Article

Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung

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Abstract

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2×10(4) oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.

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... Inadequate water and sanitation services or infrastructure at household or community level [10,18,19,21,23,33,37,38,[40][41][42]44,52,54,59,61,64,70,[72][73][74][75]78,81,82,89,100] Open defecation site near human or animal activities [10,18,23,40,43,44,61,64,69,72,74,81] Unmanaged animal waste near human or animal activities [10,26,28,31,36,40,43,44,46,50,60,66,74,81,94,100] Poor environmental hygiene, overcrowding, open slaughter, and/or a lack of garbage removal and processing services [35,38,[40][41][42]44,47,52,54,55,58,64,67,70,[72][73][74]76,78,81,82,84,88,89,91,100] Seasonality and environmental conditions for insect vector proliferation [18,28,29,[38][39][40][41]62,69,71,82,90,91,97] Unsafe food preparation, storage, sale, and/or service [19][20][21]26,29,33,38,40,46,54,55,57,62,63,68,69,72,73,81,85,90,101] Insect vector feeding behaviors and preferences, movement patterns, and living habitat predilection [19,[29][30][31]34,35,39,43,46,49,52,54,55,57,[59][60][61]64,[71][72][73]76,79,85,86,88,90,97,101] Animal contact, husbandry, and proximity to living spaces [10,18,20,21,26,28,42,44,46,47,50,52,53,57,60,65,66,90,91,94,98,100] Purposeful or accidental ingestion of contaminated insect vector by animals or humans [25,30,34,56,66,72,75,95,96] ...
... Inadequate water and sanitation services or infrastructure at household or community level [10,18,19,21,23,33,37,38,[40][41][42]44,52,54,59,61,64,70,[72][73][74][75]78,81,82,89,100] Open defecation site near human or animal activities [10,18,23,40,43,44,61,64,69,72,74,81] Unmanaged animal waste near human or animal activities [10,26,28,31,36,40,43,44,46,50,60,66,74,81,94,100] Poor environmental hygiene, overcrowding, open slaughter, and/or a lack of garbage removal and processing services [35,38,[40][41][42]44,47,52,54,55,58,64,67,70,[72][73][74]76,78,81,82,84,88,89,91,100] Seasonality and environmental conditions for insect vector proliferation [18,28,29,[38][39][40][41]62,69,71,82,90,91,97] Unsafe food preparation, storage, sale, and/or service [19][20][21]26,29,33,38,40,46,54,55,57,62,63,68,69,72,73,81,85,90,101] Insect vector feeding behaviors and preferences, movement patterns, and living habitat predilection [19,[29][30][31]34,35,39,43,46,49,52,54,55,57,[59][60][61]64,[71][72][73]76,79,85,86,88,90,97,101] Animal contact, husbandry, and proximity to living spaces [10,18,20,21,26,28,42,44,46,47,50,52,53,57,60,65,66,90,91,94,98,100] Purposeful or accidental ingestion of contaminated insect vector by animals or humans [25,30,34,56,66,72,75,95,96] ...
... (cockroach: [47,59,68,89,95,97,99]; fly: [54,58,60,61,64,67,73,82,100]). However, dung beetles were only infected experimentally [50,71]. Entamoeba spp. ...
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... The activity of dung beetles may impact on the survival of oocysts in the terrestrial environment – indirectly by breaking down the dung pat and directly by ingestion (Ryan et al 2011). Although most C. parvum oocysts ingested by dung beetles are destroyed by digestion, some pass through the intestinal tract and appear to be morphologically normal in beetle faeces. ...
... A study of the viability of Cryptosporidium oocysts after processing and burial by dung beetles, (Ryan et al 2011) showed that after five days, 13.7% of oocysts remained in dung on the surface and 86.3% had been buried. The same study also found that after five days, oocysts in buried dung had only 10% viability, compared with dung remaining at the surface where 58% of the oocysts were viable. ...
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... While other studies have found dung beetles to offer a wide range of ecosystem services related to dung removal and pathogen suppression [13,15,16,39], our studies presented here demonstrate beneficial ecosystem services offered by dung beetles in the light of developing food safety concerns. The dung beetles in our study did not display a feeding preference between pathogen-laden feces and feces that had not been inoculated with E. coli O157:H7. ...
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... Bubas bison (L. 1767) is an autumn to early spring active dung beetle that has been estimated to bury 90% of cattle dung after 5 days (Ryan et al. 2011). In comparison, O. gazella (Fabricius, 1787, which is a spring-summer active beetle, was capable of breaking up entire dung pads within 30-40 hours of introduction, despite being a smaller beetle (Bornemissza 1970). ...
