Abstract
Investigating parameters influencing natural infections with Trypanosoma cruzi via the skin, the diameters of mouthparts of different stages of triatomines vectors were measured to determine the size of the channel accessible for T. cruzi during cutaneous infection. The mean diameters of the skin-penetrating mandibles of first to fifth instar nymphs of the vector Triatoma infestans increased from 18 to 65 µm. The mean diameter in fourth instar nymphs of Dipetalogaster maxima was 86 µm. Different numbers of isolated vector-derived metacyclic trypomastigotes (10–10,000) were injected intradermally into mice. Prepatent periods, parasitemia and mortality rates were compared with those of mice obtaining 10,000 metacyclic trypomastigotes that are usually present in the first drop of faeces onto the feeding wounds of fifth and fourth instar nymphs of T. infestans and D. maxima, respectively. After injection of 50–10,000 T. cruzi, in all 42 mice the infection developed. An injection of 10 parasites induced an infection in 8 out of 15 mice. With increasing doses of parasites, prepatent periods tended to decrease. The level of parasitemia was higher after injection of the lowest dose. Except for one mouse all infected mice died. After placement of 10,000 metacyclic trypomastigotes onto the feeding wound of fifth or fourth instar nymphs of T. infestans and D. maxima, respectively, the infection rates of the groups, prepatent periods and the levels of parasitemia of T. cruzi in mice indicated that about 10–1,000 metacyclic trypomastigotes entered the skin via this route. For the first time, the present data emphasise the risk of an infection by infectious excreta of triatomines deposited near the feeding wound and the low number of invading parasites.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Triatomines are the biggest blood-sucking insects and are predominantly found between the Great Lakes of North America and Argentina (Lent and Wygodzinsky 1979). The majority of the approximately 150 species lives in the wild, as does the biggest species, Dipetalogaster maxima (Uhler 1894) (Lent and Wygodzinsky 1979; Galvão 2021). Other species live peridomestically and feed on domestic animals, e.g. hens and guinea pigs, but also invade houses (Balczun et al. 2012a; Schaub et al. 2016). Only some species are strongly adapted to houses, e.g. Triatoma infestans (Klug 1834), Rhodnius prolixus Stål 1859, Panstrongylus megistus (Burmeister 1835) and Triatoma dimidiata (Latreille 1811) (Balczun et al. 2012a; Schaub et al. 2016).
Especially during the night, triatomines approach the hosts, attracted by exhaled carbon dioxide, skin odour and temperature (Lazzari et al. 2013). Then, they swing the proboscis forward and push its tip onto the skin. The proboscis covers the thin mouthparts, the mandibles and maxillae. Teeth at the tip of the mandibles cut the skin, into which only the flexible, specifically interlocked maxillae invade, swinging in the skin by joint-free bendings, until they tap a blood capillary (Geigy and Kraus 1952; Kraus 1957; Lavoipierre et al. 1959; Wirtz 1987; Tull et al. 2020). In adults of D. maxima, the right maxilla is 12 mm long. The left one differs in structure of the tip and is usually slightly shorter (Wirtz 1987; Tull et al. 2020). Depending on the size of the blood capillary, triatomines need about 20 min to ingest 6–12 times their own body weight, e.g. up to 3.8 ml by females of D. maxima (Stadler et al. 2011; Balczun et al. 2012b). Blood ingestion of the triatomine strongly increases the rate of diuresis of the Malpighian tubules which end at the border of midgut and hindgut (Maddrell 1969). The urine flows into the rectum and thereby the rectal contents are excreted, followed by drops of clear urine (Kollien and Schaub 2000). Faeces and urine are often deposited during or shortly after feeding (reviewed by Loza-Murguía and Noireau 2010).
All species of triatomines are potential vectors of Trypanosoma cruzi (Chagas 1909) (Jurberg and Galvão 2006) which are the causative agent of Chagas disease. According to recent estimations, approximately 6 to 7 million people are infected worldwide, mostly in Latin America (WHO 2021). The biological characteristics of T. cruzi strains vary considerably, some strains developing high parasitemias in mice and killing them within 3 weeks, and others showing very low, often undetectable levels of blood trypomastigotes and inducing no deaths (reviewed by Schaub et al. 2012).
The parasite is transmitted to humans via different routes, e.g. by infectious blood, organs and food as well as by the blood-sucking triatomines (Bern et al. 2020). In contrast to other tropical vector-borne diseases, T. cruzi is not transmitted by infectious saliva of the insect but via the faeces and urine of the vectors (Schaub et al. 2016), since the infectious metacyclic trypomastigotes develop almost exclusively in the rectum of T. cruzi–infected triatomines (Kollien and Schaub 2000). There, the population of T. cruzi contains epimastigotes and spheromastigotes, both developing in five different pathways to metacyclic trypomastigotes (summarised by Schaub et al. 2016). The surface coat is changed during metacyclogenesis, enabling an evasion of the final metacyclic trypomastigotes to the complement-mediated lysis in the mammalian host (Schaub 1991). If the excreta are deposited on mucous membranes, e.g. of lips or eyes, the metacyclic trypomastigotes can easily invade the mammalian host but they are not able to invade the host’s intact skin (Eickhoff et al. 2013). This infection route is only possible if little wounds, scratches or the feeding puncture of the insect vector enable an invasion (summarised by Schuster and Schaub 2000).
