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Rock-wallabies occupy outcrops, escarpments and cliffs that incorporate structurally complex refuges; because of this, local populations present methodological challenges for monitoring purposes. We describe the development and adaptive modification of a survey technique intended to measure changes in rock-wallaby activity on a site-by-site basis. Twenty spatially independent field sites were collectively sampled across two geological strata using transects consisting of regularly spaced sampling points, with concentric series of fixed-radius circular plots at each sampling point used to count numbers of fresh/recent and/or older rock-wallaby scats. The presence/absence of fresh and/or recently deposited scats was identified as likely to be the more sensitive measure of changing use. Indexes of Activity (IoA) at each field site were subsequently obtained by transect-based sampling at 10 m intervals using 2 m fixed-radius circular plots and recording the presence/absence of fresh/recent scats therein. Derived rock-wallaby IoA ranged between 0.13 and 0.94 across the 20 sites and were approximately normally distributed. Central tendency measures associated with the IoA metrics imply some potential to develop management-themed activity thresholds. Further refinement and potential applications of the approach are discussed.

The impact of bushfire events on wild Koala (Phascolarctos cinereus) populations is poorly understood. Following the 2019/2020 bushfire season in eastern Australia, we resurveyed 123 field sites for which contemporaneous (current koala generation) pre‐fire survey data were available. Field sites were distributed across six fire grounds between Foster and Ballina on the north coast of New South Wales. At these sites, pre‐fire naive occupancy levels by koalas ranged from 25% to 71% of the sampled habitat, while post‐fire naive occupancy levels ranged from 0% to 47%. The median reduction in the naive occupancy rate by koalas when considered across all six fire grounds was 71% when standardized against pre‐fire occupancy levels. Field data provided strong corroboration between site‐based, post‐fire foliage canopy cover estimates and modelled Google Earth Engine Burnt Area Map (GEEBAM) fire‐severity categories. In terms of GEEBAM fire‐severity categories, koala survival was five times more likely in areas where forest canopies were modelled as Unburnt or Partially burnt, compared to areas where forest canopies were Fully burnt. The capacity of bushfire‐affected koala populations to recover from the 2019/20 fire season will be conditional upon size of the original population in each fire‐affected area, the enactment and implementation of supportive, recovery‐themed management regimes, future inter‐fire intervals and associated intensities. Management actions necessary to assist recovery actions are discussed.

The koala (Phascolarctos cinereus), one of the world's most iconic faunal species, was recently listed under Australian government legislation as vulnerable in the northern states of Queensland and New South Wales and in the Australian Capital Territory, but not in the southern states of Victoria and South Australia. This review synthesises empirical evidence of regional koala population trends, their conservation outlook, and associated policy challenges. Population declines are common in the northern half of the koala's range, where habitat loss, hotter droughts, disease, dog attacks and vehicle collisions are the major threats. In contrast, some southern populations are locally overabundant and are now subject to managed declines. The koala presents the problem of managing a wide-ranging species that now primarily occurs in human-modified landscapes, some of which are rapidly urbanising or subject to large-scale agricultural and mining developments. Climate change is a major threat to both northern and southern populations. The implementation of policy to conserve remaining koala habitat and restore degraded habitat is critical to the success of koala conservation strategies, but habitat conservation alone will not resolve the issues of koala conservation. There needs to be concerted effort to reduce the incidence of dog attack and road-related mortality, disease prevalence and severity, and take into account new threats of climate change and mining. Many of the complex conservation and policy challenges identified here have broader significance for other species whose population trends, and the nature of the threatening processes, vary from region to region, and through time.

Alzheimer’s disease (AD) is the most common cause of dementia among older adults. Accumulation of amyloid-β (Aβ) in the brain is considered central in AD pathogenesis and its understanding crucial for developing new diagnostic and therapeutic approaches. Recent literature suggests that ageing may induce post translational modifications in Aβ, in the form of spontaneous amino acid modifications, which enhance its pathogenic properties, contributing to its aggregation. In this study, we have investigated whether the isoaspartate (IsoD-Aβ) and pyroglutamate (pE3-Aβ) modified forms of Aβ are significantly associated with AD pathology or represent markers of ageing. Cerebral neocortex of 27 AD cases, 32 old controls (OC) and 11 young controls (YC) was immunostained for pE3-Aβ and IsoD-Aβ, quantified as protein load and correlated with other Aβ forms and p-TAU. IsoD-Aβ and pE3-Aβ were detected at low levels in non-demented controls, and significantly increased in AD (p ≤ 0.001), with a characteristic deposition of IsoD-Aβ in blood vessel walls and pE3-Aβ within neurons. Both AD and OC showed positive associations between IsoD-Aβ and Aβ (p = 0.003 in AD and p = 0.001 in OC) and between IsoD-Aβ and pE3-Aβ (p = 0.001 in AD and OC). This last association was the only significant pE3-Aβ correlation identified in AD, whereas in the control cohorts pE3-Aβ also correlated with Aβ and AβPP (p = 0.001 in OC and p = 0.010 in YC). Our analyses suggest that IsoD-Aβ accumulation starts with ageing; whereas pE3-Aβ deposition is more closely linked to AD. Our findings support the importance of age-related modifications of Aβ in AD pathogenesis. Electronic supplementary material The online version of this article (10.1186/s40478-017-0505-x) contains supplementary material, which is available to authorized users.

