A new species of Cryptosporidium (Apicomplexa: Cryptosporidiidae) from eastern grey kangaroos (Macropus giganteus)

Cryptosporidium macropodum n. sp is described. Oocysts of C. macropodum from the feces of kangaroos (Macropus spp.) are morphologically indistinguishable from other mammalian Cryptosporidium species, including C. parvum, C. hominis, C. suis, and C. canis. The oocysts are fully sporulated on excretion, lack sporocysts, and have an average width of 4.9 microm (4.5-6.0), a length of 5.4 microm (5.0-6.0), and a length:width ratio of 1.1. Phylogenetic analyses of the 18S ribosomal RNA, actin, and heat shock protein 70 (HSP70) loci demonstrate that C. macropodum is genetically distinct from all described Cryptosporidium species, including others found in marsupials. The parasite seems to be highly host-specific, because it has been found only in marsupials to date. Therefore, based on biological and molecular data, we consider C. macropodum a new species.     

Predation by red foxes limits recruitment in populations of eastern grey kangaroos

We investigated the impact of red fox (Vulpes vulpes Linnaeus 1758) predation on juvenile eastern grey kangaroos (Macropus giganteus Shaw 1790) using a replicated predator removal experiment. In two sites in Namadgi National Park, south‐eastern Australia, a persistent 1080 poisoning campaign over 18 months reduced fox density by more than 85%, and to less than 10% of the fox density in two other sites with no fox baiting. Changes in the mother : young ratios and densities of kangaroo populations were monitored twice monthly along 2‐km transects in each site from July 1993 to February 1995. Compared to nonremoval sites, where foxes were controlled, 25–40% more females retained juveniles over the period when these young became emergent from the pouch. This higher survival of emergent pouch young resulted in a significantly higher proportion of juveniles in kangaroo populations at fox control sites, which resulted in a significantly higher annual growth rate. We conclude that predation upon juveniles is an important limiting factor for kangaroo populations in Namadgi NP.     

Density dependence in foraging habitat preference of eastern grey kangaroos

For a free‐ranging forager, the suitability of a patch is dependent on population density, resource supply, resource quality, and the costs of foraging or dispersal. We quantified differences among three foraging habitats and compared this variation to temporal patterns of habitat preference by free‐ranging eastern grey kangaroos, Macropus giganteus. We investigated selection on a fine‐grained spatial scale, and asked whether habitat preference is constrained by density‐dependent mechanisms. Variation in the quantity and quality of resources among habitats was greatest during spring, when biomass and quality were highest, and differences among habitats were most pronounced. However, consistent and discernable differences among habitats were not obtained, indicating that the system fluctuated around an equilibrium state. Using isodar regressions to examine the consumer‐density relationships among habitats, open‐woodland habitat was favoured over the two open‐forest habitats for foraging. Seasonal isodars indicated that density dependence regulated preference between the three foraging habitats during autumn, spring and summer, but not during winter, when variability in resources among habitats was lowest.     

Patterns of Cryptosporidium Oocyst Shedding by Eastern Grey Kangaroos Inhabiting an Australian Watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 × 10⁶ oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum “bovine” genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium “marsupial” genotype I or “marsupial” genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.     

Foraging behaviour and dispersion of eastern grey kangaroos (Macropus giganteus) in an ideal free framework

Ideal free distribution (IFD) theory predicts that animals in competitive situations should distribute themselves among available habitat patches according to the density of conspecifics and its regulatory effect on resources. To investigate the applicability of IFD models to free-ranging herbivores, we quantified the dispersion and foraging behaviour of eastern grey kangaroos Macropus giganteus among habitat patches of differing suitability, within and outside a reservoir catchment in southern Victoria, Australia. Kangaroo densities within the catchment had a regulatory effect on resource density, while surrounding farmland maintained a higher standing crop despite higher densities of competitors. This difference was slight in autumn, however, when the system was apparently close to equilibrium. Gross bite rates of individuals foraging in farmland were lower than for individuals foraging within the catchment, and vigilance behaviour occurred more frequently in farmland habitat than any other, decreasing time devoted to feeding. Interference competition occurred in only 1.9% of focal samples, although competitive differences based on phenotype were observed. Although resource gains by individual kangaroos are likely to be influenced by other factors, including resource dynamics, predation risk and phenotypic differences, IFD theory provides a valuable analytical framework for this herbivore foraging system.     

