Bruno P. Meloni

Giardia intestinalis: Electrophoretic evidence fora species complex

The technique of allozyme electrophoresis was applied to 29 Australasian stocks and 48 clones of Giardia intestinalis from humans as a means of increasing the number of genetic markers currently available for identification and classification. Fifty different enzymes were examined and of these 26 loci were found to be suitable for use as genetic markers. The data indicate the presence of four discrete genetic groups within the sample of G. intestinalis examined. The groups had fixed genetic differences at 23-69% of loci established. The evidence suggests that G. intestinalis is a species complex. The results have important implications for the systematics of human isolates of Giardia, as well as for studies on the epidemiology and demography of giardiasis in Australia and elsewhere.

Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.

Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro.

Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 micrograms, 4-aminobenzoic acid 100 micrograms, calcium pantothenate 50 micrograms, ascorbic acid 875 micrograms, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously.

Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis : implications for reproductive biology population structure, taxonomy, and epidemiology

The nature and extent of genetic variation in Giardia was used to infer its mode of reproduction, population structure, taxonomy, and zoonotic potential. Ninety-seven isolates of Giardia duodenalis, from a defined area in Western Australia and throughout Australia and overseas, were obtained from humans, cats, cattle, sheep, dogs, goat, beaver, and rats. Enzyme electrophoresis revealed extensive genetic variation with 47 different zymodemes. The widespread occurrence of certain zymodemes and the similarity of relationships among isolates inferred from independent genetic markers suggests a clonal population structure for G. duodenalis, although occasional bouts of genetic exchange may occur. The 47 zymodemes clustered similarly in phenetic (UPGMA) and phylogenetic (Fitch-Margoliash) analyses. The level of genetic diversity in isolates from a defined geographical area in Western Australia was similar to the level of diversity in isolates from throughout Australia. These data suggest that clonal lineages within G. duodenalis are evolutionarily independent. Although there was a significant overall correlation between genetic distance separating zymodemes and occurrence in different host species, we found genetically identical isolates from humans and other animals and extensive genetic diversity between isolates from humans. We interpret this as evidence for zoonotic transmission of the parasite.

Successful in vitro cultivation of Cryptosporidium andersoni: evidence for the existence of novel extracellular stages in the life cycle and implications for the classification of Cryptosporidium.

The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.

Rodent Stroke Model Guidelines for Preclinical Stroke Trials (1st Edition)

Translational stroke research is a challenging task that needs long term team work of the stroke research community. Highly reproducible stroke models with excellent outcome consistence are essential for obtaining useful data from preclinical stroke trials as well as for improving inter-lab comparability. However, our review of literature shows that the infarct variation coefficient of commonly performed stroke models ranges from 5% to 200%. An overall improvement of the commonly used stroke models will further improve the quality for experimental stroke research as well as inter-lab comparability. Many factors play a significant role in causing outcome variation; however, they have not yet been adequately addressed in the Stroke Therapy Academic Industry Roundtable (STAIR) recommendations and the Good Laboratory Practice (GLP). These critical factors include selection of anesthetics, maintenance of animal physiological environment, stroke outcome observation, and model specific factors that affect success rate and variation. The authors have reviewed these major factors that have been reported to influence stroke model outcome, herewith, provide the first edition of stroke model guidelines so to initiate active discussion on this topic. We hope to reach a general agreement among stroke researchers in the near future with its successive updated versions.

The protective effect of hypoxic preconditioning on cortical neuronal cultures is associated with increases in the activity of several antioxidant enzymes

Preconditioning describes a variety of treatments that induce neurons to become more resistant to a subsequent ischemic insult. How preconditioned neurons adapt to subsequent ischemic stress is not fully understood, but is likely to involve multiple protective mechanisms. We hypothesized hypoxic preconditioning induces protection by a coordinated up-regulation of antioxidant enzyme activity. To test this hypothesis, we developed two in vitro models of ischemia/reperfusion, involving oxygen-glucose deprivation (OGD) where neuronal cell death was predominantly by necrosis (necrotic model) or programmed cell death (PCD model). Hypoxic preconditioning 24 h prior to OGD significantly reduced cell death from 83% to 22% in the necrotic model and 68% to 11% in the PCD model. Consistent with the hypothesis, the activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase were significantly increased by 54%, 73% and 32%, respectively, in neuronal cultures subjected to hypoxic preconditioning. Furthermore, superoxide and hydrogen peroxide concentrations following OGD were significantly lower in the PCD model that had been subjected to hypoxic preconditioning.

Albendazole: a more effective antigiardial agent in vitro than metronidazole or tinidazole

The effects of albendazole were assessed against Giardia duodenalis in vitro and compared with those of tinidazole and metronidazole. Trophozoite morphology, adherence and viability were markedly affected by albendazole, to a far greater extent than by either metronidazole or tinidazole. The results of this study, and in particular the superior potency of albendazole in vitro, are discussed with respect to its potential value as a new approach to the chemotherapy of giardiasis.

Comparative studies on the axenic in vitro cultivation of Giardia of human and canine origin: evidence for intraspecific variation

Comparative studies were carried out on the in vitro cultivation of Giardia duodenalis from dogs and humans. Cultures were initiated with trophozoites obtained by artificial excystation of cysts present in human or canine faecal specimens, or using trophozoites collected from the small intestine of dogs postmortem. 12 new human isolates of G. duodenalis were established in axenic culture from cysts present in faecal specimens, and successfully cryopreserved, an overall success rate for in vitro establishment of Giardia from cysts of approximately 44%. In contrast, not one of 24 canine isolates, whether of faecal or intestinal origin, became established in vitro. Since identical media and culture conditions were used for the cultivation of both human and canine isolates, the results may reflect strain differences. The zoonotic significance of such intraspecific variation is discussed.

In Search of Clinical Neuroprotection After Brain Ischemia. The Case for Mild Hypothermia (35°C) and Magnesium

Background and Purpose— Brain injury after stroke and other cerebral ischemic events is a leading cause of death and disability worldwide. Our purpose here is to argue in favor of combined mild hypothermia (35°C) and magnesium as an acute neuroprotective treatment to minimize ischemic brain injury. Methods and Results— Drawing on our own experimental findings with mild hypothermia and magnesium, and in light of the moderate hypothermia trials in cardiac arrest/resuscitation and magnesium trials in ischemic stroke (IMAGES, FAST-Mag), we bring attention to the advantages of mild hypothermia compared with deeper levels of hypothermia, and highlight the existing evidence for its combination with magnesium to provide an effective, safe, economical, and widely applicable neuroprotective treatment after brain ischemia. With respect to effectiveness, our own laboratory has shown that combined mild hypothermia and magnesium treatment has synergistic neuroprotective effects and reduces brain injury when administered several hours after global and focal cerebral ischemia. Conclusions— Even when delayed, combined treatment with mild hypothermia and magnesium has broad therapeutic potential as a practical neuroprotective strategy. It warrants further experimental investigation and presents a good case for assessment in clinical trials in treating human patients after brain ischemia.