate 35/50k Da, present on the surface of the parasite, which protects it against the action of the protease present in the gastric juice, facilitating its internalization in the gastric epithelium [22]. In addition, the cell adhesion molecule gp82, also present in the membrane of the trypomastigotes form of T. cruzi, assists in the binding of the parasite to the gastric mucin of the host stomach, allowing it to overcome this barrier to gain access to epithelial cells [23].

In this context, the results showed that the parasites internalization occurred in the margo plicatus region, a transition site between the non-glandular and glandular epithelium of the mice's stomach, agreeing with several studies that showed the stomach as the probable entry pathway of the parasite [14, 24, 25]. It was also found the presence of a greater number of animals with the inflammatory process and amastigotes nests in the anterior portion, although without significant difference compared to the posterior portion, corroborating studies by Dominguez [26].

It was observed that the first signs related to the penetration of metacyclic trypomastigotes in the gastric mucosa occurred after two days of infection, with the presence of an inflammatory infiltrate consisting of polymorphonuclear and eosinophils. The finding of amastigotes and mast cell nests on the fourth-day post-infection is ratified by several authors [27, 14, 25, 27], keeping these characteristics until the end of the experiment.

According to Lederkremer and Agusti [28], the access of T. cruzi to the cell can be helped by the trans-sialidase enzyme, which would help the transfer of sialic acid molecules from the host plasma membrane to the trypomastigote [29]. The presence of glycoproteins present in the parasite membrane, associated with the action of sialic acid allows the survival and infection capacity [22, 23, 30], besides the very structure of microtubules of the parasite cytoskeleton, which confers resistance to mechanical trauma and low temperatures [31, 32].

However, experimental studies by Covarrubias et al. [33] showed that pepsin present in the stomach of the host would increase the infection capacity and parasitemia rate of some strains of T. cruzi by degrading the gp90 present in the parasite membrane. All these factors inherent to the parasite and host associated with different temperatures allowed the observation of the infection of animals after administration of contaminated sugarcane juice kept at -80ºC and + 2ºC.

Research by Lino and Lino [34] showed that temperatures ranging from zero to 7ºC would be ideal for food preservation, confirming the in vivo as well as in vitro results obtained in this research at + 2ºC, because this temperature reduces the speed of chemical reactions and the reproduction of microorganisms such as bacteria, promoting their preservation and temporarily maintaining their properties and viability for food.

The maintenance of trypomastigotes’ viability in an ultra-freezer probably occurred in a process similar to the cryopreservation method [35, 36], even without the addition of cryoprotectant substances, agreeing with Coggeshall’s [35] findings, although some studies have shown that the use of glycerol would avoid the formation of ice crystals, providing a higher degree of preservation [37, 38].

Although in vitro tests have demonstrated the viability of trypomastigotes up to 120 hours at -20ºC, no infection was obtained with the mixture kept for 12 hours at this temperature. Probably, the number of surviving forms after this period is associated with slow freezing [20, 36], which favors the increase of solutes concentration and pH change, decreased the infectivity of the microorganism.

However, higher temperatures such as + 28ºC, + 60ºC, and + 80ºC prevented the survival of trypomastigotes after 12 hours of packaging, a fact also observed by Dias [17], who demonstrated the prevention of parasite transmission through contaminated food by heating above 45°C, freeze-drying, and pasteurization.

Possibly, the conditioning of the mixture in microtubes for 12 hours at 28°C promoted a strong fermentation process, favoring not only the increase in substrate concentration but also the existence of an oxygen-poor environment to provide a higher fermentation efficiency, lower the amount of sample distributed favoring not only the increase in substrate concentration but also the existence of an oxygen-poor environment to provide a higher fermentation efficiency [39]. Moreover, the presence of various contaminant microorganisms present in the feces of triatomines may have potentiated the natural fermentation reaction of the mixture, originating products such as lactic acid, acetic acid, alcohol, and CO2 [40], which led to the formation of acidic and alcoholic products that contributed to a drastic decrease in the viability of the T. cruzi population. However, Castanho et al. [11] achieved the infection of mice with sugarcane broth conditioned in 250 ml bottles for 17 hours at room temperature.

The results reinforces the hypothesis that stomach region margo plicatus as the entrance and dissemination way of the metacyclic trypomastigote into the blood circulation. Furthermore, it showed that the survivability and infection of T. cruzi conditioned at -80ºC without the use of preservatives is the least appropriate temperature for food preservation, since the exportation of food products without adequate preservation can lead to the transmission of Chagas disease to other geographical areas.

Statements and Declarations

Funding: This study was supported by Programa Institucional de Bolsas de Iniciação Científica/Conselho Nacional de Desenvolvimento Científico e Tecnológico (PIBIC/CNPq): process No. 121357/2019-8

Authors’ contribuitions: Augusto Furukawa Suzuki, Ewelyn Regina de Souza and Luciamáre Perinetti Alves Martins contributed to material preparation, data collection and analysis. Maria Angélica Spadella; Alessandre Hataka contributed to histophatological analysis. Eduardo Federighi Baisi Chagas contributes to statistical analysis. The first draft of the manuscript was written by Augusto Furukawa Suzuki and Ewelyn Regina de Souza. All authors comment on previous versions of the manuscript and read and approved the final version of the manuscript.

Ethics approval: This study was approved by the Ethics Committee on Animal Research of the Marília Medical School - protocol number 2981/19.

Conflict of interest: All authors declare no conflicts of interest related to this article

Consent to participate: Informed consent was obtained from all individual participants included in the study.

Consent for publication: The participants have consented to the submission of the study.

Availability of data and material: Not applicable

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Oral infection and survival of Trypanosoma cruzi in sugar cane juice conditioned at different temperatures

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