Interactions among social monitoring,anti-predator vigilance and group size in eastern grey kangaroos

Group size is known to affect both the amount of time that prey animals spend in vigilance and the degree to which the vigilance of group members is synchronized. However, the variation in group-size effects reported in the literature is not yet understood. Prey animals exhibit vigilance both to protect themselves against predators and to monitor other group members, and both forms of vigilance presumably influence group-size effects on vigilance. However, our understanding of the patterns of individual investment underlying the time sharing between anti-predator and social vigilance is still limited. We studied patterns of variation in individual vigilance and the synchronization of vigilance with group size in a wild population of eastern grey kangaroos (Macropus giganteus) subject to predation, in particular focusing on peripheral females because we expected that they would exhibit both social and anti-predator vigilance. There was no global effect of group size on individual vigilance. The lack of group-size effect was the result of two compensating effects. The proportion of time individuals spent looking at other group members increased, whereas the proportion of time they spent scanning the environment decreased with group size; as a result, overall vigilance levels did not change with group size. Moreover, a degree of synchrony of vigilance occurred within groups and that degree increased with the proportion of vigilance time peripheral females spent in anti-predator vigilance. Our results highlight the crucial roles of both social and anti-predator components of vigilance in the understanding of the relationship between group size and vigilance, as well as in the synchronization of vigilance among group members.     

Thermoregulation by kangaroos from mesic and arid habitats: influence of temperature on routes of heat loss in eastern grey kangaroos (Macropus giganteus) and red kangaroos (Macropus rufus)

We examined thermoregulation in red kangaroos (Macropus rufus) from deserts and in eastern grey kangaroos (Macropus giganteus) from mesic forests/woodlands. Desert kangaroos have complex evaporative heat loss mechanisms, but the relative importance of these mechanisms is unclear. Little is known of the abilities of grey kangaroos. Our detailed study of these kangaroos' thermoregulatory responses at air temperatures (T(a)) from -5 degrees to 45 degrees C showed that, while some differences occur, their abilities are fundamentally similar. Both species show the basic marsupial characteristics of relatively low basal metabolism and body temperature (T(b)). Within the thermoneutral zone, T(b) was 36.3 degrees + or - 0.1 degrees C (X + or - SE) in both species, and except for a small rise at T(a) 45 degrees C, T(b) was stable over a wide range of T(a). Metabolic heat production was 25% higher in red kangaroos at T(a) -5 degrees C. At the highest T(a) (45 degrees C), both species relied on evaporative heat loss (EHL) to maintain T(b); both panting and licking were used. The eastern grey kangaroo utilised panting (76% of EHL) as the principal mode of EHL, and while this was so for red kangaroos, cutaneous evaporative heat loss (CEHL) was significant (40% of EHL). CEHL appeared to be mainly licking, as evidenced from surface temperatures. Both species utilised peripheral vascular adjustments to control heat flow, as indicated by changes in dry conductance (C(dry)). At lower temperatures, C(dry) was minimal, but it increased significantly at T(a) just below T(b) (33 degrees C); in these conditions, the C(dry) of red kangaroos was significantly higher than that of eastern grey kangaroos, indicating a greater reliance on dry heat loss. Under conditions where heat flows into the body from the environment (T(a) 45 degrees C), there was peripheral vasoconstriction to reduce this inflow; C(dry) decreased significantly from the values seen at 33 degrees C in both kangaroos. The results indicated that, while both species have excellent thermoregulatory abilities, the desert red kangaroos may cope better with more extreme temperatures, given that they respond to T(a) 45 degrees C with lower respiratory evaporation than do the eastern grey kangaroos.     

Genetic characteristics of coastal cutthroat trout inhabiting an urban watershed

Watersheds in urban areas are often heavily degraded due to human activity, which can have negative impacts on freshwater fishes. Monitoring the genetic characteristics of urban populations can provide insights into the impact of development on aquatic ecosystems. We performed a genetic analysis of coastal cutthroat trout (Oncorhynchus clarkii clarkii) inhabiting urban tributaries in Portland, OR. By analyzing nuclear microsatellite genotypes, we were able to assess population structure, genetic diversity, and effective population size for six locations across two tributaries on opposite sides of the Willamette River. Genetic diversity was generally equivalent across all sampling locations, although populations from smaller tributaries higher in the stream network had lower levels. Levels of effective population size were low but within expected ranges for small salmonid populations. As anticipated, smaller populations had higher levels of inter-individual relatedness. The primary genetic structure divided populations on opposite sides of the Willamette River, although there was evidence of dispersal between the two groups. Our results suggest that cutthroat trout inhabiting metropolitan areas are not necessarily genetically impoverished and may exhibit characteristics typical of populations in more ‘natural’ environments. Understanding how fish, especially anadromous species, respond to urban environments is essential to evaluating the value of these areas for conservation planning.     

