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Light microscopical structure and ultrastructure of a Besnoitia sp. in the naturally infected lizard Ameiva ameiva (Teiidae) from north Brazil, and in experimentally infected mice.

A Besnoitia species of the teiid lizard Ameiva ameiva (L.), from north Brazil was established in laboratory mice and hamster by the intraperitoneal inoculation of bradyzoites in the tissue cysts. In the lizards all the cyst wall layers were closely apposed. In the mice the layers of the wall were distinguishable, and ultrastructurally the inner cytoplasmic layer contained either a tight network of endoplasmic reticulum or packed mitochondria or both. These components were less frequent or sparse in the inner cytoplasmic layer of cysts in the lizard. The only animals available for experiments in attempts to indicate the definitive host of the parasite were 3 kittens of the domestic cat and a juvenile specimen of the snake Boa constrictor raised in captivity. No evidence of infection could be detected in these animals after feeding them with the tissues of mice harbouring cysts with very large number of bradyzoites.

Animals↗

Ultrastructural studies on the sporogony of Hepatozoon spp. in Culex quinquefasciatus say, 1823 fed on infected Caiman crocodilus and Boa constrictor from northern Brazil.

Laboratory reared Culex quinquefasciatus Say, 1823 originating from the vicinity of Belem, in northern Brazil, were allowed to engorge on caimans (Caiman c. crocodilus) infected with Hepatozoon caimani (Carini, 1909) and boas (Boa constrictor) infected with H. cf. terzii (Sambon and Seligmann, 1907) both from Para State. Engorged mosquitoes killed on successive days post-feeding (p.f.) were studied by transmission electron microscopy. Images of oocysts from 13 days p.f., caiman-fed mosquitoes were also examined by scanning electron microscopy. The Hepatozoon spp. from the respective hosts differed in their ability to develop in C. quinquefasciatus. Most female mosquitoes fed on caimans, became fully engorged, and survived beyond 22 days p.f., whereas those engorged on boa became debilitated and did not survive beyond 9 days p.f. In boa-fed mosquitoes oocysts were found on the 6th day p.f. The few mosquitoes surviving to the 9th day p.f. contained either undivided oocysts or those that had already commenced sporogenic division. By 8-10 days p.f. caiman-fed mosquitoes contained uninucleate oocysts. Sporogonic divisions were observed from day 12 p.f. onwards. Although sporogenic development conformed in general with the previously reported accounts, the study allowed a more detailed examination of the plasmalemmal endocytotic system, and the consolidation of the crystalline body in specialized 'factories' of cystalline material. Sporozoite differentiation occasionally started on the 18th day p.f., but otherwise was observed only on day 22 p.f.

Alligators and Crocodiles↗

Recombinant antitoxic and antiinflammatory factor from the nonvenomous snake Python reticulatus: phospholipase A2 inhibition and venom neutralizing potential.

From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics. A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein. PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure. PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance. The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics. Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa. PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin. It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice. Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).

Amino Acid Sequence↗

Biological thermal detection in infrared imaging snakes. 1. Ultramicrostructure of pit receptor organs.

The receptor organs of snakes with "thermal vision" were studied with ultra-high-resolution scanning probe microscopy (SPM) at close to in vivo conditions to elucidate their surface morphology and materials properties critical for prospective biomimetic design of "soft matter"-based infrared (IR) sensors. The surfaces of living tissues were scanned under wet ambient conditions in physiological solution, and the resulting parameters were compared with SPM data obtained for chemically treated (formaldehyde-fixed) tissue in ambient air and TEM studies in high vacuum. We found that the microstructural parameters for the living tissue are similar to ones observed for the formaldehyde-fixed snake tissues. However, previous data obtained from TEM analysis in high vacuum underestimated actual dimensions of surface microstructures. The average spacing of the nanopit array observed within receptor surface areas, which was suggested to play a critical role in selective IR adsorption, was determined to be 520 nm. This value is close to the grating spacing required for efficient reflection of electromagnetic radiation characteristic for sunlight without affecting IR adsorbance.

Animals↗

Biological thermal detection: micromechanical and microthermal properties of biological infrared receptors.

