Ana Maria Jansen-Franken
Laboratory of Biology of Trypanosomatids, Instituto Oswaldo Cruz/Fiocruz
Although it is known that T. cruzi infects approximately 200 species of mammals, very little is known about the particularities of the interaction of this parasite with most of its hosts and wild reservoirs, either under experimental or natural conditions. Evidently, the difficulties of this type of study are obvious, but we cannot fail to emphasize that many aspects of the biology of T. cruzi still unknown would be clarified by this type of study.
One of the difficulties of studying a particular parasite-host interaction in an animal species described as “unconventional” lies in obtaining healthy colonies. Another obstacle to be overcome concerns the diagnosis for monitoring the infection of the parasite in question: it must be sensitive and specific. There are several aspects that must be considered when monitoring a parasite-host model, of which I highlight (i) the infection profile (its impact on the host) and (ii) the potential transmissibility, translated in the case of T. cruzi by high parasitemia, a feature easy to demonstrate by fresh blood tests and blood cultures. For the evaluation of these traits, it was necessary to monitor the experimental infections by T. cruzi in this species and, therefore, it was necessary to standardize a serological technique. Since there is no fluoresceinated anti-opossum conjugate on the market, we chose to use the indirect immunofluorescence technique, including an intermediate antibody (fraction against total Ig of opossum in rabbits).
The use of the Indirect Immunofluorescence Reaction (IFAT) in this “sandwich” form demonstrated (i) its usefulness in the diagnosis of Didelphis aurita infections by T. cruzi; (ii) important differences in the humoral response depending on the inoculated parasite subpopulation; (iii) the IFAT test is more sensitive than xenodiagnosis and/or blood cultures; (iv) the taxon D. aurita is able to respond with high serological titers to experimental infection by T. cruzi. These were important initial observations since marsupials were considered to have “low” ability to mount an immune response compared to placentals.
The follow-up of experimental infections of D. aurita by T. cruzi showed a unique aspect in the interaction of a mammal with T. cruzi: its ability to play the role of vector of the parasite in addition to that of reservoir. In fact, until then, no mammal had been described that simultaneously maintained the two multiplicative cycles of the parasite. Opossums are able to maintain the parasite multiplication cycle in the epimastigote form as well as the differentiation to the metacyclic form, in the light of the scent glands, in parallel with the cycle of intracellular multiplication and differentiation. Furthermore, in addition to T. cruzi, this unorthodox habitat can be colonized under experimental conditions, that is, after direct inoculation by Crithidia, Herpetomonas and Leptomonas, monogenetic trypanosomatids parasitic on insects. Opossums with scent glands parasitized by these monogeneans respond with high titers of specific antibodies, but no evidence of tissue invasion. It is, therefore, a habitat capable of maintaining parasites and/or their complement-sensitive forms indefinitely. The significance in the evolutionary path of T. cruzi in adapting to parasitism of a mammal can be seen from two perspectives: (i) the scent glands would be a primitive habitat of the parasite, a step before tissue parasitism or (ii) it would be a step of T. cruzi in the sense of becoming independent of vector transmission, as with Trypanosoma evansi. It is worth remembering that marsupials are probably the oldest hosts of T. cruzi.
Subsequent studies showed that opossums from a very young age, still in the pouch, were able to control and in some cases even eliminate experimental infections with T. cruzi isolates included in the TCII genotype (Y and FL strains) and which are extremely virulent for mice. Infections by these isolates did not result in parasitism of the scent glands and serological titers tended to be low (1:40–1:80). It was demonstrated that the absence of colonization of the scent glands by these subpopulations of the parasite was a consequence of the kinetics of the infection and not of a peculiarity of these subpopulations that, when directly inoculated there, multiplied and differentiated abundantly.
In contrast, when opossums were inoculated with strains G (49 and 327) and F, all characterized as TCI, high parasitemia could be observed for several weeks, as well as a high percentage of animals with scent gland parasitism. Infections were stable as could be demonstrated by systematically positive blood cultures and serological tests throughout the follow-up. It is worth mentioning that these isolates result, in mice, in subpatent infections and no mortality.
