Microscopy for the detection, identification and quantification of malaria parasites on stained thick and thin blood films in research settings

Summary This manual was developed to guide a move towards common standards for undertaking and reporting research microscopy for malaria parasite detection, identification and quantification. It contains procedures based on agreed quality assurance standards for research malaria microscopy defined at a consultation of: TDR, the Special Programme for Research and Training in Tropical Diseases; the Worldwide Antimalarial Resistance Network (WWARN), United Kingdom; the Foundation for Innovative New Diagnostics (FIND), Switzerland; the Centers for Disease Control and Prevention (CDC), USA; the Kenya Medical Research Institute (KEMRI) and later expanded to include Amref Health Africa (Kenya); the Eijkman-Oxford Clinical Research Unit (EOCRU), Indonesia; Institut Pasteur du Cambodge (IPC); Institut de recherche pour le Développement (IRD), Senegal; the Global Good and Intellectual Ventures Laboratory (GG-IVL), USA; the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Thailand; Queensland University of Technology (QUT), Australia, and the Shoklo Malaria Research Unit (SMRU), Thailand. These collaborating institutions commit to adhering to these standards in published research studies. It is hoped that they will form a solid basis for the wider adoption of standardized reference microscopy protocols for malaria research.

Mapping genes for osteoporosis-Old dogs and new tricks

In stark contrast to its horticultural origins, modern genetics is an extremely technology-driven field. Almost all the major advances in the field over the past 20 years have followed technological developments that have permitted change in study designs. The development of PCR in the 1980s led to RFLP mapping of monogenic diseases. The development of fluorescent-tagged genotyping methods led to linkage mapping approaches for common diseases that dominated the 1990s. The development of microarray SNP genotyping has led to the genome-wide association study era of the new millennium. And now the development of next-generation sequencing technologies is about to open up a new era of gene-mapping, enabling many potential new study designs. This review aims to present the strengths and weaknesses of the current approaches, and present some new ideas about gene-mapping approaches that are likely to advance our knowledge of the genes involved in heritable bone traits such as bone mineral density (BMD) and fracture.

Encroachment of Echinococcus granulosus into urban areas in eastern Queensland, Australia

Objective: To investigate the prevalence of Echinococcus granulosus in wild dogs (dingos and dingo-domestic dog hybrids) living in and around human habitation on Fraser Island and in townships of the Maroochy Shire, on Queensland's Sunshine Coast, Australia. Design: Wild dogs were humanely killed on Fraser Island and in the Maroochy Shire because they were deemed a potential danger to the public. Their intestines were collected and the contents examined for intestinal parasites. Procedure: Intestines were removed as soon after death as possible, packed in plastic bags and kept frozen until examination. The intestinal contents were washed, sieved and examined microscopically for the presence of helminths, which were identified and counted. Results: Intestines from 108 wild dogs, 7 foxes and 18 Fraser Island dingoes were examined. Echinococcus granulosus was only present in the wild dogs from Maroochy Shire (46.3%) with worm burdens of between 30 and 104,000. Other helminths included Spirometra erinacei, Dipylidium caninum, Taenia spp., Ancylostoma caninum and Toxocara canis. Two specimens of a trematode (Haplorchinae sp.) usually found infecting fish and seabirds were recovered from a Fraser Island dingo. Conclusion: Dingoes on Fraser Island are not infected with E. granulosus and do not pose a hydatid disease public health risk to residents or visitors. However, wild dogs examined from the Maroochy Shire do present a potential hydatid disease public health risk.

Clinical signs and duration of cyanide toxicosis delivered by the M-44 ejector in wild dogs

Sodium cyanide poison is potentially a more humane method to control wild dogs than sodium fluoroacetate (1080) poison. This study quantified the clinical signs and duration of cyanide toxicosis delivered by the M-44 ejector. The device delivered a nominal 0.88 g of sodium cyanide, which caused the animal to loose the menace reflex in a mean of 43 s, and the animal was assumed to have undergone cerebral hypoxia after the last visible breath. The mean time to cerebral hypoxia was 156 s for a vertical pull and 434 s for a side pull. The difference was possibly because some cyanide may be lost in a side pull. There were three distinct phases of cyanide toxicosis: the initial phase was characterised by head shaking, panting and salivation; the immobilisation phase by incontinence, ataxia and loss of the righting reflex; and the cerebral hypoxia phase by a tetanic seizure. Clinical signs that were exhibited in more than one phase of cyanide toxicosis included retching, agonal breathing, vocalisation, vomiting, altered levels of ocular reflex, leg paddling, tonic muscular spasms, respiratory distress and muscle fasciculations of the muzzle.