Background The goal of the study was to investigate whether cigarette smoking alters oral and nasal microbial diversity, composition, and structure. Twenty-three current smokers and 20 never smokers were recruited. From each subject, nine samples including supra and subgingiva plaque scrapes, saliva, swabs from five soft oral tissue sites, and one nasal swab from both the anterior nares were collected. 16S rRNA V3-V4 region was sequenced for microbial profiles. Results We found that alpha diversity was lower in smokers than in nonsmokers in the buccal mucosa, but in other sample sites, microbial diversity and composition were not significantly different by smoking status. Microbial profiles differed significantly among eight oral sites. Conclusions This study investigates the effect of cigarette smoking on different sites of the oral cavity and shows a potential effect of cigarette smoking on the buccal mucosa microbiota. The marked heterogeneity of the oral microbial ecosystem that we found may contribute to the stability of the oral microbiota in most sites when facing environmental perturbations such as that caused by cigarette smoking. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0226-6) contains supplementary material, which is available to authorized users.

We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.

In order to more effectively conserve Koalas, the National Koala Conservation and Management Strategy 2009 - 2014 promotes the need for reliable approaches to the assessment of Koala habitat. This work describes a point-based, tree sampling methodology that utilises the presence/absence of Koala faecal pellets within a prescribed search area around the base of trees to derive a measure of Koala activity. Confidence intervals associated with Koala activity data from 405 randomly selected field plots within which faecal pellets were recorded have been utilised to assign threshold values for three population density/habitat biomes in eastern Australia. Subject to the need for a precautionary approach to data interpretation in areas that support naturally occurring, low-density Koala populations, the approach is expected to assist field-based assessments by researchers, land managers and others interested in clarifying aspects of habitat utilisation by free-ranging Koalas, especially where identification of i...

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H-line induced significantly higher levels of IFN-γ and lower levels of IL-10 compared with those in dogs infected with L. infantum wild type (WT). Anti-Leishmania-specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H-line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H-line over 24 months post-inoculation. Dogs which had been immunized with L. infantum H-line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H-line was safe and induced a protection which is correlated with cellular immunity in dogs.

A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines invaded but were unable to survive within bone marrow-derived macrophages in vitro, whereas wild-type parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after subcutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced significant protection in mice against challenge with wild-type parasites.

Radio-tracking studies enable insights into factors that contribute to koala mortality. Two radio-tracking studies investigating the impacts of disturbance events on koalas were undertaken in different areas over the same period. Both studies employed similar techniques for koala capture, processing and monitoring. In one study, none of nine koalas died during a 5-month monitoring program following their translocation into a new habitat area, while in the second study 6 of 11 koalas died over the same period during an in situ impact-monitoring study. The two populations differed morphologically and genetically: that with the higher mortality rate notable for a smaller head and neck circumference and lower genetic diversity. Differing outcomes from the two studies lend support to a hypothesis that inbreeding and the loss of genetic information may predispose some individuals and/or populations of koalas to an elevated stress response and/or increased susceptibility to disease, the expression of which may become exacerbated in the presence of ongoing disturbance or novel stressors that can include research activities. If this is the case, the endocrinology and genetic structure of free-ranging koala populations needs to be afforded greater consideration in terms of predicting a given population’s immunological response to potential isolation and/or disturbance events.

A major mechanism whereby malaria parasites evade the host immune response to give chronic infections in patients' blood for months, or even years, is antigenic variation. In order to generate variant antigens, parasites require large multigene families. Although several gene families involved in these phenomena have been identified in the human malaria Plasmodium falciparum, to date no variant antigen gene families have been identified in malaria species that will infect widely used rodent laboratory hosts. Here we present, for the first time, to our knowledge, a large multigene family conserved in both rodent and human malarias, which is a strong candidate as a major variant antigen gene family. In each of four species of Plasmodium, three rodent malarias and the human pathogen P. vivax, homologues of the gene family were found to have a conserved three-exon structure. In the rodent malaria P. chabaudi, transcription of members of the gene family was developmentally regulated with maximum expression in late trophozoite stages, which is the developmental stage known to express variant antigen proteins.