It was observed that the increase in IgG levels in infected opossums coincided with the control of the circulating parasite population but not with the elimination of the infection. It was demonstrated, therefore, that opossums exert an expressive selective pressure that differs for the different subpopulations of T. cruzi, that is, the competence as a reservoir of this genus is not absolute, it depends on the subpopulation of the parasite. The mechanisms involved in this selective process are not yet known, but they are probably independent of the humoral immune response, although this is important in the control of circulating parasite populations in the early stages of infection, at least for some strains of the parasite.
Opossums are, among others, described as the most important reservoirs of T. cruzi due to the high prevalence of natural infection of these animals in several places inside and outside Brazil. These reports do not always correspond to reports of expressive density of triatomines in the area. This fact led us to test neonatal vertical transmission in D. aurita as an alternative mechanism of infection. Our observations showed that the high prevalence of T. cruzi infection in opossums cannot be attributed to neonatal infection: regardless of the stage or profile of the infection, acute or chronic, patent, or subpatent, females do not pass the parasite to their offspring throughout of her 13 days of pregnancy and 100 days of breastfeeding. Additionally, it was observed that pups of infected females receive, during lactation, antibodies that, in case of challenge, guarantee a 2x longer prepatency period and 4x lower parasitemias compared to offspring of uninfected females. In contrast, the duration of patent parasitemia becomes 2x longer in animals born to infected females. This last finding is interesting because it suggests increased competence as a reservoir of opossum offspring born to females infected by T. cruzi.
An evaluation of the histopathological pattern of experimentally infected D. aurita showed a predominantly lymphomacrophagic inflammatory process that was incomparably less severe than that described for other animal models. In young animals, which proved to be more sensitive than adults, inflammation was always associated with the presence of amastigote nests. Opossums inoculated at 24 days of age, still in the embryonic stage, had large nests of amastigotes scattered throughout the organism, but even these animals, still so immature, survived the infection by T. cruzi for longer than adult mice with the same inoculum. Only in this experimental group of opossums was mortality due to T. cruzi infection observed.
The oral route must be very important in nature: opossums are infected by both mouse and triatomine predation – however, when acquired through this route, although stable, infections are always subpatent and serological conversion takes longer.In experimentally infected opossums, the parasite was found in nerve cells and in smooth, striated and/or cardiac muscle fibers and the presence of trypanosomes in the scent glands did not change the general histopathological scenario (Figure 1).
The vector competence of D. aurita scent glands and, therefore, their epidemiological importance remains unknown. But the high rate of metacyclogenesis of T. cruzi described in the lumen of these, (up to 50%) suggests that yes, it may be an efficient route of parasite dispersion. It was also observed that the parasitism of the scent glands was stable, generally bilateral and subsequent in 85% of the cases to a phase of high patent parasitemia. It is worth mentioning that T. cruzi metacyclogenesis was always observed in the glandular lumen, far from the epithelium, and did not depend on parasite adhesion (Figure 2). Similarly, no hemidesmosome-like membrane specializations were observed in transmission electron microscopy.
Follow-up of experimental infections showed that scent glands are unlikely to play the role of “reservoir within the reservoir” and that stable systemic parasitism was not dependent on scent gland parasitism.
These characteristics of the interaction with T. cruzi do not extend to all representatives of the Didelphidae family: parasitism of experimentally infected Philander frenatus scent glands has never been described. This species, on the other hand, has high levels of parasitemia, high antibody titers and stable infections when inoculated with the Y strain of T. cruzi. The same occurs when inoculated with TCI isolates, which shows that this species is more eclectic as a reservoir of T. cruzi. Additionally, P. frenata recognizes a wider spectrum of parasitic peptides earlier than D. aurita, as observed by SDS-PAGE/Western blot. The histopathological picture of Philander is similar to that of opossums, although a little more severe. All this led us to suggest that the Didelphidae family, throughout its coexistence with T. cruzi, selected different strategies. Perhaps the differences between these two didelphids in the interaction with T. cruzi are due to the fact that they came into contact only after the parasite diverged.
The monitoring of experimental infections, regardless of the animal model, cannot be taken as a model of what happens in nature. However, it is essential if we want to understand, at least in part, what happens in it.