Infections via the feeding wound of the triatomine vector have been considered several times (e.g. Soares and Marsden 1986; Schuster and Schaub 2000; Eickhoff et al. 2013), but the size of wounds caused by the mouthparts of triatomines and the number of parasites invading via the feeding wound have not been determined. In the present investigation, the sizes of the mouthparts of different stages of triatomines were measured. In addition, mice were infected by intradermal injection of different doses of the metacyclic trypomastigotes or by placing metacyclic trypomastigotes of T. cruzi onto the feeding wound of a vector. A comparison of prepatent periods, parasitemias and survival time determined the number of parasites that invade the mammalian host via the feeding wound causing a patent infection.
Materials and methods
The T. cruzi strain “Chile 5”, originating from Cachiyuyo in Chile, develops high parasitemias in mice and belongs to zymodeme1 (Ebert and Schaub 1983; Schaub and Schottelius 1984), according to recent multilocus sequence typing classifications to the discrete typing unit TcI (Zingales et al. 2012; Cosentino and Agüero 2012). It was cyclically passaged between mice and triatomines or stored frozen at − 78 °C (Schaub 1988). To obtain metacyclic trypomastigotes, first instar nymphs T. infestans were fed on T. cruzi–infected mice and in the following instars on hens. After feeding of 150–200 fifth instar nymphs, infectious faeces and urine were collected and metacyclic trypomastigotes carefully isolated by DEAE-Sephacel column chromatography (Schaub 1991). Since we planned to use low numbers of parasites, transition stages with an incomplete surface coat were killed by an incubation of the samples with the same volume of serum of C57 Bl/6 mice (Schaub 1991). According to counts in Neubauer chambers, this reduced the number of isolated parasites by about 10%. Infections with freshly isolated metacyclic trypomastigotes were compared in each of the four series of experiments, each one using a new isolation.
The T. infestans strain originated from the same village as the T. cruzi strain (Böker and Schaub 1984; Kollien and Schaub 1998). D. maxima, of which field populations only occur in the Mexican fog desert on the Baja California Peninsula in Mexico (Marsden et al. 1979), was obtained from Prof. Dr. H. Mühlpfordt, Bernhard-Nocht-Institute for Naval and Tropical Diseases, Hamburg, Germany. The triatomines were reared at 26 ± 1 °C, 60–70% relative humidity, with a 18-/6-h light/dark cycle and regularly fed on hens (Schaub 1989).
Different instars of T. infestans and fourth instar nymphs of D. maxima were allowed to ingest a small amount of blood and were then killed rapidly by cutting off the proboscis or head. The total lengths of the protruded maxillae were microscopically measured in skin biopsies of the respective region. The protruded mouthparts of 4–6 nymphs of each instar were embedded on slides in Entellan® (Merck, Darmstadt, Germany) to measure microscopically the maximal outer diameter of the mandibles.
The immunodeficient Balb/c nu/nu (nude) mice originated from the Max Planck Institute of Immunobiology, Freiburg, Germany, and the immunocompetent C57 Bl/6 mice were bread at our institute. Nude mice were chosen, since nude rats showed a good dose-dependent development of parasitemias (Schaub et al. 2001). Commercial rodent diet and water, sterilised in case of the nude mice, were available ad libitum.
All procedures with mice were conducted under deep anaesthesia and animals were carefully monitored during recovery. They were anaesthetised in all experiments using a mixture of Rompun® (Bayer, Leverkusen, Germany), Ketavet® (Upjohn, Kalamazoo, Michigan) and physiological saline (3:6:18). The C57 Bl/6 mice were shaved on their backs, using an electric razor. Since T. cruzi can invade the skin through minute wounds caused by shaving (Marsden 1967), this was done 24 h before infection (Eickhoff et al. 2013). Thereby, little scratches could close. Groups of four to six young females of about 10 weeks old were used in four series of experiments. For intradermal infections, a small fold of the back skin was lifted with a curved clamp and 20 µl parasite suspension containing 10, 50, 100, 1,000 and 10,000 metacyclic trypomastigotes/mouse was injected intradermally using a 25-µl Hamilton syringe (Hamilton, Darmstadt, Germany) and a 30-gauge needle (Microlance®, Becton Dickinson, Heidelberg, Germany). This volume of a solution can be correctly injected intradermally without leaking into the subcutaneous tissue (Crowle 1976). Although the mouthparts penetrate the epidermis and dermis, we chose the much more difficult intradermal injections since we suggested a rapid change of the wound channel after rejection of the mouthparts. Five different doses were not always used within each series since not enough female mice were available or other variables were tested (effects of saliva and the isolation procedure; data not included).