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area., Author Summary A 24 month vaccine trial was conducted using 103 leishmania free dogs in an area of southeast Iran endemic for visceral leishmaniosis. The dogs were vaccinated with gentamicin-attenuated line of Leishmania infantum. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination with an attenuated line of Leishmania infantum. Nine out of 31 (29%) unvaccinated dogs, but none of those vaccinated, were positive for presence of Leishmania DNA by PCR. In western blots, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs, recognized the 21 kDa antigen of Leishmania infantum wild-type. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. The attenuated Leishmania infantum induced a significant and strong protective effect against Leishmania infantum infection in the field.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Phosphodiesterase 11A (PDE11A) is a recently identified family of cAMP and cGMP hydrolyzing enzymes. Thus far, a single splice variant designated as PDE11A1 has been reported. In this study, we identify and characterize two additional splice variants of PDE11A, PDE11A2 and PDE11A3. The full-length cDNAs are 2,141 bp for PDE11A2 and 2205 bp for PDE11A3. The ORF of PDE11A2 predicts a protein of 576 aa with a molecular mass of 65.8 kDa. The ORF of PDE11A3 predicts a protein of 684 aa with a molecular mass of 78.1 kDa. Comparison of the PDE11A2 sequence with that of PDE11A1 indicates an additional 86 aa at the N terminus of PDE11A2. Part of this sequence extends the potential cGMP binding region (GAF domain) present in PDE11A1. Compared with PDE11A2, PDE11A3 has an additional 108 N-terminal amino acids. Sequence analysis of PDE11A3 indicates the presence of another GAF domain in this region. This diversification of regulatory sequences in the N-terminal region of PDE11A splice variants suggests the interesting possibility of differential regulation of these enzymes. Recombinant PDE11A2 and -A3 proteins expressed in the Baculovirus expression system have the ability to hydrolyze both cAMP and cGMP. The K m values for cAMP hydrolysis are 3.3 μM and 5.7 μM for PDE11A2 and PDE11A3, respectively. The K m values for cGMP hydrolysis are 3.7 μM and 4.2 μM for PDE11A2 and PDE11A3, respectively. Both PDEs showed a V max ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an IC 50 of 1.8 μM, and to zaprinast, with an IC 50 of 28 μM. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with IC 50 values of 0.82 and 5 μM for dipyridamole and zaprinast, respectively.

C57BL/6J mice infected with Plasmodium berghei ANKA develop neurological dysfunction and die within 7 days of infection. We show that treatment of infected mice with a kynurenine-3-hydroxylase inhibitor prevents them from developing neurological symptoms and extends their life span threefold until severe anemia develops.

We demonstrate a digital sensing platform, termed Albumin Tester, running on a smart-phone that images and automatically analyses fluorescent assays confined within disposable test tubes for sensitive and specific detection of albumin in urine. This light-weight and compact Albumin Tester attachment, weighing approximately 148 grams, is mechanically installed on the existing camera unit of a smart-phone, where test and control tubes are inserted from the side and are excited by a battery powered laser diode. This excitation beam, after probing the sample of interest located within the test tube, interacts with the control tube, and the resulting fluorescent emission is collected perpendicular to the direction of the excitation, where the cellphone camera captures the images of the fluorescent tubes through the use of an external plastic lens that is inserted between the sample and the camera lens. The acquired fluorescent images of the sample and control tubes are digitally processed within one second through an Android application running on the same cellphone for quantification of albumin concentration in the urine specimen of interest. Using a simple sample preparation approach which takes ~5 min per test (including the incubation time), we experimentally confirmed the detection limit of our sensing platform as 5-10 μg mL(-1) (which is more than 3 times lower than the clinically accepted normal range) in buffer as well as urine samples. This automated albumin testing tool running on a smart-phone could be useful for early diagnosis of kidney disease or for monitoring of chronic patients, especially those suffering from diabetes, hypertension, and/or cardiovascular diseases.

A cautious approach to managing the impacts of disturbance on free-ranging koala (Phascolactos cinereus) populations is fundamental to effective management of this iconic species. The critique by FitzGibbon et al. (2017) of a pioneering study by Phillips (2016) on the impacts of noise on koalas argued that a departure from aspects of the methods, a disregard for disease issues, other koala mortality data and an onerous approach to mitigation of potential impact detracted from the merit of the work. In response and while acknowledging some departures in evaluation criteria, the primary outcomes arising from the study remain unchanged, concerns about unreported koala mortalities are premature, while mitigation measures proposed by Phillips (2016) have been misinterpreted. Unravelling the implications of anthropogenic disturbance on terrestrial wildlife communities is a rapidly expanding field of ecological study. The work in question provides novel descriptions of aversive behaviour by koalas, each of which remains testable in the context of disturbance ecology, thus laying the foundations for further research to be undertaken.

In the 1990s, the Pilliga forests were carrying the largest population of koalas west of the Great Dividing Range in New South Wales (NSW). Whereas the NSW koala population in its entirety was thought to be in decline, the Pilliga population stood out as potentially increasing. By 2007, anecdotal evidence suggested that the population was in decline. We undertook surveys of koalas in the Pilliga forests that repeated surveys undertaken between 1991 and 2011. We found that koalas had declined and were found in only 21% of sites in which they were observed in the initial surveys – by any measure, a 5-fold drop in occupancy in less than two decades is severe. Declines occurred evenly across the Pilliga, with persistence at a site seemingly related to a high initial density of koalas rather than to a slower rate of decline. Sites where koalas persisted were characterised as having higher temperatures and lower rainfall relative to other sites, being close to drainage lines with deeper soils and having a lower occurrence of fire. This pattern fits with the observation in the recent surveys that koalas were next to drainage lines in the western half of the Pilliga and fits with the suggestion that koalas show refugial persistence. Recovery from this point is not assured and will depend on how we manage the landscape, particularly with the threat of climate change. This will likely require active management within an adaptive management framework, such as restoration of refuges, and not simply habitat reservation.