André Luiz Rodrigues Roque
Laboratory of Biology of Trypanosomatids, Instituto Oswaldo Cruz/Fiocruz
Studies of parasite-host interaction that use the wild hosts of Trypanosoma cruzi as experimental models are rare. Even rarer are those experimentally studying the interaction of this parasite with rodents of the infraorder Hystricognathi, known as cavionorph rodents. This infra-order comprises rodents that, despite not being native to South America, seem to be, together with primates, one of the oldest groups associated with T. cruzi, behind only the autochthonous species of the orders Didelphimorphia, Cingulata, and Pilosa.
There are many reasons that discourage the experimental study of parasites in wild animals: (i) difficulties in obtaining breeding matrices, (ii) adaptation of the vivarium environment within the strict (and necessary) biosafety norms, (iii)standardization of management suitable for the maintenance of colonies; (iv) reproduction of animals, among others. The first mandatory step in this type of approach is the establishment of a colony for breeding and studying these animals. Only in this way is it possible to devise management strategies for the regular production of offspring, an essential condition for its use as an experimental model. Biological data obtained in captivity, such as longevity and reproductive patterns, are essential in assessing the role of the host in the transmission cycle of a parasite, in determining its potential as a reservoir, and in epidemiological studies involving population ecology.
Despite the clear importance of this group, evidenced by its ancient co-evolutionary relationship with T. cruzi, only two genera of caviomorph rodents have so far been experimentally monitored for their infections with this parasite: Cavia and Thrichomys. Of these, only the second is widely distributed in some regions of Brazil and can be considered a potential reservoir of the parasite.
The genus Thrichomys
The rodents of this genus belong to the Echimyidae family and are found in the Brazilian Cerrado, Caatinga and Pantanal. In the semi-arid, they are preferentially associated with mesic refuges and rocky habitats – very frequent formations in the caatinga – where they are also found in the peridomicile. In the cerrado, they are found in open areas with herbaceous extract, thickets of closed cerrado and in forests invaded by pastures, while in the swamp, they are found in small mountain ranges and regions of grasses and pastures.
The genus Thrichomys was considered monospecific for a long time, despite numerous studies showing morphometric and chromosomal differences between geographically distinct populations. It was only from the analysis of molecular, karyotypic and geographic distribution data that it was possible to conclude that the genus Thrichomys has at least five geographically separated cryptic and allopatric species (Figure 1). They are: T. fosteri (Figure 2), T. pachyurus, T. inermis, T. laurentius (Figure 3), and T. apereoides, the last 3 representing a complex of species distributed in the Brazilian Caatinga and Cerrado, along both sides of the São Francisco River.
The few studies using rodents of the genus Thrichomys as an experimental model for T. cruzi infection show that at least two species of this genus (T. laurentius and T. fosteri) are able to control and maintain T. cruzi infection, presenting patent parasitemias, effective humoral immune response and tissue damage important for the maintenance of the parasite’s wild enzootic. It is worth mentioning that these experimental studies were carried out before 2010, when the now recognized species t. fosteri was still named as T. pachyurus.
Older studies, considering this genus as still monospecific, had already demonstrated the ability of these animals to maintain stable infections by both populations considered to be parental of T. cruzi (TcI and TcII), using different isolates of the parasite, in addition to controlling parasitemia. patent and mount an effective humoral and cellular immune response.
Considering the experimental results obtained in different works, the focal distribution of the genus and the data observed in the natural infection, Thrichomys spp. they can act as (i) maintainer hosts, given their ability to maintain sub-patent parasitemias for long periods; and (ii) amplifying hosts as demonstrated by the long period of patent parasitemia under experimental conditions and positive blood culture under natural conditions. In addition, these animals can be considered generalists, withstanding anthropic pressure well in some areas and being frequently found in peri-domestic areas, presenting the potential to act as a source of infection for vectors in environments close to humans.
Cryptic species of the genus Thrichomys: importance of an accurate taxonomic diagnosis
Although common in various forms of organisms, little is known about the conditions that lead to the emergence of cryptic species and the evolutionary advantages of this process. A comparative study between two cryptic species of Thrichomys (T. laurentius and T. fosteri) shows the importance of correctly evaluating taxonomic structuring and biological differences in experimental infections by T. cruzi, as well as for other parasites. The determination of the hematological and biochemical parameters of these two species showed important physiological differences, as shown in the Table below (Table 1).