Bioinspired design of biomimetic sensors relies upon the complete understanding of properties and functioning of biological analogues in conjunction with an understanding of their microstructural organization at various length scales. In the spirit of this approach, the microscopic properties of infrared (IR) receptors of snakes with "infrared vision" were studied with scanning thermal microscopy and micromechanical analysis. Low surface thermal conductivity of 0.11 W/(m K) was measured for the IR receptor surfaces as compared to the nonspecific skin areas. This difference in surface thermal conductivity should result in a significant local temperature gradient around the receptor areas. Micromechanical analysis showed that pit organs were more compliant than surrounding skin areas with an elastic modulus close to 40 MPa. In addition, the maximum elastic modulus was detected for the outermost layer with gradually reduced elastic resistance for the interior. The porous microstructure of the underlying tissue combined with the highly branched microfibrillar network (Biomacromolecules 2001, 2, 757) is thought to be responsible for such a combination of biomaterial properties. Considering these biomaterials features, we postulated a possible design of an artificial photothermal detector inspired by the microstructure of natural receptors. This bioinspired design would include a microfabricated cavity filled with an ordered lattice of microspheres with a gradient periodicity from the surface to the interior. Such a "photonic cavity" could provide an opportunity for multiple scattering at wavelength tuned to 8-12 microm as a range of highest sensitivity.

Animals↗

MHC variation in birds and reptiles.

The major histocompatibility complex (MHC) has been studied in a multitude of mammals by now, but much less is known about its organisation and variation in other vertebrate species. The mammalian MHC is organised as a single gene cluster, but recent studies on birds suggest that this paradigm of MHC organisation has to be supplemented. The domestic chicken thus possesses two separate gene clusters which both contain MHC class I and class II B genes, and we have shown that the ring-necked pheasant Phasianus colchicus also has two unlinked clusters of class II B genes. We are studying the effect of the MHC on mate choice, survival and reproductive success in natural populations of birds and reptiles. For this reason, we are developing DNA techniques to determine the animals' MHC genotype. The amplification of the hypervariable exon 3 of the class I gene from songbirds and reptiles has provided us with species specific probes that can be used in Southern blot analysis. The first results indicate very extensive variation in all studied species, that is starlings Sturnus vulgaris, great reed warblers Acrocephalus arundinaceus and water pythons Liasis fuscus. The restriction fragment length polymorphism (RFLP) analysis also suggests that the number of MHC genes is significantly larger in these species than in pheasants and domestic chickens.

Animals↗

Developmental basis of limblessness and axial patterning in snakes.

The evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. Combined limb loss and trunk elongation is found in many vertebrate taxa, suggesting that these changes may be linked by a common developmental mechanism. Here we show that Hox gene expression domains are expanded along the body axis in python embryos, and that this can account for both the absence of forelimbs and the expansion of thoracic identity in the axial skeleton. Hindlimb buds are initiated, but apical-ridge and polarizing-region signalling pathways that are normally required for limb development are not activated. Leg bud outgrowth and signalling by Sonic hedgehog in pythons can be rescued by application of fibroblast growth factor or by recombination with chick apical ridge. The failure to activate these signalling pathways during normal python development may also stem from changes in Hox gene expression that occurred early in snake evolution.

Animals↗

A vertebrate model of extreme physiological regulation.

Investigation of vertebrate regulatory biology is restricted by the modest response amplitudes in mammalian model species that derive from a lifestyle of frequent small meals. By contrast, ambush-hunting snakes eat huge meals after long intervals. In juvenile pythons during feeding, there are large and rapid increases in metabolism and secretion, in the activation of enzymes and transporter proteins, and in tissue growth. These responses enable an economic hypothesis concerning the evolution of regulation to be tested. Combined with other experimental advantages, these features recommend juvenile pythons as the equivalent of a squid axon in vertebrate regulatory biology.

Animals↗

Physiology: postprandial cardiac hypertrophy in pythons.

Oxygen consumption by carnivorous reptiles increases enormously after they have eaten a large meal in order to meet metabolic demands, and this places an extra load on the cardiovascular system. Here we show that there is an extraordinarily rapid 40% increase in ventricular muscle mass in Burmese pythons (Python molurus) a mere 48 hours after feeding, which results from increased gene expression of muscle-contractile proteins. As this fully reversible hypertrophy occurs naturally, it could provide a useful model for investigating the mechanisms that lead to cardiac growth in other animals.