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Both indigenous and introduced Australian dung beetles (Coleoptera: Scarabaeoidea) play an important role in agricultural systems. Temperate grasslands in Australia are expected to have some of the greatest increases in temperature and reduction in precipitation due to climate change which may directly threaten dung beetles. Several biotic and abiotic factors affect the development of larvae and fitness of young dung beetles. To understand if dung beetles can continue to provide ecosystem services across temperate Australia under changing management and climate, we review what is known about their life history traits that will facilitate their adaption. We believe dung beetles will continue to provide valuable ecosystem services and have potential to aid in adapting and alleviating the impacts on crops and pastures from reduced and sporadic rainfall. However, the level of function, may be impeded due to climatic stress and a loss of diversity with thermal specialists and some indigenous species, expected to be displaced into narrower ranges. An increase in feeding competition from thermal generalists and introduced species will likely implement this movement. The evidence from naturalised introduced species indicate behavioural adaptations and/or phenotypic plasticity suggest some species will continuing to provide services that improve pasture production despite changes to climate. Ongoing monitoring of phenological shifts will inform adaptive management of this vital group.
... The vital dye exclusion/inclusion assay is known to over-estimate viability under some conditions in which damage to the parasites has not resulted in alteration in permeability, and is therefore particularly inappropriate for studies investigating the effects of disinfectants (Robertson and Gjerde, 2007). Nevertheless, over the years it has provided useful information on parasite survival under different environmental conditions in a range of studies by different research groups (e.g., Robertson et al., 1992;Merry et al., 1997;Jenkins et al., 1999;Kato et al., 2004;Nichols et al., 2004;McGuigan et al., 2006;Robertson and Gjerde, 2006;Reinoso and Bécares, 2008;Gómez-Couso et al., 2009;Ryan et al., 2011). ...
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Vital dye staining has long been used to assess viability of Cryptosporidium oocysts and Giardia cysts, with staining and enumeration in suspension. Some recent studies, however, have dried samples to microscope slides prior to staining. Here we demonstrate that this approach may considerably underestimate parasite viability in the original sample.
... In contrast, in an investigation of the fate of Cryptosporidium parvum oocysts ingested by three beetle species (Anoplotrupes stercorosus, Aphodius rufus and O. fracticornis), Mathison and Ditrich (1999) reported that the majority of oocysts were destroyed following passage through dung beetle mouthparts and gastrointestinal tract, suggesting a potentially negative influence of beetles on C. parvum transmission. A similarly negative impact of beetle activity on Cryptosporidium oocysts' viability was reported by Ryan et al. (2011), who found that oocysts' viability in feces burial by seven pairs of Bubas bison declined from 58% (control) to 10% (burial treatment). In a study of the ability of the dung beetle species Catharsius molossus to act as transport host for the pathogenic Escherichia coli strain O157: H7, only 5% of dung beetles tested positive for its presence in their gut contents, leading the authors to conclude that dung beetles appeared to play no epidemiological role in its transmission (Xu et al. 2003). ...
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... Although recently, crickets have been shown not to have such efficient conversion rates as previously claimed (Lundy & Parrella, 2015). In addition, by burying dung, insects contribute to not only to its decomposition, a key supporting service, but also to regulating services via the suppression of pathogens of both livestock and humans (Ryan et al., 2011). Insects can also provide services to commercial waste treatment, through the conversion of organic waste into nutrient-rich humus, and their larvae can provide protein-rich animal feed for chickens, pigs, and aquaculture (Diener et al., 2009). ...
... Cryptosporidium is another dung-borne gut parasite of mammals which can be at least partially controlled by dung burial, as illustrated by Ryan, Yang, Cameron, and Doube (2011). ...
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... For example, Mathison and Ditrich (1999) reported that the majority of Cryptosporidium parvum oocysts ingested by three beetle species (Anoplotrupes stercorosus, Aphodius rufus, and Onthophagus fracticornis) were destroyed following consumption. Ryan et al. (2011) also reported a decline in C. parvum oocyst viability of 48%, following feeding and subsequent burial by the beetle species Bubas bison. ...
... For example, Mathison and Ditrich (1999) reported that the majority of Cryptosporidium parvum oocysts ingested by three beetle species (Anoplotrupes stercorosus, Aphodius rufus, and Onthophagus fracticornis) were destroyed following consumption. Ryan et al. (2011) also reported a decline in C. parvum oocyst viability of 48%, following feeding and subsequent burial by the beetle species Bubas bison. ...
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... While efforts were made to simulate field conditions these inflated maxima may be due to the experiment being carried out inside a CEF where faecal deposits were isolated from stressors present in the field, and did not encounter, for example, cell wash-out following rainfall. Furthermore, under field conditions soil macrofauna such as beetles and earthworms break up faeces, which can affect the survival of E. coli (Ryan et al., 2011;Pedersen and Hendriksen, 1993). Given that the faecal material in this experiment may have been protected from some factors experienced in the field, extrapolation of our regrowth model to field conditions must be done with a degree of caution, with recognition that the experiment was undertaken to develop greater insight into what drives patterns of E. coli regrowth. ...