To obtain a natural infection route via the feeding wound of a triatomine, mice were anaesthetised and exposed to fifth instar nymphs of T. infestans. Since fifth instars of T. infestans sometimes probed more than once, we mainly used the more aggressive D. maxima of which fourth instar nymphs are of a similar size to fifth instars of T. infestans (Stadler et al. 2011). After ingestion of a small amount of blood, the triatomines were removed. The feeding site was marked with a fine pen and the mice were transferred into a moist chamber. There 10 µl parasite suspension containing 10,000 metacyclic trypomastigotes was placed onto the feeding wound. This dose is consistent with data obtained from this parasite/vector system, in which fifth instar nymphs of T. infestans deposited after blood ingestion a volume of 1–25 µl (mean 10 µl) in the first drop of faeces, containing 1–32,000 (mean 7,000) metacyclic trypomastigotes (Schaub and Lösch 1988). After 15 min, the suspension was removed using a smooth disposable cellulose sheet. Finally, the mice were transferred onto a heating plate to be kept warm until waking up. Since anaesthesia lasted for more than half an hour, oral infection by licking the skin at the application area was excluded.
Starting 12 (Balb/C nu/nu) or 16 (C57 Bl/6) days post infection, parasitemia was determined every day (Balb/C nu/nu) or every 2 days (C57 Bl/6) by examination of 100 microscopic fields (magnification 400 ×) of fresh blood preparations (Brener 1972). If one or more flagellates per microscopic field were counted, blood was diluted in physiological saline to determine the concentration in a special Neubauer chamber (height 0.02 mm). A conversion key was determined from both sets of data to compare all data in one standard. All mice, in which no blood trypomastigotes could be detected, were examined by xenodiagnosis (Meiser and Schaub 2011).
Pairwise statistic comparisons of prepatent periods and periods of survival were carried out using Student’s t-test (Statistica 9.0, Statsoft Europe, Hamburg, Germany) followed by a correlation analyses of both mean periods to the log10 of the dose. Parasitemias differed strongly within a group, allowing no statistics.
Results
In fifth instar nymphs of T. infestans, the right maxilla of the mouthparts protruding into the skin was up to 3 mm long and the left one a little bit shorter. They searched for capillaries below the 0.4–0.6-mm-thick skin of mice. The size of the mouthparts increased from instar to instar. In T. infestans, means and standard deviations of the maximal diameter of the mandibles inclusive teeths were 17.6 ± 1.8 µm in first instar nymphs, 20.4 ± 1.3 µm in second instar nymphs, 27.4 ± 1.4 µm in third instar nymphs, 39.3 ± 1.6 µm in fourth instar nymphs and 64.7 ± 4.6 µm in fifth instar nymphs (Fig. 1). In nymphs of D. maxima, the respective diameters were 37.2 ± 1.3, 44.6 ± 2.1, 63.1 ± 3.3, 86.3 ± 4.7 and 97.4 ± 6.4 µm. Comparing both species, the proboscis of T. infestans possessed more bristles at the distal end of the proboscis (Fig. S1).
Infection rates varied according to the dose of metacyclic trypomastigotes used for the intradermal infection (Table 1). In all series, the injection of 50 to 10,000 parasites/mouse was sufficient to infect all mice. Using 10 parasites, in 2 out of 4 mice in series 1, 1 out of 6 in series 3 and 4 out of 5 mice in series 4, no parasites were detected. In xenodiagnosis of mice without detectable blood trypomastigotes, no parasites developed in any of the triatomines, indicating that there had been no latent infection. Comparing infection rates in immunodeficient mice (series 1–3) and immunocompetent mice (series 4), the variation between the different series of infections was slight using high doses, indicating that infection rates were not affected by the strain of mice, but in immunocompetent mice parasites developed in less mice after injection of 10 parasites/mouse. In those groups in which 10,000 parasites had been placed onto the feeding wound, parasites could be found in all mice of series 2, 3 and 4 but in only 3 out of 5 mice of series 1 (Table 1). According to the infection rates, 50–100 parasites had invaded the mice via the wound by the mouthparts of the triatomines.
The first parasites were detected about 5 days earlier in nude mice than in immunocompetent C57 Bl/6-mice. The mean prepatent periods increased with decreasing infection doses in series 3 and 4 (Table 1). This increase was only evident in series 1 after injection of 1,000 and 100 parasites. In series 3 and 4, the mean prepatent period was correlated to the log10 of the dose of parasites (y = (− 2.18 x × log10 + 24.15); r = − 0.99; y = (− 2.48 x × log10 + 30.95); r = − 0.97, respectively). The variation was high if only 10 (series 3) or 100 parasites/mouse (series 4) had been injected. In pairwise statistic comparisons of prepatent periods within each series, in series 1 the data after injection of 1,000 and 100 parasites differed significantly (Student’s t-test; p < 0.01) and in series 3 the data of 10,000 versus 1,000 and of 1,000 versus 100. Using the data of those groups in which 10,000 parasites had been placed onto the feeding wound, in pairwise statistic comparisons of these prepatent periods to those obtained after injection of different numbers of parasites within each series, similarities are indicated if the differences were not statistically significant. This was evident in series 1 in the comparison to injections of 10 to 100 parasites/mouse, in series 2 versus 50 parasites, in series 3 versus 100 and 1,000 parasites and in series 4 versus 10,000 parasites/mouse. According to the regression equations, about 400 parasites invaded the skin in series 3 and 4. Thus, the statistics of most prepatent periods indicated individual infection doses of 10 to 1,000 parasites.