Adaptation, Physiological↗

Modeling reptile virus infection in vitro using Python regius airway organoids.

Zoonoses pose substantial global health risks, highlighting the need to better understand animal-to-human transmission. Reptiles are increasingly recognized as hosts of diverse pathogens, including numerous viruses, yet the diversity and prevalence of reptile pathogens, as well as their potential risk to humans, remain poorly understood. Here, we establish and characterize airway organoids derived from Python regius, providing an in vitro model to study reptile airway infection. Through de novo assembly of a Python regius reference genome, we characterize airway organoids at single-cell resolution, which suggests the presence of diverse cell populations including ionocytes, ciliated, secretory, goblet, endocrine, tuft, and basal cells. The organoids support productive infection with Ball Python Nidovirus (BPNV) and mount a robust epithelial antiviral response through the induction of interferon-stimulated genes, cytokines, and genes involved in chemical defense. As a proof-of-concept, treating organoids with antiviral drugs during infection reduces BPNV levels, highlighting the model's utility for drug testing. By providing a reductionist system of the serpentes airway, these organoids constitute a physiologically relevant in vitro model to study reptile viruses and host-pathogen interactions in their native host.

Animals↗

Functional site of endogenous phospholipase A2 inhibitor from python serum.

The functional site of 'phospholipase A2 inhibitor from python' (PIP) was predicted based on the hypothesis of proline brackets. Using different sources of secretory phospholipase A2 (sPLA2s) as enzyme, and [3H]arachidonate-labelled Escherichia coli as substrate, short synthetic peptides representing the proposed site were examined for their secretory phospholipase A2 (sPLA2) inhibitory activity. A decapeptide P-PB.III proved to be the most potent of the tested peptides in inhibiting sPLA2 enzymatic activity in vitro, and exhibited striking anti-inflammatory effects in vivo in a mouse paw oedema model. P-PB.III inhibited the enzymatic activity of class I, II and III PLA2s, including that of human synovial fluid from arthritis patients. When tested by ELISA, biotinylated P-PB.III interacted positively with various PLA2s, suggesting that the specific region of PIP corresponding to P-PB.III, is likely to be involved in the PLA2-PLI interaction. The effect of P-PB.III on the peritoneal inflammatory response after surgical trauma in rats was also examined. P-PB.III effectively reduced the extent of postsurgical peritoneal adhesions as compared to controls. sPLA2 levels at seventh postoperative day in the peritoneal tissue of P-PB.III-treated rats were also significantly reduced (P < 0.05) in comparison to those of the untreated controls. The present results shed additional insight on the essential structural elements for PLA2 binding, and may be useful as a basis for the design of novel therapeutic agents.

Amino Acid Sequence↗

Azithromycin metabolite identification in plasma, bile, and tissues of the ball python (Python regius).

Azithromycin is the first of a class of antibiotics classified as azalides. Six ball pythons (Python regius) were given a single dose of azithromycin at 10 mg/kg p.o. and i.v. in a crossover design. Serial blood samples were collected for unchanged azithromycin and to determine, if possible, the structure and number of circulating azithromycin metabolites. After a 4-month wash-out period, the snakes were given azithromycin p.o. as a single dose of 10 mg/kg for the study of azithromycin metabolism and metabolite tissue distribution. Bile, liver, lung, kidney, and skin samples were analyzed for the metabolites identified from the first experiment. Unchanged azithromycin accounted for 80, 68, and 60% of the total material at 12, 24, and 48 h postadministration in plasma, independent of route of administration. At both 24 and 72 h postadministration, azithromycin accounted for 70% of total azithromycin- associated material in bile. In liver and kidney, unchanged azithromycin accounted for 40% of the total azithromycin-associated material; this doubled in lung and skin. Fifteen metabolites were positively or tentatively identified in plasma, bile, or tissues of all snakes. Four of these possible metabolites: 3'-desamine-3-ene-azithromycin, descladinose dehydroxy-2-ene-azithromycin, 3'-desamine-3-ene descladinose-azithromycin, and 3'-N-nitroso,9a-N-desmethyl-azithromycin are unique to this species. Descladinose-azithromycin, 3'-N-desmethyl,9a-N-desmethyl-azithromycin, and 3'-N-desmethyl, 3'-O-desmethyl-azithromycin were the only metabolites identified in skin. Kidney tissue contained a greater number of metabolites than liver tissue, with 3'-N-didesmethyl-azithromycin being identified only in the kidney. Compared with the dog and cat, a greater number of metabolites were identified in ball python plasma. The percentage of unchanged azithromycin in bile is not different between the three species.

