Molecular characterization and identification of Hepatozoon species Miller, 1908 (Apicomplexa: Adeleina: Hepatozoidae) in captive snakes from Brazil.

Species of Hepatozoon are parasites frequently recorded in snakes. The species identification of this genus was based mostly on the gametocyte morphology and morphometric calculations. For more reliable results, molecular characterization, an initial step for the correct identification of Hepatozoon species, has been used. This study aimed to determine the prevalence and identification of Hepatozoon species in captive snakes from Brazil. To that end, morphological, morphometric, and molecular data were obtained. A total of 157 snakes; 128 venomous (Crotalus durissus) and 29 non-venomous (Epicrates crassus and Boa constrictor) were screened for Hepatozoon blood parasites. Using light microscopy, 20 (12.78%) snakes were found positive for Hepatozoon spp., of which 6/29 (20.7%) were non-venomous and 14/128 (10.9%) were venomous; all with low parasitemia. Polymerase chain reaction (PCR), performed with the primers HepF300/Hep900, confirmed all 20 (100%) samples positive for hemogregarines. Species of Hepatozoon were identified from eight sequenced samples. Two previously described species, Hepatozoon cuestensis and Hepatozoon musa, were identified. The present study is the first to report H. musa within the snake hosts E. crassus and C. durrisus. In addition, a potentially new Hepatozoon species from B. constrictor was identified.

Molecular identification of Spirometra spp. (Cestoda: Diphyllobothriidae) in some wild animals from Brazil.

Species of the genus Spirometra are diphyllobothriid tapeworms with complex life cycles and are involved in human sparganosis, a neglected disease that affects individuals worldwide. Although some species were reported in wild felids and human cases of sparganosis were described in Brazil, the biology and taxonomy of these parasites are poorly understood. In the present study, samples of diphyllobothriids (eggs and/or proglottids) obtained from the stools of wild carnivores (Leopardus pardalis and Lycalopex vetulus) and plerocercoid larvae found in a snake (Crotalus durissus) from Brazil were analysed by amplifying a fragment of the gene cytochrome c oxidase subunit 1 (cox 1). The DNA sequences obtained here for the first time from the Spirometra spp. from Brazil were used to evaluate the phylogenetic relationships with other species. Molecular data identified two species in the Brazilian samples (evolutionary divergence of 17.8-19.2%). The species were identified as Spirometra sp. 1, found in Le. pardalis, and Spirometra sp. 2 found in Ly. vetulus and C. durissus, and they differed from Asian isolates of Spirometra erinaceieuropaei (17.5-20.2% and 12.2-15.6%, respectively), a species previously considered to be distributed worldwide. Moreover, Spirometra sp. 1 is genetically distinct from Sparganum proliferum from Venezuela (19.6-20.4%), while Spirometra sp. 2 is more closely related with the Venezuelan species (6.1-7.0%). Sequences of Spirometra sp. 2 revealed that it is conspecific with the Argentinean isolate of Spirometra found in Lycalopex gymnocercus (1.9-2.2%). Taxonomic and phylogenetic aspects related to New World species of Spirometra are briefly discussed.

Massive Muscular Infection by a Sarcocystis Species in a South American Rattlesnake (Crotalus durissus terrificus).

Massive numbers of sarcocysts of a previously undescribed species of Sarcocystis were observed in the skeletal muscles throughout the body of an adult, female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of muscle fibers from 5 sampled locations. Sarcocysts were not present in cardiac muscle, smooth muscle, or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable length depending on the viewed orientation and size of the muscle fiber, and had a sarcocyst wall less than 1-µm thick. Sarcocysts were subdivided by septa and had central degeneration in older sarcocysts. Host induced secondary encapsulation or an inflammatory response was not present. By transmission electron microscopy (TEM), the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100 nanometers in width arranged in an undulating pattern and intermittently folded inward in a branching pattern. The sarcocysts contained metrocytes in different stages of development and mature bradyzoites. The nucleic acid sequence from a section of the 18S small subunit rRNA gene was most closely related to S. mucosa that uses marsupials as intermediate hosts and has an unknown definitive host. This is apparently the third report of muscular Sarcocystis infection in snakes and is the first to describe the ultrastructure of the sarcocysts and use sequencing methods to aid in identification.