Parasites as indicators of movement and population connectivity of a non-diadromous, tropical estuarine teleost: king threadfin Polydactylus macrochir

Temporal and spatial patterns in parasite assemblages were examined to evaluate the degree of movement and connectivity of post-recruitment life-history stages of a large, non-diadromous tropical estuarine teleost, king threadfin Polydactylus macrochir, collected from 18 locations across northern Australia. Ten parasites types (juvenile stages of two nematodes and seven cestodes, and adults of an acanthocephalan) were deemed to be suitable for use as biological tags, in that they were considered to have a long residence time in the fish, were relatively easy to find and were morphologically very different to each other which aided discrimination. Univariate and discriminant function analysis of these parasites revealed little difference in temporal replicates collected from five locations, suggesting that the parasite communities were stable over the timeframes explored. Univariate, discriminant function, and BrayCurtis similarity analyses indicated significant spatial heterogeneity, with BrayCurtis classification accuracies ranging from 55 to 100% for locations in north-western and northern Australia, 24 to 88% in the Gulf of Carpentaria, and 39 to 88% on the east coast of Queensland. Few differences were observed among locations separated by <200 km. The observed patterns of parasite infection are in agreement with concurrent studies of movement and connectivity of P. macrochir in that they indicate a complex population structure across northern Australia. These results should be considered when reviewing the management arrangements for this species.

Observation of a novel babesia spp. in eastern grey kangaroos (macropus giganteus) in australia

The roles and epidemiological features of tick-borne protozoans are not well elicited in wildlife. Babesia spp. are documented in many domestic animals, including cattle, horses, pigs, dogs and cats. Three cases affecting eastern grey kangaroos are described. The kangaroos exhibited neurological signs, depression and marked anaemia, and microscopic examination of blood smears revealed intraerythrocytic piroplasms. One to seven intraerythrocytic spherical, oval, pyriform and irregularly-shaped parasites consistent with Babesia spp. were seen in the blood smears and the percentage of infected erythrocytes was estimated to be approximately 7% in each case. Data suggest that the tick vector for this kangaroo Babesia sp. is a Haemaphysalis species. For Case 2, ultrastructural examination of the erythrocytes of the renal capillaries showed parasites resembling Babesia spp. and 18 of 33 erythrocytes were infected. DNA sequencing of the amplified 18S rDNA confirmed that the observed intraerythrocytic piroplasms belong to the genus Babesia. The phylogenetic position of this new kangaroo Babesia sp. (de novo Babesia macropus), as a sister species to the new Australian woylie Babesia sp., suggests a close affinity to the described Afro-Eurasian species Babesia orientalis and Babesia occultans suggesting perhaps a common ancestor for the Babesia in kangaroos. © 2012 Australian Society for Parasitology.

Fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli, including O25b-ST131, isolated from faeces of hospitalized dogs in an Australian veterinary referral centre

To determine rates of carriage of fluoroquinolone-resistant Escherichia coli and extraintestinal pathogenic E. coli (ExPEC) among dogs in a specialist referral hospital and to examine the population structure of the isolates. Fluoroquinolone-resistant faecal E. coli isolates (n232, from 23 of 123 dogs) recovered from hospitalized dogs in a veterinary referral centre in Sydney, Australia, over 140 days in 2009 were characterized by phylogenetic grouping, virulence genotyping and random amplified polymorphic DNA (RAPD) analysis. The RAPD dendrogram for representative isolates showed one group B2-associated cluster and three group D-associated clusters; each contained isolates with closely related ExPEC-associated virulence profiles. All group B2 faecal isolates represented the O25b-ST131 clonal group and were closely related to recent canine extraintestinal ST131 clinical isolates from the east coast of Australia by RAPD analysis. Hospitalized dogs may carry fluoroquinolone-resistant ExPEC in their faeces, including those representing O25b-ST131.

Demographic and functional responses of wild dogs to poison baiting

Lethal control of wild dogs - that is Dingo (Canis lupus dingo) and Dingo/Dog (Canis lupus familiaris) hybrids - to reduce livestock predation in Australian rangelands is claimed to cause continental-scale impacts on biodiversity. Although top predator populations may recover numerically after baiting, they are predicted to be functionally different and incapable of fulfilling critical ecological roles. This study reports the impact of baiting programmes on wild dog abundance, age structures and the prey of wild dogs during large-scale manipulative experiments. Wild dog relative abundance almost always decreased after baiting, but reductions were variable and short-lived unless the prior baiting programme was particularly effective or there were follow-up baiting programmes within a few months. However, age structures of wild dogs in baited and nil-treatment areas were demonstrably different, and prey populations did diverge relative to nil-treatment areas. Re-analysed observations of wild dogs preying on kangaroos from a separate study show that successful chases that result in attacks of kangaroos by wild dogs occurred when mean wild dog ages were higher and mean group size was larger. It is likely that the impact of lethal control on wild dog numbers, group sizes and age structures compromise their ability to handle large difficult-to-catch prey. Under certain circumstances, these changes sometimes lead to increased calf loss (Bos indicus/B. taurus genotypes) and kangaroo numbers. Rangeland beef producers could consider controlling wild dogs in high-risk periods when predation is more likely and avoid baiting at other times.