SUMMARYThe impact of immunization with gentamicin-attenuatedLeishmania infantum(H-line) on the immunophenotypic profile of popliteal lymph node (PLN) and peripheral blood mononuclear cells (PBMCs) of dogs was assessed by flow cytometry and immunohistochemistry. Compared with the dogs infected withL. infantumwild-type (Group WT), there was a significantly higher percentage of CD4+, CD44+T cells and CD14+, MHC-II+cells and a lower percentage of CD4+CD25+regulatory T cells in PLN of the immunized dogs withL. infantumH-line (Group H). The percentage of CD4+and CD8+T cells in PBMCs of immunized dogs was higher than that in dogs of Group WT. The CD4:CD8 ratio in PLN of dogs of Group H was significantly higher than that in dogs of Group WT. A significantly higher percentage of CD21+B cells and a lower percentage of CD79b+cells were found in PLN of the immunized dogs compared with dogs of Group WT. Immunohistochemical investigation showed no parasites in the PLN of immunized dogs whereas there were parasites in the PLN of 60% of dogs infected withL. infantumWT. In this study, the immunophenotypic profile of mononuclear cells of the immunized dogs correlates with cellular immunity.

Associations between animals of different species can take a variety of forms, ranging from commensalism where the partners live independent lives but benefit each other, to parasitism where one partner of the relationship benefits at the expense of the other, the host. In between, we have mutualism where the partners have an intimate relationship which is obligatory for both partners but is also beneficial to both. The majority of parasites in the animal kingdom are invertebrates. Amongst the protozoa, parasites of medical or veterinary importance are found amongst the amoebae (e.g. Entamoeba histolytica), the flagellates (e.g. trypanosomes) and the sporozoa (such as the malarial parasites), all of which are parasitic. The parasitic worms (helminths) are very important parasites of man and his domestic animals including the flukes (e.g. schistosomes causing bilharzia), round worms or nematodes such as Ascaris lumbricoides, the large round worm of humans, and the tapeworms or cestodes (it is the larval stages of tapeworms which are the cause of morbidity). Finally, the arthropods include important parasites such as ticks and mites (arachnids), and insects such as lice and fleas. Many insects such as mosquitoes, of course, act as vectors of parasites. Parasites are also identified as either endoparasites or ectoparasites depending on whether they are located internally or externally with respect to the host. Parasites often have incredibly complicated life cycles and employment of control measures against parasites requires detailed knowledge of these life cycles. Key Concepts: Associations between different species of animals can take various forms ranging from commensalism, through mutualism to parasitism. In commensalism both partners live independently, in mutualism there is an obligatory intimate relationship between the partners which is beneficial to both and, in parasitic relationship one partner benefits at the expense of the other. Most parasitic species are invertebrates but there are some vertebrate parasites such as the cuckoo. Parasitic species are found in both single-celled organisms, the protozoa, and the multi-celled metazoan organisms. There are important medical and veterinary parasites in all the major groups of protozoan and metazoan animals. Parasites can be found inside their host and are known as endoparasites whereas some found outside their hosts and are known as ectoparasites. Devising strategies to control parasites and avoid infection depends on an understanding of the life cycle of the parasites. Keywords: protozoa; helminths; insects; arthropods; endoparasites; ectoparasites; control; macroparasites; microparasites

Parasites must reach new hosts and have evolved a variety of strategies to achieve this. For some there is a direct life cycle, with the parasite passing from one member of the host species to another via the infective stage of the life cycle. Other parasite species have more complex life cycles, known indirect life cycles, involving intermediate host species, before final transmission to the definitive host. The host's defence against parasitisation broadly takes two forms. First there are nonimmune mechanisms such as the protection given to infection with the human malaria parasite Plasmodium falciparum by the presence of the abnormal ‘sickle’ haemoglobin. Secondly, parasites can be attacked by the full panoply of the host's acquired immune defence system. A feature of most host/parasite relationships is survival of the host but equally survival of the parasite: parasites have evolved a variety of mechanisms for evading or down-regulating the host's immune response to their advantage. The pathological consequences to the host of parasitic attack include physical damage from the host's activities to indirect consequences of the host's immune response. The problems of developing antiparasite vaccines are discussed. Key Concepts: Finding a new host is a formidable challenge for a parasite: variety of strategies evolved to achieve this end. A detailed understanding of parasite life cycles facilitates development of strategies for control of parasites of medical and veterinary importance. Host species can be very restricted for some parasites, but for some parasites many host species can be infected. Some parasites have direct simple life cycles whereas others have indirect complex lifecycles involving a vector or intermediate host. In complex life cycles the parasite's development and progression through the life cycle is adapted to the physiology and behaviour of the host. Prevalence of parasites in the host population is often over dispersed – that is most individuals have a few parasites but a few have many. In general the higher the parasite load the greater the severity of disease in the host. Defence to parasitic infection involves both nonimmune and acquired immune mechanisms. In many host/parasite relationships the host defences reduces the parasite load to low levels but fails to eliminate the parasite completely and transmission continues. Parasites have evolved a variety of strategies to survive or evade the host's defence mechanisms. Keywords: parasite; life cycles; vectors; immune response; immune evasion