Metagenomic analysis of the gut microbiota of the Timber Rattlesnake, Crotalus horridus.

Snakes are capable of surviving long periods without food. In this study we characterized the microbiota of a Timber Rattlesnake (Crotalus horridus), devoid of digesta, living in the wild. Pyrosequencing-based metagenomics were used to analyze phylogenetic and metabolic profiles with the aid of the MG-RAST server. Pyrosequencing of samples taken from the stomach, small intestine and colon yielded 691696, 957756 and 700419 high quality sequence reads. Taxonomic analysis of metagenomic reads indicated Eukarya was the most predominant domain, followed by bacteria and then viruses, for all three tissues. The most predominant phylum in the domain Bacteria was Proteobacteria for the tissues examined. Functional classifications by the subsystem database showed cluster-based subsystems were most predominant (10-15 %). Almost equally predominant (10-13 %) was carbohydrate metabolism. To identify bacteria in the colon at a finer taxonomic resolution, a 16S rRNA gene clone library was created. Proteobacteria was again found to be the most predominant phylum. The present study provides a baseline for understanding the microbial ecology of snakes living in the wild.

Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters.

Hepatozoon spp. are commonly found infecting snakes. Since the latter are parasitized by diverse forms and data in the literature show divergence, we studied Hepatozoon spp. diversity on Crotalus durissus terrificus snakes using both molecular and morphological approaches. Naturally infected animals were employed. Blood was collected, blood smears were prepared and an aliquot was stored at -20°C for DNA extraction. Five specimens of C. durissus terrificus were selected, each of them infected with one gamont type. Morphological and morphometric analyses of the found gamonts led to their grouping into three populations. For molecular characterization, seven oligonucleotide pairs that amplify distinct regions of rDNA gene were tested by adopting the PCR technique. Only the oligonucleotide pairs HepF300/Hep900 and HEMO1/HEMO2 were efficient in amplifying and distinguishing different isolates of Hepatozoon spp. from snakes. The better results were obtained when both oligonucleotide pairs were used in association. Based on the molecular and morphologic differences, three new species were proposed: Hepatozoon cuestensis sp. nov.; Hepatozoon cevapii sp. nov. and Hepatozoon massardii sp. nov. This is the first description of new Hepatozoon species from snakes, based on molecular characterization and morphological data, in South America.

Hepatozoon species of the timber rattlesnake in northern Florida: description of a new species, evidence of salivary gland oocysts, and a natural cross-familial transmission of an Hepatozoon species.

Two species of Hepatozoon, i.e., H. sauritus and H. horridus n. sp., were present in 1 of 8 timber rattlesnakes, Crotalus horridus. The narrow gamonts of H. sauritus are 15.0-19.0 x 3.5-5.0 microm, with LW 58-86 microm2 and L/W 3.2-4.7, with a narrow, rounded anterior end. The spherical to slightly ovoid oocysts produce ovoid to elongate sporocysts, 21-43 x 12-24 microm, L/W 1.20-2.7, containing on average 22.1 (10-34) sporozoites. This is the first report of a natural cross-familial transfer of a Hepatozoon species. Gamonts of H. horridus n. sp. are 13.0-17.0 x 4.0-6.0 microm, with LW 63-102 microm2 and L/W 2.6-4.0, and have broadly rounded ends. The gamont cytoplasm is vacuolated. The spherical to ovoid oocysts form spherical to elongate sporocysts 14-45 x 11-25 microm, L/W 1.0-2.3, producing an average of 13.0 (8-21) sporozoites. The salivary gland in 1 of 5 mosquitoes dissected contained 1 mature oocyst.

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis.

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

Chromatin supraorganization, DNA fragmentation, and cell death in erythrocytes of the rattlesnake, Crotalus durissus terrificus (Serpentes, Viperidae), infected with the protozoan, Hepatozoon spp. (Apicomplexa, Hepatozoidae).