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Agricultural intensification can lead to high volumes of livestock faeces being applied to land, either as solid or liquid manures or via direct defecation, and can result in reservoirs of faecal indicator organisms (FIOs) persisting within farmland. Understanding the survival of FIOs, e.g. E. coli, in agricultural environments, and in particular within different livestock faeces, is key to developing catchment management practices for the protection of ecosystem services provided by clean water. Frequently, controlled laboratory studies, under constant temperature regimes, are used to determine the impact of environmental factors on E. coli persistence in livestock faeces; however, such studies oversimplify the diurnal variations and interactions of real world conditions. The aim of this study was to investigate the survival of E. coli using a controlled environment facility, which simulated diurnal variation of temperatures typically experienced during a British spring and summer. The approach provided a comparison of E. coli persistence profiles within faeces of sheep, beef cattle and dairy cattle to allow novel interpretations of E. coli regrowth patterns in contrasting livestock faeces in the period immediately post-defecation. Thus, the coupling of a tightly controlled environment facility with high resolution monitoring enabled the development of a new non-linear, asymptotic description of E. coli proliferation in livestock faeces, with increased potential for E. coli growth observed during warmer temperatures for all livestock types. While this study focused on temperatures typical of the UK, the occurrence of a phase of E. coli regrowth has implications for microbial water quality management worldwide.
... Herbivory by non-pest insects is essential for regulating primary production in forests worldwide, consequently affecting carbon and nutrient cycling (Schowalter 2012, Metcalfe et al 2014. By removing dung from natural and anthropic environments, dung beetles indirectly suppress pathogens (Ryan et al 2011), act as gastrointestinal parasite controls (Sands and Wall 2017), and reduce the populations of horn flies in cattle (Nichols et al 2008). Soil-dwelling insects, such as termites and leaf-cutting ants, are directly linked to the carbon and nitrogen cycles, due to their effects on soil biological and physical properties and organic matter inputs from their nests (Jouquet et al 2011, Swanson et al 2019. ...
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Insects are the most abundant and diverse organisms on Earth and provide essential ecosystem services. However, Brazilian society rarely consider the importance of insects in their diverse country. Therefore, in this review, we provide an overview of ecosystem services provided by insects in Brazil. A database search returned 136 articles, published in English or Portuguese, on ecosystem services provided by insects in Brazil. The first article was published in 1982, and majority of the studies were conducted in the Atlantic Forest or the Cerrado biomes. The most frequently studied insect-provided ecosystem services were pollination, decomposition, and biological control of pests. The studies focused primarily on natural and anthropic ecosystems, and most followed an experimental approach. We noted that the term “ecosystem services” was not used frequently in studies on insects in Brazil. The information available was mostly taxon-biased. We discuss the implications of these findings in relation to reconciling economic interests and the need for insect conservation for continued provision of ecosystem services in a broader perspective. In conclusion, we argue that the scientific community should focus on understanding the ecosystem services provided by insects other than those strictly related to economic activities, and on improving communication with policymakers and citizens. As a tropical and megadiverse country, Brazil has the potential to become a protagonist in conserving and using the ecosystem services provided by insects, both locally and internationally, by providing scientific information to policymakers and citizens.
... By scattering, shredding, and burying dung, dung beetles reduce the suitability of cattle pads as breeding sites for pests and parasites that affect livestock and humans (Horgan 2001, Forgie et al. 2010, Ryan et al. 2011. Some of these pests attack animals directly to feed on their blood, whereas others are vectors of parasites, bacteria, and viruses that are pathogenic in humans and livestock (Table 3). ...
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Insects play a key role in the regulation and dynamics of many ecosystem services (ES). However, this role is often assumed, with limited or no experimental quantification of its real value. We examined publication trends in the research on ES provided by insects, ascertaining which ES and taxa have been more intensively investigated, and which methodologies have been used, with particular emphasis on experimental approaches. We first performed a systematic literature search to identify which ES have been attributed to insects. Then we classified the references retrieved according to the ES, taxonomic group and ecosystem studied, as well as to the method applied to quantify each ES (in four categories: no quantification, proxies, direct quantification and experiments). Pollination, biological control, food provisioning, and recycling organic matter are the most studied ES. However, the majority of papers do not specify the ES under consideration, and from those that do, most do not quantify the ES provided. From the rest, a large number of publications use proxies as indicators for ES, assuming or inferring their provision through indirect measurements such as species abundances, species density, species richness, diversity indices, or the number of functional groups. Pollinators, predators, parasitoids, herbivores, and decomposers are the most commonly studied functional groups, while Hymenoptera, Coleoptera, and Diptera are the most studied taxa. Experimental studies are relatively scarce and they mainly focus on biological control, pollination, and decomposition performed in agroecosystems. These results suggest that our current knowledge on the ES provided by insects is relatively scarce and biased, and show gaps in the least-studied functional and taxonomic groups. An ambitious research agenda to improve the empirical and experimental evidence of the role played by insects in ES provision is essential to fully assess synergies between functional ecology, community ecology, and biodiversity conservation under current global changes.