Evidence for a role for macrophages/monocytes is largely based on in vitro not in vivo observations. Products of activated macrophages particularly tumor necrosis factor-alpha (TNF alpha) are implicated in the killing of parasites. Access of cytokines and other factors might be through intracellular channels in the infected red blood cell. The cytotoxic elements in 'crisis' serum are uncertain but may include TNF, gamma-interferon (IFN gamma), and lipid peroxidases. TNF alpha in excess, contributes to pathology. TNF, acting as a pyrogen and raising body temperature, may moderate parasite density by killing late asexual stages. Nitric oxide and other nitrogen intermediates, products of activated macrophages and a number of other cell types, have been demonstrated both in vitro and in vivo to have a protective role. Phagocytosis of infected erythrocytes and merozoites, enhanced by the presence of immune serum in some systems, has been reported. Killing of parasites by neutrophils is enhanced by immune serum and cytokines TNF alpha, IFN gamma and lymphotoxin. A role for natural killer cells has been suggested. Evidence for antibody-dependent cellular cytotoxicity (ADCC) is controversial. Antibody-dependent cellular inhibitory activity (ADCI) (blood monocytes plus immune IgG) has been described for P. falciparum. Evidence for an important role for complement is conflicting; an involvement in the protective activity of phagocytic cells is reported. Antibody isotypes have been relatively little studied. In murine systems IgG2a may have a role early in the protective immune response followed by IgG1. In P. falciparum ADCI activity is mediated by IgG1 and IgG3, two cytophilic isotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

The host protective immune response to blood stage malaria infection was studied using Plasmodium chabaudi chabaudi (P. chabaudi) in NIH mice. It has been shown previously that CD4+ cells are critically required for protection against erythrocytic infection. Mice lacking a functional CD4+ cell compartment suffer unremitting patent primary parasitemias for at least 60 days after infection. Here, we report that the adoptive transfer of eight P. chabaudi-specific CD4+ T cell clones of either the Th1 or Th2 type to mice rendered CD4-depleted by adult thymectomy and anti-CD4 monoclonal antibody therapy fully restored the ability of recipients to control challenge infection. Control Th1 and Th2 clones specific for an unrelated antigen, ovalbumin, were unable to confer a comparable level of protection in CD4-depleted mice, even though they received regular doses of the antigen. These data demonstrate that protective immunity to asexual P. chabaudi parasites can be mediated through immune CD4+ T cell clones of either the Th1 or the Th2 subset.

SUMMARYAn attenuated line of Leishmania major (L. major H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A modification of the previously described method for the generation of attenuated L. major is described, giving rise to attenuated parasites after 8 rather than 12 subpassages. No lesions developed in BALB/c mice infected with L. major H-line, whereas L. major wild-type (WT) induced a Th2 like response with progressive lesions. Analysis of splenocyte IFN-γ and IL-4 production following stimulation with promastigotes shows that the L. major H-line preferentially induces Th1-like responses and possibly down-regulates Th2 responses in BALB/c mice. L. major H-line parasites remained localized in the skin and draining lymph node, whereas L. major WT parasites disseminated into the visceral organs of BALB/c mice. Mice infected with L. major H-line acquired some resistance against L. major WT. These results show that the attenuated cell line of L. major is not only avirulent but that it may also modulate the host immune response.

The clinicopathological changes following infection with an attenuated line of Leishmania infantum (L. infantum H-line) were evaluated in mixed breed dogs. Two groups of dogs were infected intravenously (i.v.) or intradermally (i.d.) with L. infantum H-line and two control groups were infected i.v. or i.d. with L. infantum wild-type (L. infantum WT). None of the dogs, which were infected i.v. or i.d. with L. infantum H-line, showed any abnormalities during the observation period. In contrast, two out of three dogs, which were infected i.v. with L. infantum WT, developed clinical signs of disease. In addition, no histopathological changes were seen in the liver and spleen of the dogs infected with the attenuated line of parasite, whereas the histopathological changes in the two dogs infected i.v. with L. infantum WT were severe in form and manifested by infiltration of high numbers of inflammatory cells. No promastigotes were found in cultures set up from spleens and livers of dogs infected with L. infantum H-line at 12 months post-infection, whereas promastigotes were seen in the spleen and liver cultures from 2 dogs infected i.v. with L. infantum WT. Serum levels of total IgG anti-Leishmania antibody were raised in all dogs. The antibody level in the serum of dogs infected i.v. with L. infantum WT was higher than that in dogs infected with L. infantum H-line. These results show no clinicopathological abnormalities in the dogs infected with gentamicin-attenuated L. infantum H-line. Moreover, L. infantum H-line induced IgG anti-Leishmania antibody in the dogs.