After the prepatent period, the number of parasites initially increased exponentially. In nude mice, this continued up to 5 × 108 parasites/ml blood, before they died (Fig. 2; logarithmic scale!; Figs. S2 and S3). In C57 Bl/6-mice, the number of parasites increased more slowly up to 9 × 108 trypomastigotes/ml blood and was rarely lowered (Fig. S4). In both strains of mice, the parasitemias varied considerably between the individual mice of the respective group and were higher after injection of 10 trypomastigotes. Using higher doses, the individual courses of parasitemia and the maximal number of flagellates during the infection did not correlate with the injected dose. Considering the parasitemias of those groups in which 10,000 parasites had been placed onto the feeding wound, the maximal number of blood trypomastigotes in mice of groups of series 1 and 4 indicated individual infection doses of 100–1,000 parasites. In series 2 and 3, parasitemias were high, similar to those after injection of 50 and 10 metacyclic trypomastigotes, respectively.
The survival times varied strongly. Except for one mouse that survived the infection (series 4; 10,000 parasites), most infected immunodeficient mice died within 30 days and most infected immunocompetent mice within 50 days. In series 3 and 4, the mean survival time was correlated to the log10 of the dose of parasites (y = (− 2.57 x × log10 + 29.50); r = − 0.98; y = (− 4.83 x × log10 + 56.40); r = − 0.89, respectively). In pairwise statistic comparisons of the mean survival time within each series, in series 1 the data after injection of 1,000 versus 100 parasites differed significantly (Student’s t-test; p < 0.05) and in series 3 the data of 1,000 versus 100 and of 100 versus 10 (p < 0.01 and < 0.05, respectively). In comparisons to data of those groups with “natural infections”, no statistically significant differences were evident in series 1 in the comparisons to injections of 100 and 10 parasites/mouse (but the latter comparison considered only 4 mice), in series 2 versus 50 parasites, in series 3 versus 10 parasites and series 4 versus 100 parasites/mouse. According to the regression equations, about 100 parasites invaded the skin in series 3 and 4.
Summarising all data, infection rates indicated an invasion by 50 to 100 parasites, prepatent periods and parasitemias by 10 to 1,000 parasites, and periods of survival by 10 to 100 metacyclic trypomastigotes/mouse. Taking all data into consideration, relatively low numbers of metacyclic trypomastigotes invaded the mammalian host via the minute 65- or 86-µm-wide wound by the mouthparts of a fifth instar nymph of T. infestans or fourth instar nymph of D. maxima, respectively.
Discussion
Vector-derived infections with T. cruzi are possible after drinking juices from sugarcane and guava or eating pulps of açai berries contaminated with remnants or excreta of infected triatomines and after deposition of such excreta on mucous membranes and skin wounds (Nóbrega et al. 2009; Eickhoff et al. 2013; Silva-dos-Santos et al. 2017). Placing varying numbers of metacyclic trypomastigotes onto the conjunctivae or oral mucosa of mice, a minimum of 640 and 1250 trypomastigotes, respectively, induced an infection of all mice (Kirchhoff and Hoft 1990). Placing excreta of triatomines containing 50, 500 or 5,000 metacyclic trypomastigotes inside the oropharynx or directly over a needle prick into the skin, the 50% infective dose for cutaneous challenge was 100-fold higher than that of oral challenge, indicating that oral mucosal transmission was more efficient than cutaneous transmission (Eickhoff et al. 2013).
Early investigations of the invasion of T. cruzi by placing infectious T. cruzi on the skin seemed to indicate an invasion of the intact skin (e.g. Dias 1932), but were later attributed to the previous shaving of the skin. The possibility that the feeding wound of the vector’s mouthparts offer an entry was investigated by placing 2,500 T. cruzi in 50 µl excreta of triatomines for 45 min onto the intact skin of a mouse and onto the feeding wound of a third instar nymph of D. maxima (Soares and Marsden 1986). This resulted in no infection for the intact skin and an infection rate of 24% for the feeding wound. The authors emphasised that a triatomine nearly never places its faeces directly onto its own feeding lesion and that this route of infection with T. cruzi must be uncommon (Soares and Marsden 1986). Placing excreta of triatomines containing 50–5,000 metacyclic trypomastigotes over the feeding wound of a triatomine, mice never became infected (Eickhoff et al. 2013). However, the developmental stage and species of the triatomines are not mentioned. According to the present measurements, the sizes of the mouthparts differ considerably, probably affecting the risk of infection, a topic of future investigations. In our system, all mice became infected after a 5- and 15-min exposure of excreta containing 15,000 metacyclic trypomastigotes onto the feeding site of fifth instar nymphs of T. infestans (Schuster and Schaub 2000).