Thesis
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Protozoan parasites of the genus Cryptosporidium can cause severe gastrointestinal symptoms, primarily diarrhoea, in e.g. young calves and humans. Some species in the genus, including C. parvum, have a broad host range, are transmittable between animals and humans and can cause significant disease in humans, cattle and sheep. In contrast, pigs primarily host the species-specific C. suis and C. scrofarum, while C. parvum is only observed occasionally. However, studies of Cryptosporidium infections in pigs are scarce and studies of commercial outdoor-reared organic pigs are lacking. Outdoor housing can lead to contact with wildlife and thus potential exposure to additional Cryptosporidium species compared with conventional indoor housing. Cattle and conventionally reared pigs are known to have high Cryptosporidium prevalences and young animals can excrete large amounts of oocysts. Waste from these farm animals is spread on agricultural land, and this, together with year-round contamination of pastures housing some age groups of Danish organic pigs, could contribute to contamination of nearby water sources by runoff. This thesis examined the occurrence of Cryptosporidium species in organic pigs and the fate of oocysts following manure management and treatment. In three sub-studies, the prevalence, intensity and zoonotic potential of Cryptosporidium on three organic pig farms and any season and age-related variations in these were determined; leaching of Cryptosporidium parvum oocysts through soil to which raw and separated liquid pig slurry had been applied was compared; and the inactivation of Cryptosporidium parvum oocysts in cattle slurry with added aq. ammonia was determined. To assess age-related Cryptosporidium (and Giardia) prevalence, infection levels, seasonal differences, genetic variation and zoonotic potential in naturally infected, organically reared pigs, oocyst excretion was monitored based on quarterly sampling (September 2011-June 2012) of piglets (n=152), starter pigs (n=234), fatteners (n=230) and sows (n=240). Faeces samples were screened and oocysts quantified by immunofluorescence microscopy, and 56 subsamples from Cryptosporidium-infected pigs were successfully analysed by PCR amplification and partial sequencing of the small subunit (SSU) 18S rRNA and the heat shock protein (hsp70) gene, respectively. Cryptosporidium infection was observed in 40.9% (350/856) of pigs, of which 8.2% (70/856) were dual-infected with Cryptosporidium and Giardia. The infection intensity ranged from 200 to 10,551,200 OPG (mean 99,062 OPG), and surprisingly, dual-infected pigs tended to excrete lower levels of oocysts than pigs harbouring only Cryptosporidium. Of the successfully genotyped isolates, 67.9% (38/56) were C. scrofarum and 32.1% (15/56) C. suis. The C. scrofarum-infected pigs excreted fewer oocysts (mean OPG: 54,848 ± 194,508) than pigs infected with C. suis (mean OPG: 351,035 ± 351,035). The prevalence, infection intensity and presence of Cryptosporidium species varied significantly between age groups (prevalence: 53.3% piglets, 72.2% starter pigs, 40.4% fatteners, 2.9% sows). Piglets had the highest infection intensity and excreted on average 1177-fold more OPG than sows. Piglets almost exclusively hosted C. suis, while starter pigs and fatteners predominantly hosted C. scrofarum. The overall prevalence, infection intensity and species distribution was stable throughout this sampling year. As organic pigs are partly reared outdoors, environmental contamination with Cryptosporidium is inevitable. Although, sporadic cases of C. suis and C. scrofarum are reported in humans, the contribution from organic pigs in this study to the epidemiology of cryptosporidiosis in humans is apparently of minor importance. Accordingly, our findings suggest that the public health risk associated with Cryptosporidium originating from organically outdoor reared pigs seems to be negligible in Denmark. The potential for contamination of land drains and groundwater by vertical soil transport of viable C. parvum oocysts present in slurry was studied using simulated rainfall events on intact soil columns with surface-applied raw slurry or with injected raw or separated liquid slurry. In weekly samplings, C. parvum oocysts were detected in leachate from all soil columns, regardless of slurry type and application method, although the recovery rates were low (<1%). Soil columns with injected liquid slurry leached nearly four-fold and 10-fold more oocysts than soil columns with injected and surface-applied raw slurry, respectively. Among leachate samples containing oocysts, 61.1% yielded viable oocysts (determined by inclusion and exclusion of vital dyes), with the number positively correlated (r=0.63) with total number of oocysts found. Thus, the risk of groundwater contamination depends on organic matter present in the slurry and application method to soil. The potential for C. parvum oocyst inactivation in cattle slurry following addition of approximately 60 mM aq. ammonia and the effect of temperature over time (4, 10 and 20 °C) was studied in a laboratory set-up. The results showed that addition of ammonia to slurry significantly affected oocyst inactivation over time, with an estimated 34.3% reduction in presumably viable oocysts (4', 6 diamidino-2-phenylindole positive (DAPI+) and propidium iodide negative (PI-)), a 43.4% increase in presumably non-viable oocysts (DAPI+ PI+) and a 37.5% increase in potentially viable (DAPI- PI-) oocysts at day 14 post