The size of red blood cells (larger in T. laurentius) and the amount of red blood cells (larger in T. fosteri) are important physiological differences observed between these species. It is known that animals with a higher number of red blood cells per unit volume and in smaller sizes, have a greater total surface area of red blood cells and, consequently, are more efficient in transporting oxygen from the lungs to the tissues. This is the case, for example, for thoroughbred racehorses as compared to draft horses. In the same line of reasoning, when in danger, muscle oxygenation is faster and escape is more efficient in T. fosteri than in T. laurentius. These differences may reflect a physiological adaptation to the type of habitat occupied by each of them in nature. T. laurentius are found in rocky habitats, full of refuges where the animal, in a situation of danger, can locate itself without having to travel long distances. T. fosteri are, in turn, found in small mountain ranges and regions of grasses and pastures where, in a situation of danger, their respiratory and circulatory capacity are essential in the search for a refuge that is almost always distant.
In view of the experimental infection by T. cruzi, important differences were observed in the pattern of infection between the cryptic species T. fosteri and T. laurentius, which are reflected in the ability of these animals to act as reservoirs in their respective localities. T. laurentius is more resistant to infection than T. fosteri, expressed by lower parasitemias and less tissue damage, regardless of the isolate studied.
- laurentius is widely distributed in the caatinga where it shares its rocky habitat with triatomine vectors of the parasite. These animals can frequent human dwellings and have already been found infected in peridomiciliary environments around the PARNA Serra da Capivara/PI, a former endemic region of the disease. In addition to the results obtained in the experimental infection of these animals, to the high prevalence of positivity in blood cultures in the field, T. laurentiusplays an important role as a reservoir in its wild environment.
In addition to being able to maintain the sylvatic cycle of T. cruzi and act as a maintainer host, T. fosteri seems to have a greater capacity to act as an amplifying host than T. laurentius. Experimentally infected T. fosteri showed longer patent periods and parasitemias, in addition to more severe tissue damage (especially in the heart). This pattern of infection makes it more susceptible to predation in a habitat where refuges are scarce and the number of predators is large. It is worth mentioning that, although the Pantanal is not currently an endemic area for Chagas’ disease, there is an active enzootic cycle in the region, with descriptions of different DTUs of the parasite infecting small wild mammals and accidental contact with humans can result in the emergence of cases of the parasite. illness.
Ultimately, it is worth mentioning that studies on the pathogenicity of experimental T. cruzi infection in caviomorphs obtained so far show that the antiquity of the parasite-host relationship (as proposed between caviomorphs and T. cruzi) does not necessarily evolve to a harmonic interaction, but it can evolve to one that favors the transmissibility of the parasite, regardless of its degree of pathogenicity to the host. In the case of Thrichomys, both analyzed species present significant cardiac damage, which is certainly reflected in the capacity and speed of tissue oxygenation. Thus, in nature, the infected animal would have its ability to escape predators compromised by cardiac injury, predisposing it to be more easily preyed upon. When being preyed upon, and in the presence of the parasite in the blood or other tissues of the rodent, the predator may become infected, in one of the oldest and most efficient mechanisms of dispersion of this parasite in nature.
Bezerra CM, Cavalcanti LP, Souza Rde C, Barbosa SE, Xavier SC, Jansen AM, Ramalho RD, Diotaiut L. Domestic, peridomestic and wild hosts in the transmission of Trypanosoma cruzi in the Caatinga area colonised by Triatoma brasiliensis. Mem Inst Oswaldo Cruz. 2014 109(7): 887-98.
Herrera HM, Rademaker V, Abreu UG, D’Andrea PS, Jansen AM. Variables that modulate the spatial distribution of Trypanosoma cruzi and Trypanosoma evansi in the Brazilian Pantanal. Acta Trop. 2007 (1): 55-62.
Nascimento FF, Lazar A, Menezes AN, Durans Ada M, Moreira JC, Salazar-Bravo J, D’Andrea PS, Bonvicino CR. The role of historical barriers in the diversification processes in open vegetation formations during the Miocene/Pliocene using an ancient rodent lineage as a model. PLoS One. 2013 8(4):e61924. doi: 10.1371/journal.pone.0061924.
Rademaker V, Herrera HM, Raffel TR, D’Andrea PS, Freitas TP, Abreu UG, Hudson PJ, Jansen AM. What is the role of small rodents in the transmission cycle of Trypanosoma cruzi and Trypanosoma evansi (Kinetoplastida Trypanosomatidae)? A study case in the Brazilian Pantanal. Acta Trop. 2009 111(2): 102-7. doi: 10.1016/j.actatropica.2009.02.006.