In the present investigation, we placed 10,000 metacyclic trypomastigotes onto the feeding wound for 15 min and 19 out of 21 mice became infected. Therefore, parasites invade rapidly or the little feeding lesion stays open for invading parasites for some minutes. In humans, this period could be even longer, because on the contrary to mice, humans have a relatively hard and inelastic layer of ceratinocytes on the surface of the skin (Bucher and Wartenberg 1989). In addition, triatomine saliva and the histamine in a triatomines faeces cause allergic and itching reactions in humans (Harington 1958; Lumbreras et al. 1959; Costa et al. 1981; Marshall et al. 1986; Moffitt et al. 2003). Therefore, it is quite probable that faeces plus T. cruzi are scratched or smeared into the feeding wound if a triatomine deposits its faeces close to its feeding wound while or shortly after feeding and that parasites invade the skin by that route. The period of time between finishing blood ingestion and the deposition of faeces is the relevant factor. Many species and stages of triatomines defaecate before finishing feeding, others several minutes after having left the host (e.g. Wood 1951; Zeledón et al. 1977; Schaub and Lösch 1988; Trumper and Gorla 1991; Almeida et al. 2003; Loza-Murguía and Noireau 2010; Reisenman et al. 2011). The latter triatomines are less relevant for the direct transmission of the parasite. T. cruzi–infected triatomines seem to defaecate earlier after blood ingestion than uninfected specimens. However, for some T. cruzi–triatomine systems, such effects are not reported (reviewed by Verly et al. 2020; Guarneri and Schaub 2021; Schaub 2021).
If the number of T. cruzi invading via the wound of the mouthparts should be determined, a correlating response is required. Using immunocompetent mice and intraperitoneal and subcutaneous inoculations of 1, 10, 100 and 1,000 metacyclic trypomastigotes, only the period of time until the mice died was related to the dose of infection (Mshelbwala and Ormerod 1973). In addition, the number of infected mice was correlated to the infection dose, but even at the highest dose, not all mice became infected. In our system, the prepatent period and periods of time until the mice died were only correlated to the dose of parasites in series 3 (immunodeficient mice). In series 4 (immunocompetent mice), all infected mice except for one infected mouse died. Mice remaining uninfected were only present in the groups inoculated with 10 trypomastigotes (7 out of 15 mice). The prepatent period especially seemed to reflect the number of parasites infecting the mice, more evident in series 3. This was more strictly evident in infections of nude rats using 10–10,000,000 blood trypomastigotes (Schaub et al. 2001). In the present investigation, the variation of parasitemias within a group was high after receiving a low dose. This also occurred after subcutaneous infections of mice with 100 vector-derived metacyclic trypomastigotes/mouse, at least partly due to psychoneuroimmunological effects within groups of mice (Schuster and Schaub 2001a, b). The infections of the present investigation were not affected by the isolation procedure of metacyclic trypomastigotes: In 2 groups receiving parallel an intradermal injection of excreta containing 100 and 10,000 metacyclic trypomastigotes, the prepatent periods were similar to those groups receiving the isolated parasites (Heide 1999). In addition, using nymphs of D. maxima 1 day after resection of salivary glands of nymphs, no differences were evident to the infections via the feeding wound of normal nymphs, indicating no effect of the saliva of this species. However, in future investigations, nymphs of T. infestans after resection of salivary glands should be included. Effects of saliva occur in the transmission of Leishmania by sand flies (reviewed by Kamhavi et al. 2018).
In future investigations, the invasion via the wounds of different nymphal instars should be compared. According to the infection rates, prepatent periods and parasitemias, about 10 to 1,000 flagellates invade via the similarly sized feeding wound of fifth instar nymphs of T. infestans and fourth instar nymphs of D. maxima and these are able to cause a patent infection. The small number of parasites invading a host is important since many infection experiments were performed with high doses of T. cruzi, e.g. 105 trypomastigotes (e.g. Ribeiro dos Santos and Hudson 1981; Mateus et al. 2019). Choosing infection doses of about 100 trypomastigotes/mouse, the variation of parasitemias within a group of mice is high but the effects reflect natural conditions.
Data availability
Not applicable.
Code availability
Not applicable.