treatment when compared with untreated raw slurry. However, only 40.4% presumably non-viable oocysts were present in the slurry despite addition of ammonia (mean concentration 89.7 ± 1.22 mM). No differences in inactivation were noted in correlation to incubation temperature in raw slurry or slurry added ammonia. Our findings indicate that ammonia may be used to reduce pathogen concentrations in slurry prior to application to agricultural land, although the exact concentration and exposure time needed to kill all Cryptosporidium oocysts were not determined in the present study. In conclusion, environmental contamination with Cryptosporidium oocysts from Danish organic pigs appear to be a minor concern for human health, but animal waste from these pigs is a potential source of high environmental contamination with Cryptosporidium oocysts all year round. Viable oocysts in slurry can reach the groundwater by vertical transport. However, the risk of waterborne human cryptosporidiosis from animal waste might be reduced substantially by treating slurry with aq. ammonia prior to application to soil.
Article
Water is a major route of transmission for Cryptosporidium and oocysts commonly occur in surface and recreational waters as a consequence of fecal contamination from Wildlife or anthroponotic sources. There are many characteristics possessed by Cryptosporidium oocysts that allow them to persist in aquatic environments, including recreational waters, and to bypass water treatment processes. These types of events lead to outbreaks of cryptosporidiosis, caused by direct exposure to contaminated recreational water (such as swimming pools) or by drinking contaminated potable water. Previous chapters have discussed the epidemiology of Cryptosporidium in relation to waterborne transmission and also the sources and presence of oocysts in drinking and recreational waters. This chapter will review the processes contributing to the removal and inactivation of Cryptosporidium oocysts from surface waters and wastewaters, including natural processes that occur in surface waters and engineered processes used for the production of drinking water or for the treatment of wastewater.
Article
Dung beetles are described as key ecosystem service providers. Because of ecosystem benefits from their activity, approval has been granted to import dung-burying beetles for release into pasture systems in New Zealand. Concerns remain, however, about the impact of dung beetle tunnelling and burying activity on the leaching of contaminants through soil. To assess the quality of water leaching from soil cores containing dung beetles, a trial was conducted using 12 intact Allophanic soil core lysimeters under in situ soil moisture and temperature regimes. Four lysimeters received dung and beetles (Geotrupes spiniger and Onthophagus taurus), four received dung and no beetles and four (controls) received no dung and no beetles. The quality of water was assessed by monitoring leachate samples for Escherichia coli and total N and P for 6 months. Key findings of the study were that concentrations of E. coli, N and P leaching through the lysimeters treated with dung beetles were not significantly different from soils without dung beetles. In addition, where dung was buried there was a significant decrease in E. coli and total N and P in surface soil compared with the lysimeters receiving dung only. Results from this study suggest dung beetle tunnelling and burying activity does not negatively impact the quality of water leaching through Allophanic soil.
Article
Abstract Because of outbreaks of cryptosporidiosis in humans, Cryptosporidium has become a public health concern. Commercial swine operations can be a source of this protozoan parasite. Although the species distribution of Cryptosporidium is likely dominated by C. suis, a fraction may be comprised of other Cryptosporidium species infectious to humans such as C. parvum. To better understand the survival dynamics of Cryptosporidium oocysts associated with swine operations, two experiments were performed to determine die-off rates of C. parvum oocysts in a swine waste lagoon (2009 and 2010) and its spray field (2010 and 2011). Sentinel chambers containing a lagoon effluent suspension of C. parvum oocysts were submerged in the lagoon, and triplicate chambers were removed over time; oocysts were extracted, and assayed for viability. For comparative purposes, inactivation rates of Ascaris suum eggs contained in sentinel chambers were also determined. For two spray field experiments air-dried and sieved surface soil was placed in sentinel chambers, hydrated, and inoculated with a lagoon effluent suspension of C. parvum oocysts. Sentinel chambers and control oocysts in PBS contained in microcentrifuge tubes were buried 1.5 cm below the soil surface in three blocks. Triplicate chambers and controls were removed over time; oocysts were extracted, and assayed for viability. Based on the first order decay equation, days to reach 99% die-off (T99) were determined. T99-values determined for the two lagoon experiments were 13.1 and 20.1 weeks, respectively. A T99-value for Cryptosporidium parvum in the spray field was significantly longer at 38.0 weeks than the control oocysts in PBS at 29.0 weeks. The waste lagoon and spray field system of manure management at this large-scale farrowing operation appeared to reduce the load of Cryptosporidium oocysts before they can be hydrologically transported off the operation, and reduces their likelihood of contaminating surface waters and threatening public health.