Tooth wear in eastern grey kangaroos (Macropus giganteus) and western grey kangaroos (Macropus fuliginosus), and its potential influence on diet selection, digestion and population parameters

As teeth wear in eastern and western grey kangaroos, the pattern of enamel ridges changes. This affects the amount of available cutting edges and consequently the masticatory efficiency. These kangaroos also have molar progression. There is evidence for the independence of rates of tooth wear and molar progression, and variation is found in the maximum amount of wear attained by teeth both within and between populations. This variation has the potential to affect diet selection, digestion and population parameters.     

Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor

An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 10⁵ oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa) molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa) molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.     

Cryptosporidium parvum: structural components of the oocyst wall

Cryptosporidium parvum, an enteropathogenic parasite, infects a wide range of mammals including man and constitutes a substantial veterinary and medical threat due to its ubiquitous distribution and the stability of the oocyst stage. The oocyst wall of C. parvum is known to be extremely resistant to chemical and mechanical disruption. Isolated oocyst walls are shown by both thin sectioning and negative staining transmission electron microscopy to possess a filamentous array on the inner surface. This filamentous array can be greatly depleted by digestion with proteinase K and trypsin, but pepsin has less effect. Ultrasonication of the untreated oocyst walls produced almost no fragmentation, but extension of the suture resulted in inward spiraling of the wall to generate ellipsoid and cigar-shaped multilayer bodies, with the filamentous array still present. When ultrasonicated, proteinase K-digested oocyst walls progressively fragmented into small sheets. These wall fragments, depleted of filaments, are shown by negative staining to possess a pronounced linearity, indicative of an integral highly complex lattice structure.     

Near real-time detection of Cryptosporidium parvum oocyst by IgM-functionalized piezoelectric-excited millimeter-sized cantilever biosensor

Piezoelectric-excited millimeter-sized cantilever (PEMC) biosensors were fabricated and functionalized with immunoglobulin M (IgM) for the detection of Cryptosporidium parvum oocyst in a flow configuration at 1 mL/min. The detection of 100, 1000, and 10,000 oocysts/mL was achieved with a positive sensor response in less than 1 min. Bovine serum albumin (BSA) was used as a blocking agent in each experiment and was shown to eliminate non-specific binding. The sensor's resonance frequency response correlates with C. parvum oocyst concentration logarithmically. The oocyst attachment rate was found to increase by an order of magnitude in increasing concentration from 100 to 10,000 oocysts/mL. The significance of these results is that IgM-functionalized PEMC sensors are highly selective and sensitive to C. parvum oocyst and therefore, have the potential to accurately identify and quantify C. parvum oocyst in drinking water.     

Mother–offspring distances reflect sex differences in fine‐scale genetic structure of eastern grey kangaroos

Natal dispersal affects life history and population biology and causes gene flow. In mammals, dispersal is usually male‐biased so that females tend to be philopatric and surrounded by matrilineal kin, which may lead to preferential associations among female kin. Here we combine genetic analyses and behavioral observations to investigate spatial genetic structure and sex‐biased dispersal patterns in a high‐density population of mammals showing fission–fusion group dynamics. We studied eastern grey kangaroos (Macropus giganteus) over 2 years at Wilsons Promontory National Park, Australia, and found weak fine‐scale genetic structure among adult females in both years but no structure among adult males. Immature male kangaroos moved away from their mothers at 18–25 months of age, while immature females remained near their mothers until older. A higher proportion of male (34%) than female (6%) subadults and young adults were observed to disperse, although median distances of detected dispersals were similar for both sexes. Adult females had overlapping ranges that were far wider than the maximum extent of spatial genetic structure found. Female kangaroos, although weakly philopatric, mostly encounter nonrelatives in fission–fusion groups at high density, and therefore kinship is unlikely to strongly affect sociality.     

Evaluation of Cryptosporidium parvum oocyst recovery efficiencies from various filtration cartridges by electrochemiluminescence assays

AIMS: To evaluate four types of filtration cartridges for their capacities, efficiency for capture and release of Cryptosporidium parvum oocysts for detection. METHODS AND RESULTS: Filtration cartridges included in this evaluation were IDEXX Filta-Max, Gelman Envirochek HV, Corning CrypTest, and Filterite Sigma+. Various dosages of C. parvum oocysts were spiked into water samples with a wide range of turbidity (10-50 NTU). Electrochemiluminescence assays were employed to enumerate viable or total number of C. parvum oocysts in these eluates. Among the cartridges tested, Filta-Max consistently showed higher oocyst recovery efficiency, especially with large volume, highly turbid water samples. CONCLUSIONS: Filta-Max filter is the best performer because of its higher oocyst recovery efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall sensitivities of various C. parvum oocyst detection assays in water samples can be improved if highly efficient oocyst recovery filtration cartridges such as Filta-Max are incorporated in sample preparation.