References
Almeida CE, Francischetti CN, Pacheco RS, Costa J (2003) Triatoma rubrovaria (Blanchard, 1843) (Hemiptera-Reduviidae-Triatominae) III: patterns of feeding, defecation and resistance to starvation. Mem Inst Oswaldo Cruz 98:367–371
Balczun C, Meiser CK, Schaub GA (2012) Triatomines as vectors of American trypanosomiasis. In: Mehlhorn H (ed) Parasitology research monographs, vol 3. Arthropods as vectors of emerging diseases. Springer-Verlag, Berlin, pp 275–299
Balczun C, Pausch JK, Schaub GA (2012) Blood digestion in triatomines – a review. Mitt Dtsch Ges Allg Angew Entomol 18:331–334
Bern C, Messenger LA, Whitman JD, Maguire JH (2020) Chagas disease in the United States: a public health approach. Clin Microbiol Rev 33:e00023-e119. https://doi.org/10.1128/CMR.00023-19
Böker CA, Schaub GA (1984) Scanning electron microscopic studies of Trypanosoma cruzi in the rectum of its vector Triatoma infestans. Z Parasitenkd 70:459–469
Brener Z (1972) Therapeutic activity and criterion of cure on mice experimentally infected with Trypanosoma cruzi. Rev Inst Med Trop Sao Paulo 4:389–396
Bucher O, Wartenberg H (1989) Zytologie, Histologie und mikroskopische Anatomie des Menschen, 11th edn. Verlag Hans Huber, Bern
Cosentino RO, Agüero F (2012) A simple strain typing assay for Trypanosoma cruzi: discrimination of major evolutionary lineages from a simple amplification product. PLoS Negl Trop Dis 6(7):e1777. https://doi.org/10.1371/journal.pntd.0001777
Costa CHN, Costa MT, Weber JN, Gilks GF, Castro C, Marsden PD (1981) Skin reactions to bug bites as a result of xenodiagnosis. Trans R Soc Trop Med Hyg 75:405–408
Crowle AJ (1976) Delayed hypersensitivity in the mouse. Adv Immunol 21:197–264
Dias E (1932) Expérinces sur la transmission du Trypanosoma cruzi de l`insecte au vertébré. CR Hebdom Soc Biol Fil 111:490–492
Ebert F, Schaub GA (1983) The characterization of Chilean and Bolivian Trypanosoma cruzi stocks from Triatoma infestans by isoelectrofocusing. Z Parasitenkd 69:283–290
Eickhoff CS, Dunn BA, Sullivan NL, Hoft DF (2013) Comparison of the infectivity of Trypanosoma cruzi insect-derived metacyclic trypomastigotes after mucosal and cutaneous contaminative challenges. Mem Inst Oswaldo Cruz 108:508–511
Galvão C (2021) Taxonomy. In: Guarneri AA, Lorenzo MG (eds) Triatominae – the biology of Chagas disease vectors. Springer Nature, New York, pp 15–38
Geigy R, Kraus C (1952) Rüssel und Stechakt von Rhodnius prolixus. Acta Trop 9:272–276
Guarneri AA, Schaub GA (2021) Interaction of triatomines, trypanosomes and microbiota. In: Guarneri AA, Lorenzo MG (eds) Triatominae – the biology of Chagas disease vectors. Springer Nature, New York, pp 345–386
Harington JS (1958) Histamine and histidine in excreta of the blood-sucking bug Rhodnius prolixus. Nature 178:268
Heide B (1999) Auswirkungen von vektorspezifischen Faktoren auf die transkutane Infektion mit Trypanosoma cruzi (Chagas 1909) (bzw. den Infektionsverlauf) in der Maus und die Interaktion von murinen Langerhanszellen mit dem Flagellaten. Dissertation, Freie Universität Berlin, Germany; https://refubium.fu-berlin.de/handle/fub188/3515
Jurberg J, Galvão C (2006) Biology, ecology, and systematics of Triatominae (Heteroptera, Reduviidae), vectors of Chagas disease, and implications for human health. Denisia 19:1096–1116
Kamhavi S, Valenzuela JG, Coutinho-Abreu IV, Brodskyn CI (2018) Role of sand fly saliva on Leishmania-infection and the potential of vector salivary proteins as vaccines. In: Boulanger N (ed) Skin and arthropod vectors. Academic press, London, pp 96–119
Kirchhoff LV, Hoft DF (1990) Immunisation and challenge of mice with insect-derived metacyclic trypomastigotes of Trypanosoma cruzi. Parasite Immunol 12:65–74
Kollien AH, Schaub GA (1998) The development of Trypanosoma cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation. J Euk Microbiol 45:59–63
Kollien AH, Schaub GA (2000) The development of Trypanosoma cruzi in Triatominae. Parasitol Today 16:381–387
Kraus C (1957) Versuch einer morphologischen und neurologischen Analyse des Stechaktes von Rhodnius prolixus Stål 1858. Acta Trop 14:35–87
Lavoipierre MMJ, Dickerson G, Gordon RM (1959) Studies on the methods of feeding of blood-sucking arthropods I - the manner in which triatomine bugs obtain their blood-meal, as observed in the tissue of the living rodent, with some remarks on the effects of the bite on human volunteers. Ann Trop Med Parasitol 53:235–250
Lazzari CR, Pereira MH, Lorenzo MG (2013) Behavioural biology of Chagas disease vectors. Mem Inst Oswaldo Cruz 108:34–47
Lent H, Wygodzinsky PW (1979) Revision of the Triatominae (Hemiptera, Reduviidae), and their significance as vectors of Chagas’ disease. Bull Am Museum Nat Hist 163:123–520