Technical Report
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This project assessed the pasture growth and environmental benefits of dung beetles to the southern Australian cattle industry using Bubas bison as an example of the deep-tunnelling dung beetles introduced to Australia by CSIRO. B. bison is one of a group of four deep-tunnelling dung beetle species (including Geotrupes spiniger, Onitis caffer, Copris hispanis) that are well suited to southern Australia but are currently established over only a small portion of their potential range. The environmental and agricultural benefits of such species had not been examined previously. There were three field experiments. The first demonstrated that complete dung burial by B. bison on one occasion increased pasture production (dry matter) by about 30% and that increased production has persisted for 3+ years after dung burial. Experiment 2 demonstrated that even small feral beetle populations increased pasture production. Experiment 3 provided an explanation by showing that dung burial by B. bison increased earthworm populations, the permeability of soil to water and the levels of nitrate, available phosphate, sulphur, carbon and organic matter in the subsoil. We recommend that monitoring continue for 2 years, that paddock-scale validation be undertaken and that the four species of deep-tunnelling dung beetles be established throughout their potential range in southern Australia.
Article
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Inclusive cross sections of η production by e+e- annihilation for c.m. energies between 4.0 and 5.0 GeV are presented. The η production is shown to be correlated with the production of a weakly decaying particle, indicating that its main source is F production. At the 4.42 GeV resonance it is correlated with a low energy photon, suggesting or production. A mass determination of the F is made at 4.42 GeV using the F → ηπ decay channel.
Article
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At least 325 water-associated outbreaks of parasitic protozoan disease have been reported. North American and European outbreaks accounted for 93% of all reports and nearly two-thirds of outbreaks occurred in North America. Over 30% of all outbreaks were documented from Europe, with the UK accounting for 24% of outbreaks, worldwide. Giardia duodenalis and Cryptosporidium parvum account for the majority of outbreaks (132; 40.6% and 165; 50.8%, respectively), Entamoeba histolytica and Cyclospora cayetanensis have been the aetiological agents in nine (2.8%) and six (1.8%) outbreaks, respectively, while Toxoplasma gondii and Isospora belli have been responsible for three outbreaks each (0.9%) and Blastocystis hominis for two outbreaks (0.6%). Balantidium coli, the microsporidia, Acanthamoeba and Naegleria fowleri were responsible for one outbreak, each (0.3%). Their presence in aquatic ecosystems makes it imperative to develop prevention strategies for water and food safety. Human incidence and prevalence-based studies provide baseline data against which risk factors associated with waterborne and foodborne transmission can be identified. Standardized methods are required to maximize public health surveillance, while reporting lessons learned from outbreaks will provide better insight into the public health impact of waterborne pathogenic protozoa.
Article
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A viability assay for oocysts of Cryptosporidium parvum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples.
Article
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Vegetated buffer strips were evaluated for their ability to remove waterborne Cryptosporidium parvum from surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log10 reductions for spiked C. parvum oocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm3. Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborne C. parvum (1- to 2-log10 reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log10 reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of ≤20% and a length of ≥3 m should function to remove ≥99.9% of C. parvum oocysts from agricultural runoff generated during events involving mild to moderate precipitation.
Article
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Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding of C. parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces−1, with a 54% probability of detecting just one oocyst g of feces−1. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces−1. The estimated environmental loading rate of C. parvum ranged from 3,900 to 9,200 oocysts cow−1 day−1, which is substantially less than a previous estimate of 1.7 × 105 oocysts cow−1 day−1 (range of 7.7 × 104 to 2.3 × 105 oocysts cow−1 day−1) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.
Article
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The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P < 0.01). Inactivation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day(-1).
Article
A modified enhanced chemiluminescent assay was assessed for its ability to detect oocysts of Cryptosporidium parvum using either X-ray film or a photomultiplier for imaging. The photomultiplier was able to detect a minimum theoretical number of 6.25 purified oocysts and did not appear to have a problem with the detection of false positives. However, because of the plate format of this system, it is not possible to confirm results by microscopy. When X-ray film was utilised for imaging, false positives were detected on 16% occasions, but as few as two oocysts could be detected by this system. X-ray film was also used in the screening of environmental samples previously assessed by microscopy. Although 23% of the negative samples were considered by BEC to be positive, all those samples which contained oocysts were also found to be positive by this system. Until such time as this technique can be refined by increasing the avidity of the monoclonal antibody, enhanced chemiluminescence might provide a useful method for initial screening of environmental and water samples for Cryptosporidium oocysts, with subsequent confirmation by microscopy of samples considered to be positive.