The genus Callomys
Callomys callosus as an experimental model of Chagas disease
Sonia Gumes Andrade e Isis F. Magalhães Santos
Laboratory of Experimental Chagas’ Disease,
Gonçalo Moniz Research Center/Fiocruz
Email: firstname.lastname@example.org; email@example.com
Callomys callosus RENGER, 1830 (Rodentia-Cricetidae) (Figure 1) is a South American wild rodent similar to the mouse (Mus musculus), distributed in Argentina, Bolivia, Paraguay and in the central-west, northeast and south regions of Brazil, until Paraná. From pastoral habits, it can adapt to different environments. It can be observed in wild and domestic environments (domestic and peri-domicile), maintaining close contact with humans. These animals have an average lifespan of 715.2 days for females and 822.5 days for males.
C. callosus was described in the 1970s as a natural reservoir of Trypanosoma cruzi. Studies on the biological behavior of Callomys in Brazil were carried out in the Central region where it was observed that the colonies had nocturnal habits and sedentary life in their natural habitat. Based on these observations, the first colony adapted to laboratory conditions was initiated, with three pregnant females, which over a year and six months produced 401 individuals.
Callomys presents reproductive behavior characterized by an estrous cycle of six days (polyestrus) with a gestation period between 20 to 30 days. Mating is monogamous and the couple produces 8 to 10 litters per year.
This animal has been used as an experimental model, because in addition to being adaptable to laboratory conditions, it participates in the cycle of transmission of pathogenic microorganisms to humans, being a model widely used in studies of viral infections.
The fact that this rodent was found harboring T. cruzi and constituting a wild reservoir of the parasite, made it to be investigated for the ability to maintain T. cruzi and be adapted to the laboratory being currently used in studies of experimental infection with different strains of T. cruzi.
It is interesting to verify how this animal behaves, when infected by T. cruzi strains, kept in the laboratory, in comparison with the mouse infection, not only regarding the development of histopathological lesions but also regarding the activation of peritoneal macrophages of these animals against to infection. Strains Y, F (which are strains already used in the laboratory) and wild strains M226 (isolated from naturally infected C. callosus) and Costalimai (isolated from Triatoma costalimai) were used in this study. C. callosus showed parasitemic levels superimposable to those of mice, with strains Y and F, however, while mice had high mortality, all Callomys survived the infection, showing regression of histopathological lesions between 40 and 60 days. With the two strains of T. cruzi of wild origin (M226 and Costalimai), the mice did not develop patent parasitemia, however they presented intense tissue lesions, while the C. callosus developed low parasitemia and absence of lesions, indicating an adaptation and greater resistance to the wild strains. The histopathological and ultrastructural study allowed to demonstrate peculiar aspects of the infection of this rodent. C. callosus, when infected by the F strain, or the Colombian strain, both representatives of Biodeme Type III, Z1, develops intense inflammatory lesions in skeletal muscle and myocardium, with a predominance of macrophages, which assume an epithelioid aspect, lymphocytes and fibroblasts. Simultaneously, an intense fibrogenic process develops, which begins early (26 to 30 days), with marked fibroblast proliferation and thickening of the intercellular matrix, with collagen deposition. From 50 to 60 days of infection, there is regression of inflammation and fibrosis, leaving only residual focal infiltrates. Interstitial collagen deposits show densification and the matrix shows signs of fibrolysis, indicating regression of the fibrogenic process.
The control of histopathological alterations and parasitism have been linked to the role of cytokines such as IFNγ, which is elevated in serum in the acute phase of infection, associated with increased release of H2O2, corresponding to the period preceding the parasitemia peak. The increase in TNF and TGF-β is possibly associated with the control of parasite multiplication, in the development of early fibrogenesis, as well as in the modulation of these alterations.
With this special profile, C. callosus becomes an interesting model for the study of possible immunomodulatory mechanisms that may be involved in the fibrogenesis of chagasic myocarditis, since it naturally has an adaptation mechanism in the parasite-host relationship that may be important to assess its role as a natural reservoir of T. cruzi(Figure 2).