The effects of short-term disturbances that result in changes to movement patterns and/or behaviour of wildlife are poorly understood. In this study the movements of seven koalas were monitored before, during and after a five-day music festival. During the monitoring program koalas occupied home-range areas of 0.6–13 ha with one or more core areas of activity. Aversive behaviour in the form of evacuation of known ranging areas was demonstrated by three koalas that had core areas within 525 m of the approximate centre of the festival area, the associated responses comprising movements that were perpendicular to and away from staging areas where music was played. Responses contained within known ranging areas were observed in three other koalas whose core areas were located up to 600 m away. The type of response appeared related to the proximity of koala home ranges to music-staging areas, while the maximum distance associated with an aversive response was 725 m. Six of the radio-tracked koalas returned to their home-range areas following the conclusion of festival activities. While the specific stimulus eliciting aversive behaviour was not identified, responses in all instances were initiated during the musical phase of the festival event. The potential for short-term disturbances such as music festivals to significantly influence the ranging patterns of koalas warrants recognition of possible longer-term ecological consequences for planning and management purposes.

Interactions between antigen-presenting dendritic cells (DCs) and T cells are essential for the induction of an immune response. However, during malaria infection, DC function is compromised and immune responses against parasite and heterologous antigens are reduced. Here, we demonstrate that malaria infection or the parasite pigment hemozoin inhibits T cell and DC interactions both in vitro and in vivo, while signal 1 intensity remains unaltered. This altered cellular behaviour is associated with the suppression of DC costimulatory activity and functional T cell responses, potentially explaining why immunity is reduced during malaria infection., Author Summary Malaria is a major infectious disease, affecting 500 million people and causing 2.7 million deaths each year. The severity of malaria is, in part, due to the failure of the host immune system to effectively clear an infection and generate protective immunity. Dendritic cells (DCs) are central to the immune system; by presenting components of pathogens to circulating T cells, they are able to initiate a highly specific immune response to clear an infection. Importantly, the quality of the interaction between T cell and DCs can affect the functional outcome of the immune response. However, previous work has demonstrated that DCs are modified by malaria parasites, resulting in inefficient priming of the adaptive immune system. Here, we have visualised the interactions between DCs and T cells in the context of malaria and demonstrate that infection is able to prevent priming of immune responses by antagonising these cell–cell contacts. Importantly, the failure to form long-lasting interactions is not due to reduced presentation of antigens by the DC, suggesting that other mechanisms may be involved. These studies provide a visual insight into the mechanism by which parasites may suppress immunity and highlight the importance of early cellular interactions in the immune response.

Functionally related homologues of known genes can be difficult to identify in divergent species. In this paper, we show how multi-character analysis can be used to elucidate the relationships among divergent members of gene superfamilies. We used probabilistic modelling in conjunction with protein structural predictions and gene-structure analyses on a whole-genome scale to find gene homologies that are missed by conventional similarity-search strategies and identified a variant gene superfamily in six species of malaria (Plasmodium interspersed repeats, pir). The superfamily includes rif in P.falciparum, vir in P.vivax, a novel family kir in P.knowlesi and the cir/bir/yir family in three rodent malarias. Our data indicate that this is the major multi-gene family in malaria parasites. Protein localization of products from pir members to the infected erythrocyte membrane in the rodent malaria parasite P.chabaudi, demonstrates phenotypic similarity to the products of pir in other malaria species. The results give critical insight into the evolutionary adaptation of malaria parasites to their host and provide important data for comparative immunology between malaria parasites obtained from laboratory models and their human counterparts.

An attenuated line of Leishmania mexicana (the L. mexicana H-line) has been established by culturing in vitro under gentamicin pressure. BALB/c mice infected with the L. mexicana H-line developed a CD4(+)Th1-like response, indicated by the cytokine profile of their splenocytes stimulated by L. mexicana wild-type (WT) promastigotes. This profile is sustained after these mice are challenged with L. mexicana WT. Control mice infected with L. mexicana WT alone developed a CD4(+)Th2-like cytokine profile. In mice immunized with L. mexicana H-line and then challenged with WT-line, were eliminated when immunizing H-line parasites persisted in the skin and draining popliteal lymph nodes (PLNs). In experiments in which mice were inoculated with attenuated and WT parasites at the same time, either at the same site or on separate sides of the mouse, growth of the WT parasites was significantly contained and controlled, indicating a possible therapeutic role for the attenuated parasites.

Central nervous system tissue of mice infected withPlasmodium bergheiANKA (PbA) exhibits similar histopathological features to those in post-mortem human cerebral malaria (CM) tissue. In this study, the neurochemical characteristics of PbA-infected and control mice were compared. Substance P-containing neurones were almost completely lost from the cortex and striatum of PbA-infected mice seven days after inoculation, whereas the intensity of calbindin immunolabelling was increased compared with controls. Neuropeptide Y- and somatostatin-containing neurones were dramatically reduced in number only in the cortex of day 7 post-inoculation mice compared with controls. This neurochemical pattern in mice with CM is similar to that previously reported to be produced in rats by quinolinic acid. Since the level of quinolinic acid is known to be raised in the brains of PbA-infected mice, the results would be consistent with a role for quinolinic acid in the production of brain damage in fatal murine CM.