Forms of the protozoan of the Hepatozoon genus are detected free in the circulation and also within some of the erythrocytes of infected snakes. In healthy snakes, DNA fragmentation and cell death usually affect a few circulating erythrocytes in agreement with the long life span expected for these cells. In the present study we investigated whether infection by Hepatozoon spp. affected the incidence of DNA fragmentation and cell death in erythrocytes from the rattlesnake, Crotalus durissus terrificus. Methods such as the kinetics of Feulgen-DNA hydrolysis, and the TUNEL and comet assays, previously used for the study of chromatin organization and DNA fragmentation in erythrocytes of healthy snakes, were used. The results indicated that Hepatozoon spp. increased the DNA fragmentation and chromatin condensation typical of cell death in circulating erythrocytes of C. d. terrificus, including cells that do not harbour the parasite. The Hepatozoon infection is thus suggested to accelerate destruction of erythrocytes in the rattlesnake, not only affecting cells harbouring the parasite, but also in those without it.

Description of the gamonts of a small species of Hepatozoon sp. (Apicomplexa, Hepatozoidae) found in Crotalus durissus terrificus (Serpentes, Viperidae).

A small species of the genus Hepatozoon found in a specimen of Crotalus durissus terrificus from the Botucatu region, São Paulo State, Brazil is described. The morphologic alterations induced in the snake's erythrocytes by the presence of this parasite are described. Morphology and morphometric analyses were performed using the Qwin Lite 2.5 computerized image analysis system (Leica). The Hepatozoon possessed a small and short body (8.1+/-0.5 microm long and 3.8+/-0.4 microm wide), with round extremities. The cytoplasm varied from pale blue to basophilic and had no granulations. Its nucleus was large, occupied a large area of the cytoplasm, and was irregular in shape and not condensed. Despite its small size, this parasite induced important changes in the host cell. Total parasitemia observed was 56.6%.

Occurrence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes, Viperidae) in Brazil.

The objective of the present study was to investigate the prevalence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in the snake Crotalus durissus terrificus (Serpentes, Viperidae). Fifty animals were evaluated for the presence of oocysts of Cryptosporidium sp. at the time of arrival and 30 and 60 days later. Intestinal washings with saline solution (1% body weight), fecal samples, and organ scrapings were collected during the study. Oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by a density gradient centrifugation technique. Smears were made with the sediment and submitted to modified acid-fast and auramine-rhodamine staining. Cryptosporidium-positive smears were used as controls for the experimental findings. The overall prevalence of Cryptosporidium sp. oocysts was 14%. Among the positive snakes, oocysts were detected only in the intestinal washing in two specimens, only in the feces in four specimens, and in both materials at least once in one specimen. The positive snakes were predominantly from Santa Maria da Serra city State of São Paulo (57.1%). We also observed that all of the examinations that presented positive results were obtained at least 27 days after the capture of the animals.

Hepatozoon species (Apicomplexa: Hepatozoidae) of the corn snake, Elaphe guttata (Serpentes: Colubridae) and the pigmy rattlesnake, Sistrurus miliarius barbouri (Serpentes: Viperidae) in south Florida.

Hepatozoon guttata n. sp. is described from the corn snake (Elaphe guttata guttata) from south Florida. Gamonts average 14.6 x 4.6 (13-17 x 3.5-6) and are not recurved, with nucleus situated in the second quarter of the gamont. Erythrocyte cytoplasm rarely appears partially dehemoglobinized. The infected cells are usually distorted and are longer than the uninfected erythrocytes but do not differ in width; nuclei are smaller in length and width than those of uninfected cells. Sporogony in Aedes aegypti occurs within the head and the thorax but rarely in the abdomen. Oocysts are spherical to usually ovoid, 87.2 x 75.9 (45-155 x 40-152.5) and contain on average 7.1 (2-32) sporocysts. Sporocysts are spherical to ovoid, 34.8 x 31.0 (20-55 x 17.5-47.5), with 45.7 (14-89) sporozoites contained within. The pigmy rattlesnake (Sistrurus miliarius barbouri) in south Florida is parasitized by H. sistruri n. sp. Gamonts average 14.1 x 5.6 (12.6-15.8 x 4.7-6.3) in breadth and are not recurved, with the nucleus always situated in the second quarter of the gamont commonly at the midbody. Erythrocyte cytoplasm is not dehemoglobinized. The infected cells are always distorted and similar in length to the uninfected cells but with greater width and enlarged nuclei. Sporogony in A. aegypti occurs within head, thorax, and abdomen. Oocysts are spherical to usually ovoid, 163.6 x 154.7 (92-245 x 82-240) and contain 27.4 (12-42) sporocysts. Sporocysts are round to usually ovoid, 39.7 x 33.5 (25-50 x 20-50), with 45.7 (19-70) sporozoites.