Article
Populations of beef cattle represent a potential non-point source of environmental contamination for Cryptosporidium parvum if on-farm management practices fail to minimize transport from bovine manure to adjacent water sources. Characterizing this risk of contamination requires several parameters to be estimated, the most important being a valid and precise estimate of the oocyst loading rate per animal unit. The oocyst loading rate is defined in this study as the total number of oocysts excreted by a cohort of adult beef cows during a 24[emsp4 ]h period. We propose a methodology for estimating this parameter for low prevalent populations whereby the majority of individuals are test negative. Under specific degrees of confidence and at the population scale, this methodology generates estimates for maximal oocyst loading based on the sensitivity of the diagnostic test and the point prevalence and intensity of fecal shedding from a cross-sectional survey of the target population.Our cross-sectional survey on California beef cows generated a prevalence of infection of 1.1 % (6/557) and an intensity of oocyst shedding ranging from 219 to 5,491 oocysts/g, with a geometric mean of 835 oocysts/g from six positive cows. Negative binomial estimate of the percent recovery of the diagnostic assay was 0.235. Based on this percent recovery and using approximately 19.4[emsp4 ]mg of feces per assay, the DT90 of our assay, defined as the concentration of oocysts at which our diagnostic assay had a 90 % probability of detecting one or more oocysts in a sample, was 755 oocyst/g feces. At a 95 % confidence level, the estimated maximum number of oocysts being excreted in the feces of California beef cows ranged from 4.8 to 14.4 oocysts/g feces/cow, or 7.7104 to 2.3105 oocysts/beef cow/day.
Article
It was established in Part 1 that potential impediments to movement of colloids through soil can be subdivided into straining and filtration, depending on whether a particle has a dimension similar to pores (leading to physical trapping) or much smaller. Information about size distributions of particles was also presented in Part 1. Owing to the dependence of colloid or particle capture processes on the relative size distributions of the particles and pores, information about pore sizes distributions has been derived here (Part 2) from hydrological equations. Various approaches to extending this mathematical treatment to indicate when particles would be trapped by necks or irregularities in the pore are also presented. Results suggest that protozoan microorganisms (such as Cryptosporidium and Giardia) are the only colloidal contaminants with sufficiently large diameter to have their movement restricted by physical straining in the soil pores when water drains from the soil under gravity, but if the soil is very wet (near to saturation) even these will move without restriction.
Article
There are 10 valid species of Cryptosporidium and perhaps other cryptic species hidden under the umbrella of Cryptosporidium parvum. The oocyst stage is of primary importance for the dispersal, survival, and infectivity of the parasite and is of major importance for detection and identification. Because most oocysts measure 4–6 μm, appear nearly spherical, and have obscure internal structures, there are few or no morphometric features to differentiate species and in vitro cultivation does not provide differential data as for bacteria. Consequently, we rely on a combination of data from three tools: morphometrics, molecular techniques, and host specificity. Of 152 species of mammals reported to be infected with C. parvum or an indistinguishable organism, very few oocysts have ever been examined using more than one of these tools. This paper reviews the valid species of Cryptosporidium, their hosts and morphometrics; the reported hosts for the human pathogen, C. parvum; the mechanisms of transmission; the drinking water, recreational water, and food-borne outbreaks resulting from infection with C. parvum; and the microscopic, immunological, and molecular methods used to detect and identify species and genotypes.
Article
Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotype and subtype levels. These tools have been increasingly used in characterizing the transmission of Cryptosporidium spp. in humans and animals. Results of these molecular epidemiologic studies have led to better appreciation of the public health importance of Cryptosporidium species/genotypes in various animals and improved understanding of infection sources in humans. Geographic, seasonal and socioeconomic differences in the distribution of Cryptosporidium spp. in humans have been identified, and have been attributed to differences in infection sources and transmission routes. The transmission of C. parvum in humans is mostly anthroponotic in developing countries, with zoonotic infections play an important role in developed countries. Species of Cryptosporidium and subtype families of C. hominis have been shown to induce different clinical manifestations and have different potential to cause outbreaks. The wide use of a new generation of genotyping and subtyping tools in well designed epidemiologic studies should lead to a more in-depth understanding of the epidemiology of cryptosporidiosis in humans and animals.