We report here the cloning, expression, and characterization of human PDE11A1, a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits ≤50% amino acid identity with the catalytic domains of all other PDEs, being most similar to PDE5, and has distinct biochemical properties. The human PDE11A1 cDNA isolated contains a complete open reading frame encoding a 490-amino acid enzyme with a predicted molecular mass of 55,786 Da. At the N terminus PDE11A1 has a single GAF domain homologous to that found in other signaling molecules, including PDE2, PDE5, PDE6, and PDE10, which constitutes a potential allosteric binding site for cGMP or another small ligand. Tissue distribution studies indicate that PDE11A mRNA occurs at highest levels in skeletal muscle, prostate, kidney, liver, pituitary, and salivary glands and testis. PDE11A is expressed as at least three major transcripts of ≈10.5, ≈8.5, and ≈6.0 kb, thus suggesting the existence of multiple subtypes. This possibility is further supported by the detection of three distinct proteins of ≈78, ≈65, and ≈56 kDa by Western blotting of human tissues for PDE11A isoforms. Recombinant human PDE11A1 hydrolyzes both cGMP and cAMP with K m values of 0.52 μM and 1.04 μM, respectively, and similar V max values. Therefore, PDE11A represents a dual-substrate PDE that may regulate both cGMP and cAMP under physiological conditions. PDE11A is sensitive to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) as well as zaprinast and dipyridamole, inhibitors that are generally considered relatively specific for the cGMP-selective PDEs, with IC 50 values of 49.8 μM, 12.0 μM, and 0.37 μM, respectively.

The expression of antisense RNA is a powerful means of extinguishing unwanted gene activity in the eukaryotic cell (see this volume). This approach has worked particularly well in plantst4 and animals5 in which antisense-producing gene constructs can be inserted into the germ cell. In man, germ cell manipulation is not yet possible. The problem arises as to how somatic cells can be targeted with genes generating antisense RNAs. Apart from retroviral vectors, a multitude of techniques exist for introducing DNA into eukaryotic somatic cells (ref. 6 and references therein). During the last few years, we and others7J have been interested in adapting the cellular mechanisms of receptor-mediated endocytosis of macromolecules for the importation of DNA molecules into cell^.^-^* The principle of the method is to covalently connect a polycation, usually polylysine, to a ligand for a receptor on the cell surface. DNA, usually in the form of cloned genes, is then bound ionically to the polylysine. During importation of the DNA by receptor-mediated endocytosis the ligand binds to the receptor, and during internalization of both of these entities the DNA is thought to become colocalized, first in the coated pits and later in the endosome. The further fate of the DNA can be particularly well studied when reporter genes such as the firefly luciferase gene or the bacterial P-galactosidase gene, driven from a viral enhancer/promoter, are employed. It can be predicted that DNA in the endosome is directed to the lysosomal compartment, where it can be expected to be destroyed by resident nucleases. Despite this, apparently a small amount of DNA does escape destruction, so that reporter gene activity is observed in a few cells of a cell population (see below). Because the genes are expressed, it is probably safe to assume that some of the DNA has reached the cell nucleus. When transferrin is used as a ligand, the transfection procedure is called “transferrinfection.” Consistent with the postulated chain of events during transferrinfection, we find that the addition of the lysosomatropic agent chloroquine, which prevents acidification and concomitant activation of lysosomal degradative enzymes, greatly enhances expression of the chosen reporter gene.“ This enhancement can be spectacular, as for instance in the human erythroleukemic cell line K562 in which subsequent to the

The sphere organelles (spheres) of Xenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in the Xenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified in Xenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification in Xenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.

A cluster of Xenopus laevis U7 snRNA genes has been isolated and sequenced. The gene structure is more compact than, but otherwise comparable to, the major U snRNA genes since the distal sequence element (DSE) is located only 4 nt upstream of the PSE. The corresponding RNA is present in the oocyte and accumulates early in oogenesis.

Background: Dendritic cells (DCs) are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the host’s systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. Results: Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell) membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. Conclusions: Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy.

Tree species preferences of a koala population inhabiting a small area of forest and woodland in the Campbelltown area, south-west of Sydney, were investigated over a two-year period. In total, 2499 trees from 45 independent field sites were assessed, with tree species preferences determined on the basis of a comparative analysis of proportional data relating to the presence/absence of koala faecal pellets. The results established that grey gum (Eucalyptus punctata) and blue-leaved stringybark (E. agglomerata) were most preferred by koalas in the study area, but only when growing on shale-based substrates. The preferential utilisation of E. punctata and E. agglomerata on substrates derived from shales, compared with that recorded for the same species on sandstones, suggests that their use by koalas was influenced by differences in nutrient status between substrates. Regression analyses further identified a trend for use of at least one of the preferred species (E. punctata) to be more commonly associated with larger trees. Results are discussed in terms of their relevance to issues of resource availability and the need to reconsider, by way of a hierarchical approach, the use of food trees by koalas generally. The presence of E. punctata and E. agglomerata and their occurrence in conjunction with shale-based substrates are considered to be important limiting factors affecting the present-day distribution and abundance of koalas in the Campbelltown area.

