Hemoparasites of the genus Trypanosoma ( Kinetoplastida : Trypanosomatidae ) and hemogregarines in Anurans of the São Paulo and Mato Grosso do Sul States – Brazil

Wild animals are exposed to numerous pathogens, including hemoparasites. The Trypanosoma and hemogregarine group are frequently reported as parasites in anurans (frogs, tree frogs and toads). The identification of these hemoparasites is usually made through stage observation of their morphology in the peripheral blood of the host. There are no studies, however, based on the biological cycle of these hemoparasites. The objective of the present study was to evaluate the presence of hemogregarines and Trypanosoma spp. in anurans captured in the States of São Paulo and Mato Grosso do Sul – Brazil and to perform the morphological and morphometric characterization of these hemoparasites. The species of anurans examined were: Dendropsophus nanus, D. minutus, Leptodactylus chaquensis, L. podicipinus, L. labyrinthicus, L. fuscus, Bufo granulosus, B. schneideri, Phyllomedusa hypocondrialis, Trachicephalus venulosus, Scinax fuscovarius and Hypsiboas albopunctatus. Of the total of 40 animals studied, four (10%) were positive for hemogregarines and eight (20%) were positive for Trypanosoma spp. Hemogregarine gamonts showed variable morphology and, in addition to intra-erythrocytic forms, extra-erythrocytic forms were also observed. Extremely different forms of Trypanosoma were observed, as described in the literature, with the broad and oval forms being the most common.


INTRODUCTION
The Trypanosoma genus and the hemogregarines have been reported among the hemoparasites infecting anurans.Biological and molecular characterization data are almost inexistent for these parasites in these animals and species nomination is difficult.The term, hemogregarine, is used to describe blood parasites collectively belonging to the suborder Adeleorina, Phylum Apicom-plexa (Jakes et al. 2003).The main genera of this group are the Hepatozoon (Family Hepatozoidae) and Haemogregarina (Family Haemogregarinidae).Differentiation between these genera is generally made through the observation of the sporogonic stage of the parasite in the vector.However, most descriptions have been based only on the blood stage in the host, while the biological cycle of the species is not well elucidated.The only completely described species of this genus is Hepatazoon catesbianae, a parasite of Rana catesbeiana that, An Acad Bras Cienc (2009) 81 (2) 200 DENISE D.M. LEAL et al. in addition to its forms in the vertebrate host, a sporogonic development in the Malpighian tubule of the insect vector (Desser et al. 1995) has also been described.In Brazil, there is only one report on hemogregarine in the toad (Bufo crucifer) (Souza and Filho 1974).
The genus Trypanosoma belongs to the Kinetoplastida order and the family Trypanosomatidae.Species classification of anurans Trypanosoma is confusing, due to the great polymorphism present in this genus and the absence of studies on the biological cycle.Since numerous species have been described, it is plausible that the same parasite has received different names in different hosts and different geographic localizations.Moreover, exogenous influences, such as temperature, maturity and density of the population of hosts and environment pollution can influence the different shapes of the hemoparasite (Bardsley and Harmsen 1973, Mackenzie 1999, Silva et al. 2005).
The possible vectors of Trypanosoma spp.
have not yet been identified, but according to previous descriptions, they may include hematophagous dipteran and leeches (Barrow 1953, Woo 1969, Desser et al. 1975).Species of Phlebotomus have been shown to be vectors of this genus in frogs from China (Feng and Chao 1943) and in California (Ayala 1971).In Brazil, a recent report has described the infection of this genus in anurans.Ferreira et al. (2007) observed the morphology of the trypanosomes of different biomes in Brazil, and also genetically studied the genera of 25 isolated species of anurans.The authors demonstrated that the restriction of the parasite to the host is not absolute and showed that some genotypes of Trypanosoma are shared by more than one species of different genera and, sporadically, by species from distinct families of anurans.Some genotypes were also seen to be restricted to certain localities and were different from those of other trypanosomes previously studied in the rest of the world.
The knowledge of the hemoparasites of anurans is of importance, since these animals are in a process of extinction and also are extremely sensitive to environmental variations (Silvano and Segalla 2005).The objective of this work was to observe the occurrence of hemogregarines and Trypanosoma spp. in anurans, and to report the morphological and morphometrical characterization of the hemoparasites found.