Article
A total of 477 faecal samples from pre-weaned sheep from 5 different farms in the south west of Western Australia were screened for the presence of Cryptosporidium and Giardia using PCR. There were substantial differences in prevalence between the farms and overall prevalence was 24.5% and 11.1%, respectively for Cryptosporidium and Giardia. At the 18S locus, 66 Cryptosporidium positives were identified, the majority of which were C. bovis (n=52), followed by the cervid genotype (n=10) and C. parvum (n=2). At a second diagnostic locus, using C. parvum and C. hominis-specific qPCR primers, 63 C. parvum positives were identified, some of which were co-infections with C. bovis. The C. parvum/C. hominis qPCR was more sensitive than the nested 18S PCR at detecting C. parvum. This may be due to the low numbers of oocysts present, as quantitation data indicated that all the C. parvum detected were present in low numbers (1-10 oocysts). It may also be that using C. parvum-specific primers is necessary to determine the true prevalence of C. parvum. Amongst Giardia positive isolates, G. duodenalis genotype E (livestock) was the most prevalent (36/53), with G. duodenalis genotype A detected in five positive isolates. There were also 11 mixed A and E infections detected. The findings of the present study indicate that pre-weaned lambs are not an important source of zoonotic Giardia genotypes in Australia but may be an important source of zoonotic Cryptosporidium.
Article
Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 micrograms, 4-aminobenzoic acid 100 micrograms, calcium pantothenate 50 micrograms, ascorbic acid 875 micrograms, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously.
Article
The fate of oocysts of Cryptosporidium parvum ingested by dung beetles and the possible role these beetles serve in the dissemination of cryptosporidiosis were tested on the following species: Anoplotrupes stercorosus, Aphodius rufus, and Onthophagus fracticornis. Ten specimens of each species were offered cattle dung supplemented with 5.9 x 10(6) oocysts of C. purvum. After 24 hr of feeding, the beetles were examined for the presence of oocysts on their external surfaces, in their gastrointestinal tracts, and in feces passed during the experiment. Results indicate that although many oocysts pass safely through the mouthparts and gastrointestinal tracts of the beetles, the majority of them are destroyed. Coprophagous insects can, therefore, be considered an important aspect in the ecology of gastrointestinal diseases of man and livestock, as both agents of control and dissemination.
Article
Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotype and subtype levels. These tools have been increasingly used in the characterization of the transmission of Cryptosporidium spp. This review addresses the most recent developments in molecular epidemiology of cryptosporidiosis. The recent development of subtyping tools has led to better understanding of the population genetics and transmission of Cryptosporidium in humans. The population structure of C. parvum and C. hominis is apparently more complicated than previously suggested, with the likely existence of both clonal and panmictic populations. Thus, the transmission of C. parvum (genotype II) in humans is shown to be different in different areas, with zoonotic transmission important in certain places and anthroponotic transmission in others. The use of molecular tools has also led to the identification of geographic and temporal differences in the transmission of C. parvum and C. hominis, and better appreciation of the public health importance of other Cryptosporidium species/genotypes and the frequency of infections with mixed genotypes or subtypes. Factors involved in the transmission of human cryptosporidiosis are difficult to examine using conventional methods. The use of molecular tools has been helpful in the assessment of the zoonotic potential of various Cryptosporidium spp. and sources of human infections, and has started to play a significant role in the characterization of transmission dynamic in endemic and epidemic areas.
Article
The widespread usages of molecular epidemiological tools have improved the understanding of cryptosporidiosis transmission. Much attention on zoonotic cryptosporidiosis is centered on Cryptosporidium parvum. Results of genotype surveys indicate that calves are the only major reservoir for C. parvum infections in humans. The widespread presence of human-adapted C. parvum, especially in developing countries, is revealed by recent subtyping and multilocus typing studies, which have also demonstrated the anthroponotic transmission of C. parvum subtypes shared by humans and cattle. Developing and industrialized countries differ significantly in disease burdens caused by zoonotic species and in the source of these parasites, with the former having far fewer human infections caused by C. parvum and little zoonotic transmission of this species. Exclusive anthroponotic transmission of seemingly zoonotic C. parvum subtypes was seen in Mid-Eastern countries. Other zoonotic Cryptosporidium spp. are also responsible for substantial numbers of human infections in developing countries, many of which are probably transmitted by anthroponotic pathways. The lower pathogenicity of some zoonotic species in some populations supports the occurrence of different clinical spectra of Cryptosporidium spp. in humans. The use of a new generation of molecular diagnostic tools is likely to produce a more complete picture of zoonotic cryptosporidiosis.
Introduced dung beetles in Australia 1967-2007: current status and future directions, LandCare, Australia and the Orica Community Foundation
  • P B Edwards
Edwards, P.B., 2007. Introduced dung beetles in Australia 1967-2007: current status and future directions, LandCare, Australia and the Orica Community Foundation. <http://www.landcareonline.com/Edwards%20Penny%20BOOK%-20ALL%20PAGES-final.pdf>.