Neutralization of bacterial endotoxins by frog antimicrobial peptides

The ability of skin antimicrobial peptides of the southern bell frog, Litoria raniformis, to neutralize in vitro the endotoxin, proinflammatory lipopolysaccharide (LPS) complex, from two different gram‐negative bacterial pathogens, human pathogen Escherichia coli (0111:B4) and frog pathogen Klebsiella pneumoniae, was investigated. The LPS neutralization activity of the natural mixture of skin antimicrobial peptides was measured using chromogenic Limulus amebocyte lysate assays. These skin antimicrobial peptides neutralized the LPSs from both pathogens at physiologically relevant concentrations (IC₅₀ < 100 µg/mL) showing their potential for non‐specific LPS neutralization in vivo in the skin of infected frogs and for development of anti‐endotoxin agents.     

Characteristics of the interaction of a treponemal hemolysin with rabbit erythrocytes

The mechanism of action on rabbit red cells of Treponema hyodysenteriae hemolysin was studied using volume analysis and release of hemoglobin. While fixation of the hemolysin on the erythrocytes is temperature independent, it appears that hemolysis is temperature dependent. The kinetics of hemolysis proceed according to a sigmoid curve characterized by a prelytic lag. The duration of the prelytic lag varies inversely with the quantity of hemolysin but the rate and the maximum value of hemolysis are directly proportional to the quantity of hemolysin. The effect of sucrose and trypan blue on the hemolysin and the red cells suggest that erythrocyte lysis is likely to be induced by the hemolysin in a way different from that known for other hemolytic agents.     

Analysis of the Structural Gene Encoding a Hemolysin in Vibrio mimicus

An environmental isolate of V. mimicus, strain E-33, has been reported to produce and secrete a hemolysin of 63 kDa. The hemolysin is enterotoxic in test animals. The nucleotide sequence of the structural gene of the hemolysin was determined. We found a 2,232 bp open reading frame, which codes a peptide of 744 amino acids, with a calculated molecular weight of 83,903 Da. The sequence for the structural gene was closely related to the V. cholerae el tor hlyA gene, coding an exocellular hemolysin. The amino terminal amino-acid sequence of the 63 kDa hemolysin, purified from V. mimicus, was determined by the Edman degradation method and found to be NH₂-S-V-S-A-N-N-V-T-N-N-N-E-T. This sequence is identical from S-152 to T-164 predicted from the nucleotide sequence. So, it seems that the mature hemolysin in V. mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da. Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal. Our results suggest that, like V. cholerae hemolysin, two-step processing also exists in V. mimicus hemolysin.     

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A₂ but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A₂ were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Macrophage activation in vivo and in vitro

Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, N-acetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C₃ receptor. In vivo stimulation triggered the capacity to internalize up to 45% of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C₃ receptor may be the most useful parameter.     

Altitudinal distribution of chytrid (Batrachochytrium dendrobatidis) infection in subtropical Australian frogs

The disappearance of amphibian populations from seemingly pristine upland areas worldwide has become a major focus of conservation efforts in the last two decades, and a parasitic chytrid fungus, Batrachochytrium dendrobatidis, is thought to be the causative agent of the population declines. We examined the altitudinal distribution of chytrid infections in three stream‐dwelling frog species (Litoria wilcoxii, L. pearsoniana and L. chloris) in southeast Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of high‐altitude frog populations, infection prevalence and intensity would be greatest at higher altitudes. Overall, 37.7% of the 798 adult frogs we sampled were infected with B. dendrobatidis, and infections were found in all the populations we examined. Contrary to our initial hypothesis, we found no consistent evidence that high‐altitude frogs were more likely to be infected than were lowland frogs. Further, the intensity of fungal infections (number of zoospores) on high‐altitude frogs did not differ significantly from that of lowland frogs. Batrachochytrium dendrobatidis appears to be capable of infecting frogs at all altitudes in the subtropics, suggesting that all populations are at risk of decline when conditions favour disease outbreaks. We did find evidence, however, that chytrid infections persist longer into summer in upland as compared with lowland areas, suggesting that montane amphibian populations remain susceptible to disease outbreaks for longer periods than do lowland populations. Further, we found that at high altitudes, temperatures optimal for chytrid growth and reproduction coincide with frog metamorphosis, the life‐stage at which frogs are most susceptible to chytrid infections. While these altitudinal differences may account for the differential population‐level responses to the presence of B. dendrobatidis, the reason why many of southeast Queensland's montane frog populations declined precipitously while lowland populations remained stable has yet to be resolved.     