An assessment of the tree species preferences of koalas inhabiting forest and woodland communities growing on Quaternary deposits in the Port Stephens area, New South Wales, was undertaken between November 1994 and March 1996. Using a plot-based methodology, 3847 trees were sampled, comprising 15 Eucalyptus species and 17 species of non-eucalypt. Evidence of tree use by koalas, specifically the presence of koala faecal pellets, was recorded from beneath 10 Eucalyptus species and 9 species of non-eucalypt. Tree species preferences were determined by analyses of log- likelihood ratios derived from data based on the presence/absence of koala faecal pellets, rather than on gross counts. This approach confirmed significant variation in the levels of utilisation amongst and between different tree species, and that two in particular – swamp mahogany (E. robusta) and drooping red gum (E. parramattensis) – were most preferred. Increases in the levels of use of other tree species were also positively associated with the presence of E. robusta and/or E. parramattensis. Levels of utilisation of E. robusta and E. parramattensis did not alter significantly in response to changes in their respective densities, suggesting that the relative abundance of both was important in terms of understanding the carrying capacity of vegetation communities utilised by koalas. The results have established the success with which an enumerative approach to the interpretation of faecal pellet data can be utilised to clarify the tree species preferences of koalas. Application of the approach for habitat assessment and mapping purposes is also discussed.

A human lung cDNA expression library was screened by using a rabbit antiserum specific for a human Clara cell 10 kDa protein. The cDNA from two positive clones was sequenced by the dideoxy chain termination method. The nucleotide and primary amino-acid sequence deduced therefrom are presented. The N-terminal amino-acid sequence of the Clara cell 10 kDa protein, purified from bronchoalveolar lavage, was also determined. The deduced and experimentally determined sequences were identical where data for both were available. From the amino-acid composition, deduced and experimentally determined amino-acid sequences, it was determined that the 10 kDa protein in bronchoalveolar lavage consists of two identical 70-amino-acid long polypeptide chains joined by two cystine residues. The size of mRNA for the protein was found to be about 0.6 kb and the monomeric nascent protein, obtained by in vitro translation of lung mRNA was about 7.3 kDa in size. The 10 kDa protein recovered from bronchoalveolar lavage has 61% sequence identity with rabbit uteroglobin, the two proteins have common predicted secondary structures with marked surface differences when comparing predicted and actual structure determined by X-ray diffraction. The differences imply similarity of structure but, not identity of function.

Background: Intestinal parasitic infections are a public health problem in developing countries such as Mexico. As a result, two governmental programmes have been implemented: a) "National Deworming Campaign" and b) "Opportunities" aimed at maternal care. However, both programmes are developed separately and their impact is still unknown. We independently investigated whether a variety of socio-economic factors, including maternal education and employment levels, were associated with intestinal parasite infection in rural school children.\ud \ud Methods: This cross-sectional study was conducted in 12 rural communities in two Mexican states. The study sites and populations were selected on the basis of the following traits: a) presence of activities by the national administration of albendazole, b) high rates of intestinal parasitism, c) little access to medical examination, and d) a population having less than 2,500 inhabitants. A total of 507 schoolchildren (mean age 8.2 years) were recruited and 1,521 stool samples collected (3 per child). Socio-economic information was obtained by an oral questionnaire. Regression modelling was used to determine the association of socio-economic indicators and intestinal parasitism.\ud \ud Results: More than half of the schoolchildren showed poliparasitism (52%) and protozoan infections (65%). The prevalence of helminth infections was higher in children from Oaxaca (53%) than in those from Sinaloa (33%) (p < 0.0001). Giardia duodenalis and Hymenolepis nana showed a high prevalence in both states. Ascaris lumbricoides, Trichuris trichiura and Entamoeba hystolitica/dispar showed low prevalence. Children from lower-income families and with unemployed and less educated mothers showed higher risk of intestinal parasitism (odds ratio (OR) 6.0, 95% confidence interval (CI) 1.6–22.6; OR 4.5, 95% CI 2.5–8.2; OR 3.3, 95% CI 1.5–7.4 respectively). Defecation in open areas was also a high risk factor for infection (OR 2.4, 95% CI 2.0–3.0).\ud \ud Conclusion: Intestinal parasitism remains an important public health problem in Sinaloa (north-western Mexico) and Oaxaca (south-eastern Mexico). Lower income, defecation in open areas, employment status and a lower education level of mothers were the significant factors related to these infections. We conclude that mothers should be involved in health initiatives to control intestinal parasitism in Mexico.

The balance between T helper type 1 (Th1) and T helper type 2 (Th2) cells determines the outcome of many important diseases. Using cloned murine T cell lines, evidence is provided that Th 1, but not Th 2, cells can be activated by specific antigens or a T cell mitogen, concanavalin A, to produce large amounts of nitric oxide (NO). Furthermore, NO can inhibit the secretion of interleukin (IL)-2 and interferon-γ by Th 1 cells but has no effect on IL-4 production by Th 2 cells. Th 1 and Th 2 cells can, thus, be distinguished by their differential production of and susceptibility to NO. NO exerts a self-regulatory effect on Th 1 cells which are implicated in immunopathology.