Abbreviata terrapenis (Nematoda: Physalopteridae): an accidental parasite of the banded rock rattlesnake (Crotalus lepidus klauberi).

The nematode, Abbreviata terrapenis (Physalopteridae) was found in 16 (6%) of 267 banded rock rattlesnakes (Crotalus lepidus klauberi) from Arizona and New Mexico. Abbreviata terrapenis in C. lepidus represents an accidental parasite in that "infection" was acquired by the ingestion of lizard prey. Feeding captive snakes on wild-caught lizards poses a risk of introducing nematodes to the snakes.

Megaselia scalaris (Diptera: Phoridae) causing myiasis in Crotalus durissus terrificus (Serpentes: Viperidae) in Brazil.

We describe a case of myiasis in Crotalus durissus terrificus (Laurenti) caused by Megaselia scalaris (Loew). The snake was found in Anhembi, Sao Paulo, Brazil, with a lesion measuring 25 mm in diameter where the larvae of M. scalaris had penetrated the ribs. The opportunistic behavior of the larvae of M. scalaris is discussed.

The in vitro development of the pentastomid Porocephalus crotali from the infective instar to the adult stage.

The in vitro development of the pentastomid Porocephalus crotali from the infective, seventh (VII) instar, dissected from the tissues of rat intermediate hosts, to the adult male (X) and female (XI) instars, normally resident in the lung of rattlesnake definitive hosts, is described. The culture medium comprised washed human blood cells, resuspended in bovine serum (50:50, v/v), with 20% minimum essential medium and antibiotics. Two batches of approximately 100 pentastomids, maintained at a density of 2 worms/ml, were cultured in 500 ml bottles in an atmosphere of 5% CO2 in air at 28 degrees C. Culture bottles were rotated slowly on a Rollacell system and the medium was replenished every 2-3 days. Growth was monitored at various intervals by weighing worms, measuring cast cuticles with attached moulted hooks recovered from the medium, and by dissecting worms for measurements of various organ systems. Three moults separated the infective and adult male instar, whereas 4 moults were necessary in the case of females. In both cultures natural mortality was about 10% over a 160-day period, by which time 33-51% of females and 62-70% of males had reached the terminal instar. Some males attained full sexual maturity with seminal vesicles loaded with viable sperm: such males were indistinguishable from males recovered from naturally infected rattlesnakes. However, females never achieved full size, even after 200 days, and although copulation did not occur in vitro, some females became patent.

First record of Pachysentis canicola (Acanthocephala:Oligacanthorhynchida) and the occurrence of Mesocestoides sp. tetrathyridia (Cestoidea:Cyclophyllidea) in the western diamondback rattlesnake, Crotalus atrox (Serpentes:Viperidae).

This paper represents the first documentation and description of Pachysentis canicola cystacanths and the first report of a Pachysentis sp. in a paratenic reptile from the Americas. The western diamondback rattlesnake (Crotalus atrox) is also listed as a new host for tetrathyridia of the cyclophyllidean cestode (Mesocestoides sp.) and for the cystacanths of the oligacanthoryhynchid acanthocephalan (P. canicola). Six rattlesnake specimens were examined from Nolan County (32.30 degrees N, 100.39 degrees W), Texas. Four snakes (67%) were found parasitized with between 3 and 16 (mean 7) encapsulated tetrathyridia and 1 host additionally was infected with 6 P. canicola cystacanths within its mesentery.

Intracapsular asexual proliferation of Mesocestoides sp. tetrathyridia in the gastrointestinal tract and mesenteries of the prairie rattlesnake (Crotalus viridis viridis).

Nonproliferous and proliferous tetrathyridia (Mesocestoides sp.) have been reported from natural and experimental infections in reptiles and rodents. Multiple tetrathyridia (1+) in individual capsules (host-derived) were observed for both types of tetrathyridia and in both types of animals. To determine whether proliferous tetrathyridia in this genus could replicate in host capsules of the prairie rattlesnake (Crotalus viridis viridis), 11 snakes were inoculated and maintained in 2 environmental chambers (25 C and 30 C). One snake at each temperature was removed at 4-wk intervals and killed. Sections of the gastrointestinal tract and adjacent mesenteries were histologically examined. The increase in the numbers of tetrathyridia per capsule was significant at both 25 C (P = 0.007) and 30 C (P < 0.001). Identification of 7 intracapsular tetrathyridia with binary scoleces served as collaborative evidence. This paper presents the first significant evidence for asexual replication of the proliferous tetrathyridia (Mesocestoides sp.) in a reptilian host.