MATERIALS AND METHODS
Forty amphibian specimens, captured in São Paulo State (Botucatu, Fartura and Chavantes) and Mato Grosso do Sul State (Pantanal-Corumbá) were studied.The animals were captured from October to November, 2005 and October to November, 2006.The species belong to the family Hylidae (tree frog), family Leptodactylidae (frog) and family Bufonidae (toad).Animals were collected by hand, blood was taken from their hearts and blood smears were prepared.Smears were fixed in absolute methanol for 3 minutes and stained with 10% Giemsa for 30 minutes.The blood smears were examined with an optical microscope, 1,000X magnification, for the diagnosis of hemoparasites.The morphometrical analysis of the parasites was performed using a computerized system of image analysis (Lite Qwin 3,01 -Leica).
The variables recorded for hemogregarines were: Body Area (BA), Body Length (BL) and Body Width (BW); Area (NA), length (NL) and width of the nucleus of the parasite (NW).For the Trypanosoma genus, the following traits were analyzed: Body Area (BA), Body Length excluding the free flagellum (BL) and width of the parasite (BW); Width at the widest part of body including the undulating membrane (W); Area of the nucleus (NA); Distance from the end anterior to the middle of the nucleus (A-N); Distance from the posterior end to middle of the nucleus (P-N); Distance from the anterior end to the kinetoplast (K-A); Distance from the posterior end to the kinetoplast (K-P); Distance from the kinetoplast to the middle of the nucleus (K-N); Distance of the kinetoplast to the posterior end of the nucleus (K-Np); Total length including free flagellum (TF); Length of the undulating membrane (LMO) and Free flagellum (FF).
The experiments related to this paper have been approved by the Unesp Bioscience Institute's Ethics Committee approval.The animals were captured according to the IBAMA (Brazilian Institute of Environment and Renewable Natural Resources) license no.099/99-DIFAS.And they were sacrificed with overdose of anaesthetic.

RESULTS
Of the 40 animals examined, 10% were positive for hemogregarines and 20% were positive for Trypanosoma spp.(Fig. 1).Among all positive samples one was chosen as a representative as shown in this paper.The hemogregarines were generally observed inside the red blood cells.Gamonts were observed with variable morphology; some presented a broad shape, others had a narrow and elongated shape (Fig. 2a) or a pear drop aspect.The position of the host cell nucleus was central or slightly displaced towards one of the extremities.The cytoplasm of the parasite varied from eosinophilic to basophilic.The nuclear chromatin was sometimes condensed, or was loose or fragmented.The displace-ment of the nucleus of the erythrocyte was observed in the presence of some gamonts (Fig. 2b).Smaller forms of the parasite were also seen, in which the nucleus occupied a central position and the nucleus chromatin was condensed.In these parasites, the displacement of the nucleus of the erythrocyte did not occur (Fig. 2c).However, an extraerythrocytic shape was observed, which contained the nucleus located centrally, loose chromatin and an eosinophilic cytoplasm (Fig. 2d).The details of each hemogregarine and its respective hosts are listed in Figure 3.
The Trypanosoma forms observed were extremely polymorphic, with oval, elongated or slender shapes.Some parasites presented rounded extremities in the region previous to the kinetoplast (Fig. 4a), or were slender in the region after the kinetoplast.The presence of a surface costate was sometimes observed in the body of the parasite (Fig. 4b).The nucleus, when present, had a varying format that was round to elongate.The kinetoplast, when visible, was located before, after or in the region of the nucleus.The undulating membrane, when presented, was seen extended along the whole body of the parasite, or along half the body or only at the extremity of the Trypanosoma sp.The flagellum, when presented, had dimensions that varied from short to long (Fig. 4c and 4d).The measurements of each described form and its respective hosts are detailed in Figure 5.
Due to a lack of more complete data in the literature, although some forms were similar to those already described, small morphological differences did not allow the identification of the Trypanosoma species.