Loza-Murguía M, Noireau F (2010) Vectorial capacity of Triatoma guasayana (Wygodzinsky & Abalos) (Hemiptera: Reduviidae) compared with two other species of epidemic importance. Neotrop Entomol 39:799–809
Lumbreras H, Flores W, Escallón A (1959) Allergische Reaktionen auf Stiche von Reduviiden und ihre Bedeutung bei der Chagas-Krankheit. Z Trop Med 10:6–19
Maddrell SHP (1969) Secretion by Malpighian tubules of Rhodnius. The movements of ions and water. J Exp Biol 51:71–77
Marsden PD (1967) Trypanosoma cruzi infections in CFI mice. II. Infections induced by different routes. Ann Trop Med Parasitol 61:62–67
Marsden PD, Cuba CC, Alvarenga NJ, Barreto AC (1979) Report on a field collection of Dipetalogaster maximus (Hemiptera, Triatominae) (Uhler, 1894). Rev Inst Med Trop Sao Paulo 21:202–206
Marshall N, Liebhaber M, Dyer Z, Saxon A (1986) The prevalence of allergic sensitization to Triatoma protracta (Heteroptera: Reduviidae) in a Southern California, USA, community. J Med Entomol 23:117–124
Mateus J, Guerrero P, Lasso P, Cuervo C, González JM, Puerta CJ, Cuéllar A (2019) An animal model of acute and chronic Chagas disease with the reticulotropic Y strain of Trypanosoma cruzi that depicts the multifunctionality and dysfunctionality of T cells. Front Immunol 10:918. https://doi.org/10.3389/fimmu.2019.00918
Meiser CK, Schaub GA (2011) Xenodiagnosis. In: Mehlhorn H (ed) Parasitology research monographs, vol 1. Nature helps… How plants and other organisms contribute to solve health problems. Springer-Verlag, Berlin, pp 273–299
Moffitt JE, Venarske D, Goddard J, Yates AB, deShazo RD (2003) Allergic reactions to Triatoma bites. Ann Allergy Asthma Immunol 91:122–128
Mshelbwala AS, Ormerod WE (1973) Measurement of the infectivity of Trypanosoma cruzi in faeces of Rhodnius by comparison of dose-response curves. J Gen Microbiol 75:339–350. https://doi.org/10.1099/00221287-75-2-339
Nóbrega AA, Garcia MH, Tatto E, Obara MT, Costa E, Sobel J, Araujo WN (2009) Oral transmission of Chagas disease by consumption of açaí palm fruit. Brazil Emerg Infect Dis 15(4):653–655. https://doi.org/10.3201/eid1504.081450
Reisenman CE, Gregory T, Guerenstein PG, Hildebrand JG (2011) Feeding and defecation behavior of Triatoma rubida (Uhler, 1894) (Hemiptera: Reduviidae) under laboratory conditions, and its potential role as a vector of Chagas disease in Arizona, USA. Am Soc Trop Med Hyg 85:648–656
Ribeiro dos Santos R, Hudson L (1981) Denervation and the immune response in mice infected with Trypanosoma cruzi. Clin Exp Immunol 44:349–354
Schaub GA (1988) Developmental time and mortality of larvae of Triatoma infestans infected with Trypanosoma cruzi. Trans R Soc Trop Med Hyg 82:94–96
Schaub GA (1989) Trypanosoma cruzi: quantitative studies of development of two strains in small intestine and rectum of the vector Triatoma infestans. Exp Parasitol 68:260–273
Schaub GA (1991) Rapid isolation of metacyclic trypomastigotes of Trypanosoma cruzi from faeces and urine of the vector. Acta Trop 50:51–58
Schaub GA (2021) An update in the knowledge of parasite-vector interactions of Chagas disease. Res Rep Trop Med 12:63–76. https://doi.org/10.2147/RRTM.S274681
Schaub GA, Lösch P (1988) Trypanosoma cruzi: origin of metacyclic trypomastigotes in the urine of the vector Triatoma infestans. Exp Parasitol 65:174–186
Schaub GA, Schottelius J (1984) Identification of trypanosomes isolated from North Chile. Z Parasitenkd 70:3–9
Schaub GA, Hölscher C, Mossmann H (2001) Development of high numbers of blood trypomastigotes of Trypanosoma cruzi in nude rats. Parasitol Res 87:245–247
Schaub GA, Meiser CK, Balczun C (2012) Interactions of Trypanosoma cruzi and triatomines. In: Mehlhorn H (ed) Parasitology research monographs, vol 2. Progress in parasitology. Springer, Berlin, pp 155–178
Schaub GA, Vogel P, Balczun C (2016) Parasite-vector interactions. In: Walochnik J, Duchêne M (eds) Molecular parasitology – protozoan parasites and their molecules. Springer-Verlag, Wien, pp 431–489
Schuster JP, Schaub GA (2000) Trypanosoma cruzi: skin penetration kinetics of vector-derived metacyclic trypomastigotes. Int J Parasitol 30:1475–1479
Schuster JP, Schaub GA (2001) Trypanosoma cruzi: the development of estrus cycle and parasitemia in female mice maintained with or without male pheromones. Parasitol Res 87:985–993
Schuster JP, Schaub GA (2001) Experimental Chagas disease: the influence of sex and psychoneuroimmunological factors. Parasitol Res 87:994–1000
Silva-dos-Santos D, Barreto-de-Albuquerque J, Guerra B et al (2017) Unraveling Chagas disease transmission through the oral route: gateways to Trypanosoma cruzi infection and target tissues. PLoS Negl Trop Dis 11(4):e0005507. https://doi.org/10.1371/journal.pntd.0005507