Experimental modes of Caryospora bigenetica (Apicomplexa: Eimeriidae) infection in swine and the effects of temperature and salinity on parasite infectivity in porcine tissue.

Four crossbred pigs (Sus scrofa) were inoculated orally with Caryospora bigenetica oocysts derived from snake and mouse feces, and with C. bigenetica infected mouse tissue. One pig also was given i.m. injections of methylprednisolone acetate. All four pigs displayed clinical signs including erythema, edema, and lethargy. Caryocysts were observed histologically in numerous tissues including ear, tongue, jowl, shoulder, loin, intercostal, ham, hock, and feet. The four pigs each were butchered into six commercial cuts: shoulder, loin, side, ham, hock, and feet. Raw 10 g samples from each cut were bioassayed by pepsin digestion and s.c. inoculation into 12 Swiss-Webster mice (Mus musculus) and 12 cotton rats (Sigmodon hispidus). Seventeen of 24 mice and cotton rats exhibited clinical signs and C. bigenetica tissue infections. Remaining portions of the six commercial cuts were temperature or saline treated, and 10 g samples were bioassayed in 16 mice and 12 cotton rats. No clinical sign or tissue infection was observed in these animals. Our study presents evidence that swine can be infected with C. bigenetica by ingesting oocysts present in snake feces or mouse feces (following inoculation of mice with snake-derived oocysts) or by ingesting C. bigenetica infected rodent tissue, that endogenously produced C. bigenetica oocysts are not excreted in the feces of swine, and that C. bigenetica in pork can be rendered noninfective by freezing at -20 degrees C (-4 degrees F) for 21 days, frying at 84 degrees C (183 degrees F) for 17 min, microwaving at 88 degrees C (190 degrees F) for 17 min, grilling at 82 degrees C (180 degrees F) for 48 min, baking at 95 degrees C (203 degrees F) for 230 min, boiling at 100 degrees C (212 degrees F) for 60 min, or by curing at 4 degrees C (39 degrees F) for 20 days.

Experimental transmission of Caryospora bigenetica Wacha et Christiansen, 1982 (Apicomplexa: Eimeriidae) from a rattlesnake, Crotalus atrox, to rodents and pigs.

Caryosporan oocysts were found in the feces of a diamond rattlesnake, Crotalus atrox, kept in ZOO Ustí nad Labem, Czech Republic. Comparison of oocyst structure with hitherto described Caryospora species from snakes revealed that they were Caryospora bigenetica Wacha et Christiansen, 1982. The rattlesnake, Crotalus atrox, represents a new host for C. bigenetica. The common vole (Microtus arvalis) and gerbil (Meriones unguiculatus) were successfully infected with a mixture of C. bigenetica oocysts and sporocysts following oral inoculation. All experimentally infected animals displayed clinical signs of dermal coccidiosis, including swellings of the face, ears, footpads, and scrota or vagina. Gerbils developed more severe clinical signs of infection and had a higher mortality than voles. Developmental stages of C. bigenetica were found in connective tissue of the nose, cheeks, ears, scrotum or vagina. Transmission of C. bigenetica caryocysts between common voles and mice, between mice and common voles, common voles and gerbils, and common voles and pigs was demonstrated. This study demonstrates that C. bigenetica can be transmitted by predation or cannibalism between different species of rodents and pigs.

Development of third-stage Physaloptera larvae from Crotalus viridis rafinesque, 1818 in cats with notes on pathology of the larvae in the reptile. (Nematoda, Spiruroidea).

The prairie rattlesnake, Crotalus viridis, was found to be commonly infected with third-stage Physaloptera larvae. A total of 112 larvae were fed to 3 laboratory raised cats. Adult worms recovered 42 and 59 days postinfection were identified as P. rara. Observations were made on the pathology of the larvae in the snake.