Cutaneous acariasis in the African clawed frog (Xenopus laevis)

Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.     

Induction of ovarian follicular development in the subadult frogRana tigrina using luteinizing hormone releasing hormone-acetate

In the subadultRana tigrina administration of 2 μg luteinizing hormone releasing hormone-acetate/frog six days a week for 4 weeks in April resulted in the formation of medium (in all 8 frogs) and large sized (in 4 out of 8 frogs) yolky oocytes and, concomitant increases in the oviductal mass. The ovarian and oviductal masses showed a 10-fold increase over the control frogs. In untreated frogs the ovaries were transparent and contained first growth phase oocytes only. The oviducts were also infantile. The pituitary sections were stained using antisera raised in rabbit against the β-subunit of human luteinizing hormone and human follicle stimulating hormone. Immunoreactivity, staining intensity, cytoplasmic granulation and, cell, nuclear and cytoplasmic areas of gonadotrophs (B₂ cells) increased significantly in luteinizing hormone releasing hormone treated frogs. The above findings suggest that pituitary-ovarian axis in the subadultRana tigrina is responsive to luteinizing hormone releasing hormone and that long-term treatment with the hormone induces cytomorphological changes in the gonadotrophs which result in the conversion of inactive cells into secretory cells. This is accompanied by precocious vitellogenic growth of oocytes in the subadult frogs.     

Burrowing in frogs

More than 95% of burrowing Anura dig hindfeet first into the soil, a pattern unique to frogs among terrestrial vertebrates. The postero-laterally placed hindlimbs and associated musculature of frogs are preadaptations for hindfeet digging. One fossorial, backwards burrower, Glyphoglossus molossus (Microhylidae), has morphological modifications of the hindlimb for positioning the spade-like metatarsal tubercle and for increasing the force of the lower leg during digging. In contrast, in the headfirst burrower Hemisus marmoratus (Ranidae) there is extensive reorganization of the pectoral-cranial morphology compared to that: of a non-burrowing confamilial species. A model links the shifts in the pectoral morphology in Hemisus marmoratus to specific action patterns of headfirst: burrowing. Finally, data on stomach contents, natural history and energy utilization of frog species are presented to demonstrate the interrelationships of distinct loco. motor patterns with specific feeding strategies.     

Purification and characterization of Treponema hyodysenteriae hemolysin

A hemolysin produced by Treponema hyodysenteriae ATCC27164 was purified from broth filtrates by acetic and (NH₄)₂SO₄ precipitations followed by ion exchange chromatography on diethylaminoethyl-Sephacel and gel filtration using Ultrogel AcA44. The purified hemolysin displayed only one band on polyacrylamide gel electrophoresis. By gel filtration the molecular weight was estimated as 74,000 daltons. The isolated hemolysin was oxygen resistant, heat labile and was not inactivated over a wide range of pH values. Further analysis indicated that this hemolysin was probably a polypeptide or a protein associated with lipids and nucleotides. Its action on rabbit erythrocytes which did not require any divalent cations could not be related to a lipolytic or proteolytic activity.     

Distribution of 32P-labelled endotoxin in the frog.

Distribution and elimination of 32P-labelled endotoxin were studied in normal frogs and in frogs pretreated with a high dose of unlabelled endotoxin 24 hr previously. Like in other animals, the spleen and the liver of the frogs were found the most active R.E.S. organs. The alimentary tract too exhibited considerable R.E.S. activity. Pretreatment with unlabelled endotoxin resulted in a decrease of 32P-endotoxin uptake by the liver, the spleen and the alimentary tract. Evidence was obtained that the well-known effects of endotoxin on the R.E.S. of the mammals can be demonstrated in the frog as well.     

Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K_m creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with ³²P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of ³²P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.     

The role of calcium in endotoxin-induced release of calcitonin gene-related peptide (CGRP) from rat spinal cord

In the present study, the role of calcium in endotoxin-induced CGRP release was studied. 2 .5-50 μg/mL endotoxin and 1 -10 mmol/L caffeine caused concentration-dependent increase of CGRP release from rat spinal cord in vitro. However, no additive effect could he found when caffeine and endotoxin were concomitantly incubated. By using capsaicin, Ca2 -free medium, Omega-Conotoxin, nifedipine, W-7, ryanodine, MgCl2, Tris-ATP, rutheni-um red, the results indicate that the release of CGRP evoked by endotoxin from the sensory fibers of rat spinal cord is dependent on extracellular calcium. After entering into the cell through the N-type calcium channel, calcium binds to calmodulin, and triggers calcium release from intracellular calcium store by activating the caffeine-sensitive but ryan-odine-insensitive mechanism.     

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.     

Tolerance and Other Biological Properties of Vibrio parahaemolyticus Endotoxins

Biological properties of endotoxins prepared from three strains of Vibrio parahaemolyticus were compared with reference to commercially prepared Salmonella typhi endotoxin. Endotoxin assays performed in rabbits included dermal Shwartzman reactivity, pyrogenicity, heat stability, and ability to induce tolerance as well as cross-tolerance. Mice were used for endotoxin LD₅₀ determinations. Results showed V. parahaemolyticus endotoxins were similar to that of S. typhi strain O901. Induction of tolerance to V. parahaemolyticus strain 11590 endotoxin resulted in complete cross-tolerance to S. typhi endotoxin, and vice versa. Partial cross-tolerance to S. typhi endotoxin was demonstrated with rabbits rendered tolerant to endotoxin from V. parahaemolyticus strains Sak-3 and FC1011. Absorption spectra, nitrogen, phosphorus and carbohydrate analyses revealed additional similarities between endotoxins from V. parahaemolyticus and endotoxin from a member of the Enterobacteriaceae.     

Isolation and some properties of beta-hemolysin produced by Nocardia asteroides

An intracellular beta-hemolysin capable of lysing human, sheep and cow erythrocytes but not cells from some other animals was isolated from the cell walls of the three developmental cell-forms of Nocardia asteroides and characterised. The spherical cell-forms contained the highest amounts of the hemolysin (100 h.u./mg protein) and the least LD₅₀ for mice suggesting that this may be the cell-form most pathogenic to susceptible animals. The hemolysin has the properties of a protein, was pH stable and sensitive to both catabolite repression and temperature. The activity of the hemolysin was enhanced by Ca⁺⁺ and Na⁺ ions. The hemolysin was immunogenic in rabbits.     

Behavioural elements reflect phenotypic colour divergence in a poison frog

The coexistence of both aposematic and cryptic morphs as different anti-predator strategies within a species seems to be an unusual phenomenon in nature. The strawberry poison frog, Oophaga pumilio, shows an astonishing colour diversity among populations in western Panama. In this study we selected a red and a green colour morph from two Panamanian islands (Isla Solarte and Isla Colón) for behavioural observations and measurements of conspicuousness. We found that red frogs were more visible to both conspecific frogs and potential predators than green frogs. Interestingly the difference in conspicuousness was most pronounced at the substrate that males used as principal calling places. Red males were more active and spent more time foraging than green males, which spent more time hidden. The association between conspicuousness of colouration and behaviour results in a more aposematic and a more cryptic anti-predator strategy. This is the first study which links differences in conspicuousness between animals on their natural backgrounds to differences in foraging as well as anti-predator behaviour and discusses the results in light of previous findings of toxicity analyses and potential costs and benefits of aposematism. To this end, our study adds a novel perspective for explaining extreme colour diversity between populations within an initially aposematic species.