Administration, Oral↗

Reptilian echocardiography: insights into ontogeny and phylogeny?

We describe the echocardiographic findings in a large reptile-the carpet python. If ontogeny recapitulates phylogeny, the study of reptilian hearts may provide insights into human cardiac development. In addition, the reptilian heart has unique structural and physiological adaptations that may broaden our perspective on evolutionary cardiac adaptation.

Adaptation, Physiological↗

Fine structure of the development of Sarcocystis singaporensis in Python reticulatus from macrogamont to sporulated oocyst stage.

Three, 4-month old reticulated pythons (Python reticulatus) hatched from eggs laid by a newly caught female from Singapore Island, were fed on muscles of Sarcocystis singaporensis-infected Rattus rattus caught in Singapore. Snakes were sacrificed five, six and eight days later. The infected tissues were studied by transmission electron microscope. The present communication summarizes findings on macrogamont and oocyst stages. In the premature stages, rough endoplasmic reticulum consolidate into a large rectangular array; the electron-dense wall-forming-like bodies reveal a laminar structure. Macrogamont parasitophorous vacuoles became filled with granular matrix and electron-dense strands, which later on consolidate into a coat around the fertilized zygote. The oocyst wall is constructed from several formed membranes combined with deposited substance. All development to the sporulated oocyst stage occurs in the mucosal epithelium.

Animals↗

Ultrastructural study of the development of Sarcocystis singaporensis sarcocysts in the muscles of its rat host.

Laboratory rats fed sporocysts of Sarcocystis singaporensis (Zaman & Colley, 1975) Zaman & Colley, 1976 originating from Singapore were euthanized 22, 23, 33 and 80 days later. Sporocysts were extracted from feces of either naturally or laboratory-infected Python reticulatus. Electron microscopically examined longue and esophageal muscles yielded images of successive developing stages of sarcocysts. The primary wall evolved from a continuous thin layer into folds and later, into villar protrusions. At all stages the wall was interrupted by pinocytotic-like indentations. Young sarcocysts contained only metrocytes, they divided by endodyogeny into daughter metrocytes. The first bradyzoites appeared only 33 d.p.i. Sarcocysts by 80 d.p.i. were enclosed in a fully differentiated primary wall and contained almost entirely bradyzoites.

Animals↗

Hepatozoon cf. terzii (Sambon & Seligman, 1907) infection in the snake boa constrictor constrictor from north Brazil: transmission to the mosquito Culex quinquefasciatus and the lizard Tropidurus torquatus.

Specimens of Hepatozoon-infected Boa constrictor constrictor were obtained from localities in Pará State, north Brazil. Gametocytes in erythrocytes of the peripheral blood measured 10 x 2.5-16.2 x 3.7 microns. They were similar to those described as Haemogregarina terzii by Sambon & Seligmann (1907) in B. c. constrictor, in that they did not distort the infected erythrocyte, and their dimensions approximated those given by Carini (1947). Lungs and liver of infected snakes contained actively dividing meronts of a single type, and cysts containing two to six cystozoites were also present in the liver. Our initial feeding of Culex quinquefasciatus on infected snakes consistently resulted in a heavy death-rate of the engorged mosquitoes, with only a few surviving till the 9th day post feeding. These contained numerous oocysts which were undivided or in early stages of division. A fifth and final experiment, however, provided a few mosquitoes surviving up to 21 days post infection (dpi), and these contained fully sporulated oocysts measuring 190-200 microns in diameter and containing over 60 sporocysts of 19-30 microns in diameter. The number of sporozoites in each sporocyst was estimated as approximately 50. The nature of the parasite's sporogonic cycle in the mosquito thus justifies inclusion of this haemogregarine in the genus Hepatozoon. Two wild-caught specimens of the lizard Tropidurus torquatus were fed with mosquitoes containing fully developed oocysts (21 dpi). When sacrificed, three months later, large numbers of dizoic, tetrazoic and hexazoic cysts were demonstrated in their livers. Cystozoites released from these cysts were shown to possess a conspicuous refractile body.

Animals↗