DISCUSSION
In the present study, the frequency of hemoparasites obtained in anurans was relatively high, especially for Trypanosoma spp.(20%).For hemogregarines, the frequency was lower than 10%, but prominent nevertheless.Mohammed andMansour (1963, 1966a, b, c) observed a higher frequency of hemogregarines in toads than was seen in the present study (30.5%).With regard to the frequency of Trypanosoma in anurans, the results differed according to the group studied.Lehmann (1959) diagnosed 44.4% of R. boyli as infected with Trypanosoma, while Ayala (1970) found 15.6% of B. boreas parasitized; Werner et al. (1988) observed prevalences that varied from 1.5% to 26.9% in toads, according to the species of Trypanosoma, and Woo and Bogart (1984) found 16.7% of tree frogs to be infected with Trypanosoma.
The results obtained in our study, however, are similar to another study on terrestrial and aquatic anurans species, originating from Canada, in which a greater prevalence of animals was observed to be infected by Trypanosoma spp.than by hemogregarines (Barta and Desser 1984).In a study on anurans of Costa Rica, results were similar (Desser 2001).Although it was not possible to compare the results of the animals collected in São Paulo with those collected in the Mato Grosso do Sul, due to the differences among the species collected in the two states, the number of positive animals was greater in the Mato Grosso do Sul State than in the São Paulo State.One of the explanations for the lower number of parasites could be the strong association of these parasites with the environment.In environments that are more ecologically modified, animals tend to have a reduced number of hemoparasites (Mackenzie 1999, Silva et al. 2005), possibly explaining the decreased number seen in the State of São Paulo, although other factors must be taken in consideration, such as climate and density of vectors.
Hemogregarines and Trypanosoma observed showed variability of morphological forms.Sixteen different forms of hemogregarines were detected in four species of anurans and 28 different forms of Trypanosoma spp. in seven species of anurans.Generally, these parasites could not be compared to those already described in the literature, since the previous descriptions of these protozoa are outdated and insufficient, due to the lack of recent studies on these parasites from anurans.According to the literature, hemogregarines are commonly found in frogs (Ranidae) and toads (Bufonidae) in the whole world, whereas in tree frogs (Hylidae) there are no records of these hemoparasites (Smith 1996).In the present study, animals of the family Leptodactylidae and of the family Hylidae were observed infected with hemogregarines; according to Smith (1996), arborous anurans may be less subjected to these hemoparasites.For the Bufonidae, where accounts of hosting hemogregarines are common (Smith 1996), we found only one positive species (Chaunus schneideri) (Fig. 3).
It was not possible to classify the species based only on the forms and dimensions of the hemogregarines found; therefore, these forms may represent the same species of parasite or several different species.Since the biological cycles of these hemogregarines have not been observed in the vector, it was not possible to classify generically the parasites as being Hepatozoon or Haemogregarina.
An Acad Bras Cienc ( 2009) 81 (2)     With regard to the forms of Trypanosoma spp.found, a great polymorphic variability prevailed, similarly to descriptions made in the literature.We experienced difficulty in classification, even utilizing the morphology and morphometrical data.Miyata (1978) described many specific details of each species; species found sometimes had a similar morphology to those species described in the literature, but differed in some corporal dimensions.These authors raised the following question: "Are the great majority of Trypanosoma observed new forms or a phase of development of the parasite?",since few studies have been carried out with hemoparasites in anurans.We observed that, in the descriptions made by Miyata (1978), some characteristics could be used to separate these parasites, such as the po-sition of the kinetoplast, the position of the nucleus and the stretch of the free flagellum, amongst others.In spite of all these parameters, it is very difficult to characterize these species, since these structures were sometimes not visible.

Parasite dimensions Hosts
Of the animals positive for hemogregarines and Trypanosoma, some were infected by the two hemoparasites: L. chaquensis, L. podicipinus e P. hypocondrialis.The great pleomorphism observed could indicate distinct species, but we could not discard the possibility of the existence of different stages in the same species.Moreover, different species of anurans can be parasitized by the same species of Trypanosoma and this could modify, or not, the morphology of the species according to the host.Although T. rotatorium is possibly the most described species in the literature, none of the forms of Trypanosoma observed in the present study corresponded faithfully to this species.According to our results, we may conclude that for the characterization of these species of Trypanosoma and hemogregarines in anurans, molecular studies of the blood stages associated with ecological and biological aspects are necessary.In addition, the possible impacts of these infections in these animals and the importance of the presence of hemoparasites as an indicator of the environmental conditions need to be considered.

Fig. 3 -
Fig. 3 -Hemogregarine morphometrical parameters observed in four species of anurans captured in São Paulo and Mato Grosso do Sul States -Brazil.

Fig. 5 -
Fig. 5 -Trypanosoma spp.morphometrical parameters observed in seven species of anurans captured in São Paulo and Mato Grosso do Sul States -Brazil.