Diet of dingoes and other wild dogs in peri-urban areas of north-eastern Australia

Knowledge of the resource requirements of urban predators can improve our understanding of their ecology and assist town planners and wildlife management agencies in developing management approaches that alleviate human-wildlife conflicts. Here we examine food and dietary items identified in scats of dingoes in peri-urban areas of north-eastern Australia to better understand their resource requirements and the potential for dingoes to threaten locally fragmented populations of native fauna. Our primary aim was to determine what peri-urban dingoes eat, and whether or not this differs between regions. We identified over 40 different food items in dingo scats, almost all of which were mammals. Individual species commonly observed in dingo scats included agile wallabies, northern brown bandicoots and swamp wallabies. Birds were relatively common in some areas but not others, as were invertebrates. Dingoes were identified as a significant potential threat to fragmented populations of koalas. Dietary overlap was typically very high or near-identical between regions, indicating that peri-urban dingoes ate the same types or sizes of prey in different areas. Future studies should seek to quantify actual and perceived impacts of, and human attitudes towards, peri-urban dingoes, and to develop management strategies with a greater chance of reducing human-wildlife conflicts.

Signalling in Malaria Parasites the Malsig Consortium

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

Phylogenetics of Myrmica ants and their social parasites

Understanding the overwhelming diversity of life calls for complex organisational schemes. The field of systematics may thus be seen as the cornerstone of evolutionary biology. In the last few decades, systematics has been rejuvenated through the introduction of molecular methods such as DNA barcoding and multi-gene phylogenetic approaches. These methods may shed new light on established taxonomic ideas and problems. For example, the classification of ants has aroused much debate due to reinterpretation of morphological characters or contradictions between molecular data and morphology. Only in the last few years a consensus was reached regarding the phylogeny of ant subfamilies. However, the situation remains deplorable for lower taxonomic ranks such as subfamilies, tribes and genera. This thesis describes the systematics and evolution of the Holarctic ant genus Myrmica and the tribe to which it belongs, Myrmicini. Using barcoding, molecular-phylogenetic data and divergence time estimations, it addresses questions regarding the taxonomy, morphology and biogeography of this group. Furthermore, the interrelationships between socially parasitic Myrmica species and their hosts (other species in the genus) were inferred. The phylogeny suggests that social parasitism evolved several times in Myrmica. Finally, this thesis investigated whether coevolution shaped the phylogeny of socially parasitic Maculinea butterflies that live inside Myrmica colonies. No evidence was found for coevolution.

Population structure and evolution in the ant Plagiolepis pygmaea and its two social parasites Plagiolepis xene and Plagiolepis grassei

Social behaviour affects dispersal of animals and is an important modifier of genetic population structures. The female sex is often philopatric, which maintains coancestry within the breeding groups and promotes cooperative behaviours. This enables also inclusive fitness returns from altruism and explains why some individuals sacrifice personal reproduction for the good of others in social insects such as ants. However, reduced dispersal and population substructuring at the level of colonies may also entail inbreeding, loss of genetic diversity, and vulnerability. In addition, the most vulnerable ants are species that are evolved to parasitize colonies of other ants, and which compromise between abilities to disperse and the efficiency to parasitize the host. On the other hand, certain social organisations of ant colonies may facilitate a species to disperse outside its natural range and become a pest. Altogether, knowledge on genetic structuring of ant populations, as well as the evolution of their life histories can contribute to conservation biology and population management. The aim of this thesis was to investigate population structures and phylogenetic evolution of the ant Plagiolepis pygmaea and its two obligatory, workerless social parasites (inquilines) P. xene and P. grassei with genetic markers and DNA sequence data. The results support the general assumption that populations of inquiline parasites are highly fragmented and genetically vulnerable. Comparison of the two parasites suggests that differences in their relative abundance may follow from their interaction with the host, i.e. how well the species is adapted to reproduce in the host colonies. The results also indicate that the most recent free living ancestor to these two parasite species is their common host. This is considered to provide evidence for the controversial issue of sympatric speciation. Further, given that the level of adaptations to parasitic life history depends on the evolutionary time since the free-living ancestor, the results establish a link between species rarity and its evolutionary age. The populations of the host species P. pygmaea displayed significantly reduced dispersal both among the females (queens) and males, and high levels of inbreeding which may enhance worker altruism. In addition, the queens were found to mate with multiple males. Given the high relatedness between the queens and their mates, this occurs probably for non-genetic reasons, e.g. without benefits associated in genetically more diverse offspring. The results hence caution that the contribution of non-genetic factors to the prevailing mating patterns and genetic population structures should not be underestimated.

A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax

Malaria causes a worldwide annual mortality of about a million people.Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition,our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.