Servio Urdaneta-Morales and Leidi Herrera
Laboratory of Trypanosoma Biology of mammals, Sección de Parasitologia, Instituto de Zoologia Tropical, Facultad de Ciências, Universidad Central de Venezuela, Caracas, Venezuela
Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) is a hemoflagellate that causes American trypanosomiasis in exclusively neotropical mammals belonging to 8 orders, which act as reservoirs in wild niches or in artificial ecotopes where they can infect humans and their domestic animals.
For the time being, this parasitosis is presented primarily as a zoonosis transmitted by fecal contamination with metacyclics, by more than 100 species of strictly hematophagous insects (Hemiptera, Reduviidae, Triatominae, “vinchucas, chipos, barbeiros”). We have carried out research to determine the experimental behavior of strains isolated from wild reservoirs captured in urban and rural environments in Venezuela, obtaining the following results:
- Adult opossums (Didelphis marsupialis) from urbanized areas of Caracas, capital of Venezuela, showed histologically few pseudocysts with amastigotes and trypomastigotes in the heart, with mild lymphoplasmohistiocytic inflammation and lysis. Metacyclics obtained by xenodiagnosis of donor opossums, inoculated into NMRI mice, produced parasite nests with severe inflammation in cardiac and skeletal muscle, and mild inflammation in liver and colonic smooth muscle, as well as parasites without infiltrate in lung, pancreas and brain; thereby showing an expansive and greater change in the histotropism found in the original reservoir;
even when this parasitosis is considered a pansystemic tissue infection, few studies have been carried out to determine the behavior of the parasite in Organs genital organs, adipose tissue, cartilage and bone tissue, as well as in the eyeball. Using the same protocol described, we obtained the following results: instillation through the external meatus of the vagina and penis (isolated from Rattus rattus) and inoculation into the scrotum (isolated from marsupialis) with blood from infected mice produces systemic parasitism with invasion of the heart, skeletal muscle , skin, duodenum, pancreas and ovary in vaginally instilled animals (Figure 1); penile infection was detected by xenodiagnosis; Intrascrotal inoculation also produces infection in the testis and vas deferens (smooth muscle). These results show that the contact of infected blood with the genital mucosa can produce blood and tissue parasitism, which emphasizes the possibility of transmission by coitus through menstrual blood with T. cruzi and that the infection routes that we used experimentally are as effective in terms of intra-peritoneal;
- it is demonstrated that adipose tissue is a suitable environment for the parasite by inoculating NMRI mice with metacyclics from isolates characterized by RAPD as Z1 (= cruzi I) obtained from Panstrongylus geniculatus, D. marsupialis, and R. rattus collected in urban and rural areas. Pseudocysts with amastigotes or trypomastigotes were present in macrophages and adipocytes of mice associated with lymphocytic infiltrates, showing that populations of the parasite can usually colonize fatty tissues, regardless of the host species or geographic area from which the isolates were obtained. It is therefore recommended to study this tissue in research on the characterization of this parasitic disease;
- Metacyclic compounds obtained from xenodiagnosis in mice infected with isolates of the indicated donor species produced, in a murine model, amastigote nests, intermediate stages and trypomastigotes in chondroblasts, osteoblasts, chondrocytes, osteocytes, fibroblasts and cartilage and bone marrow macrophages (Figure 2). The perichondrium and periosteum showed scarce mononuclear infiltrate. Our results demonstrate the need to carry out previous discards of the presence of cruzi, to avoid transmission through bone marrow transplants or bone implants;
- Research on other alternative routes of transmission of cruzi in relation to the usual fecal route led us to investigate the presence of the parasite or its genetic material in ocular tissues. Metacyclics from isolates from Brazil and Venezuela, typified as T. cruzi I or II, were inoculated in the murine model described and in specimens of Trichomys apereoides(Rodentia, Echimyidae), a very old caviomorph restricted to Brazil and Paraguay. The complete ocular globule of each animal was studied histologically and the T. cruzi k-DNA was extracted and amplified by PCR. Nests with amastigotes were found in corneal stromal fibroblasts and adjacent myocytes in mice inoculated with a D. marsupialis isolate; k-DNA (330 bp) from T. cruzi was determined in ocular tissues of both models. These results demonstrate that the ocular environment is a possible appropriate microniche for the parasite and highlights the risk of transmission of T. cruzithrough ocular fluids and parasitized corneal transplants.