Soares VA, Marsden PD (1986) Penetracao do Trypanosoma cruzi atraves do ponto da picada de triatomineos. Rev Soc Bras Med Trop 19:165–166
Stadler A, Meiser CK, Schaub GA (2011) “Living syringes”: use of haematophagous bugs as blood samplers from small and wild animals. In: Mehlhorn H (ed) Parasitology research monographs, vol 1. Nature helps… How plants and other organisms contribute to solve health problems. Springer-Verlag, Berlin, pp 243–271
Trumper EV, Gorla DE (1991) Density-dependent timing of defaecation by Triatoma infestans. Trans R Soc Trop Med Hyg 85:800–802
Tull T, Henn F, Betz O, Eggs B (2020) Structure and function of the stylets of hematophagous Triatominae (Hemiptera: Reduviidae), with special reference to Dipetalogaster maxima. Arthropod Struct Dev 58:100952. https://doi.org/10.1016/j.asd.2020.100952
Verly T, Costa S, Lima N, Mallet J, Odêncio F, Pereira M, de Carvalho Moreira CJ, Britto C, Pavan MG (2020) Vector competence and feeding-excretion behavior of Triatoma rubrovaria (Blanchard, 1843) (Hemiptera: Reduviidae) infected with Trypanosoma cruzi TcVI. PLoS Negl Trop Dis 14(9):e0008712. https://doi.org/10.1371/journal.pntd.0008712
WHO (2021) Chagas disease (American trypanosomiasis). Available from: https://www.who.int/health-topics/chagas-disease#tab=tab_1. Accessed 8 November 2021
Wirtz HP (1987) Eindringen der Mundwerkzeuge von Raubwanzen durch eine Membran (Hemiptera: Reduviidae). Entomol Gen 12:147–153
Wood SF (1951) Importance of feeding and defecation times of insect vectors in transmission of Chagas’ disease. J Econ Entomol 44:52–54
Zeledón R, Alvarado R, Jirón LF (1977) Observations on the feeding and defecation patterns of three triatomine species (Hemiptera: Reduviidae). Acta Trop 34:65–77
Zingales B, Miles MA, Campbell DA et al (2012) The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol 12:240–253. https://doi.org/10.1016/j.meegid.2011.12.009
Acknowledgements
The authors thank Dr. H. Mossmann for supplying the nude mice and Petra Wahl for the measurements of the mouthparts of the triatomines. We are most grateful to Jacinta Essling for the correction of the English version of the manuscript and Dr. Martin Kroll for his valuable help in statistics.
Funding
Open Access funding enabled and organized by Projekt DEAL. The support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged (project Scha 339/16–1).
Author information
Authors and Affiliations
Contributions
Conceptualisation: Barbara Waldeck, Günter Schaub; methodology: Barbara Waldeck; formal analysis and investigation: Barbara Waldeck; writing — original draft preparation: Barbara Waldeck, Günter Schaub; writing — review and editing: Barbara Waldeck, Günter Schaub; funding acquisition: Günter Schaub; resources: Günter Schaub; supervision: Günter Schaub.
Corresponding author
Ethics declarations
Ethics approval
The experiments were performed in accordance with German animal welfare registration (No. 23.8720, 20.A.9).
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Conflict of interest
The authors declare no competing interests.
Additional information
Section Editor: David Bruce Conn
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary file1 (JPG 680 KB)
Fig. S1 Mouthparts of afifth instar nymph of T. infestans withmandibles and maxillae protruded out of the proboscis (identical magnificationas in Fig. 1).
Supplementary file2 (DOC 785 KB)
Fig. S2: Development of Trypanosoma cruzi in and period ofsurvival of immunodeficient mice after intradermal injection of 10 to 1,000metacyclic trypomastigotes and afterplacementof 10,000 parasites onto the feeding wound of Triatoma infestans (series 1).
Supplementary file3 (DOC 326 KB)
Fig. S3: Development of Trypanosoma cruzi in and period ofsurvival of immunodeficient mice after intradermal injection of 50 metacyclictrypomastigotes and afterplacementof 10,000 parasites onto the feeding wound of Triatoma infestans (series 2).
Supplementary file4 (DOC 682 KB)
Fig. S4: Development of Trypanosoma cruzi in and period ofsurvival of immunocompetent mice after intradermal injection of 10 to 10,000metacyclic trypomastigotes and afterplacementof 10,000 parasites onto the feeding wound of Dipetalogaster maxima (series 4). (1 Mouse survived after injectionof 10,000 parasites).
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Waldeck, B., Schaub, G.A. “Natural infections” with Trypanosoma cruzi via the skin of mice: size of mouthparts of vectors and numbers of invading parasites. Parasitol Res 121, 2033–2041 (2022). https://doi.org/10.1007/s00436-022-07516-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-022-07516-5