Peter Daniel Stuart

Silicon, endophytes and secondary metabolites as grass defenses against mammalian herbivores

Grasses have been considered to primarily employ tolerance in lieu of defense in mitigating damage caused by herbivory. Yet a number of mechanisms have been identified in grasses, which may deter feeding by grazers. These include enhanced silicon uptake, hosting of toxin-producing endophytic fungi and induction of secondary metabolites. While these mechanisms have been individually studied, their synergistic responses to grazing, as well as their effects on grazers, are poorly known. A field experiment was carried out in 5 × 5 m outdoor enclosures to quantify phytochemical changes of either endophyte-infected (E+) or endophyte-free (E-) meadow fescue (Schedonorus pratensis) in response to medium intensity (corresponding with densities of ca. 1200 voles/ha for 5 weeks during 3 months) or heavy intensity (ca. 1200 voles/ha for 8 weeks during 3 months) grazing by a mammalian herbivore, the field vole (Microtus agrestis). A laboratory experiment was then conducted to evaluate the effects of endophyte infection status and grazing history of the grass diet on vole performance. As predicted, grazing increased foliar silicon content, by up to 13%. Grazing also increased foliar levels of phosphorous and several phenolic compounds, most notably those of the flavonols isorhamnetin-diglycoside and rhamnetin derivative. Silicon concentrations were consistently circa 16% higher in E+ grasses than in E-grasses, at all levels of grazing. Similarly, concentrations of chlorogenic acid derivative were found to be consistently higher in E+ than in E- grasses. Female voles maintained on heavily grazed grasses suffered higher mortality rates in the laboratory than female voles fed ungrazed grass, regardless of endophyte infection status. Our results conclusively demonstrate that, in addition to tolerance, grasses employ multi-tiered, effective defenses against mammalian grazers.

Food resources and intestinal parasites as limiting factors for boreal vole populations during winter

Processes limiting the growth of cyclic vole populations have stimulated considerable research and debate over several decades. In Fennoscandia, the peak density of cyclic vole populations occurs in fall, and is followed by a severe winter decline. Food availability and intestinal parasites have been demonstrated to independently and synergistically limit wildlife populations. The purpose of this study was to directly compare competing food and parasite hypotheses on the limitation of overwintering high-density vole populations. Moreover, we evaluated the ability of food limitation and nematode infection to interact and thereby intensify population declines. A two-factor experiment with food supplementation and antihelminthic medication was conducted on replicated, enclosed field vole (Microtus agrestis) populations in central Finland over one full boreal winter. Population abundance, survival, and demographic attributes were monitored through live trapping. Vole feces were concurrently examined for the eggs of Heligmosomidae nemadotes and oocysts of eimerian coccidians. We found that vole density declined in all treatment groups throughout winter. However, food supplementation mitigated this decline through positive effects on reproduction, and voles in food-supplemented populations were generally in better physiological condition than non-supplemented voles. Food supplementation and antihelminthic treatment reduced the prevalence of Heligmosomidae nematodes, while neither food nor medication affected the prevalence of eimerians, or infection intensity of either parasite group. Although food supplementation and antihelminthic medication aided in the clearance of Heligmosomidae nematodes, their prevalence did not influence vole population growth, and this parasite group is therefore unlikely to contribute to the cyclic winter decline of boreal vole populations. Instead food resources acting alone were the primary factor limiting vole population growth.

Food provisioning alters infection dynamics in populations of a wild rodent

While pathogens are often assumed to limit the growth of wildlife populations, experimental evidence for their effects is rare. A lack of food resources has been suggested to enhance the negative effects of pathogen infection on host populations, but this theory has received little investigation. We conducted a replicated two-factor enclosure experiment, with introduction of the bacterium Bordetella bronchiseptica and food supplementation, to evaluate the individual and interactive effects of pathogen infection and food availability on vole populations during a boreal winter. We show that prior to bacteria introduction, vole populations were limited by food availability. Bordetella bronchiseptica introduction then reduced population growth and abundance, but contrary to predictions, primarily in food supplemented populations. Infection prevalence and pathological changes in vole lungs were most common in food supplemented populations, and are likely to have resulted from increased congregation and bacteria transmission around feeding stations. Bordetella bronchiseptica-infected lungs often showed protozoan co-infection (consistent with Hepatozoon erhardovae), together with more severe inflammatory changes. Using a multidisciplinary approach, this study demonstrates a complex picture of interactions and underlying mechanisms, leading to population-level effects. Our results highlight the potential for food provisioning to markedly influence disease processes in wildlife mammal populations.

Investigating the role of wild carnivores in the epidemiology of bovine neosporosis.

Neospora caninum is a protozoan parasite, primarily associated with bovine abortion. The only definitive hosts discovered to date are carnivores. This study aimed to identify the role of mammalian carnivores in the epidemiology of bovine neosporosis. A sample bank of serum, fecal and brain samples was established: American mink (Mustela vison), red foxes (Vulpes vulpes), pine martens (Martes martes), badgers (Meles meles), stoats (Mustela erminea), otters (Lutra lutra) and feral ferrets (Mustela putorius). Approximately 1% of mink and 1% of fox samples were positive by IFAT. According to PCR analysis of DNA extracted from brain tissue, 3% of the mink, 4% of the otters and 6% of the foxes examined were infected with N. caninum. All fecal samples tested negative for N. caninum DNA (n = 311), suggesting that the species that tested positive were intermediate not definitive hosts. This is the first time that tissues from mustelids have tested positive for N. caninum. The need to test 2 relatively large (~200 mg) targeted parts of the brain to avoid false negatives was also identified. The relatively low prevalence of N. caninum in Irish carnivores suggests that the local ecology of a species has an important influence on its epidemiological role.

Diet Quality Limits Summer Growth of Field Vole Populations

Marked variation occurs in both seasonal and multiannual population density peaks of northern European small mammal species, including voles. The availability of dietary proteins is a key factor limiting the population growth of herbivore species. The objective of this study is to investigate the degree to which protein availability influences the growth of increasing vole populations. We hypothesise that the summer growth of folivorous vole populations is positively associated with dietary protein availability. A field experiment was conducted over a summer reproductive period in 18 vegetated enclosures. Populations of field voles (Microtus agrestis) were randomised amongst three treatment groups: 1) food supplementation with ad libitum high protein (30% dry weight) pellets, 2) food supplementation with ad libitum low protein (1% dry weight; both supplemented foods had equivalent energy content) pellets, and 3) control (no food supplementation), n = 6 per treatment. Vole density, survival, demographic attributes and condition indicators were monitored with live-trapping and blood sampling. Highest final vole densities were attained in populations that received high protein supplementation and lowest in low protein populations. Control populations displayed intermediate densities. The survival rate of voles was similar in all treatment groups. The proportion of females, and of those that were pregnant or lactating, was highest in the high protein supplemented populations. This suggests that variation in reproductive, rather than survival rates of voles, accounted for density differences between the treatment groups. We found no clear association between population demography and individual physiological condition. Our results demonstrate that dietary protein availability limits vole population growth during the summer growing season. This suggests that the nutritional quality of forage may be an underestimated source of interannual variation in the density and growth rates of widely fluctuating populations of herbivorous small mammals.

Serological Survey of Rodent-Borne Viruses in Finnish Field Voles

In northern Europe, rodent populations display cyclic density fluctuations that can be correlated with the human incidence of zoonotic diseases they spread. During density peaks, field voles (Microtus agrestis) become one of the most abundant rodent species in northern Europe, yet little is known of the viruses they host. We screened 709 field voles, trapped from 14 sites over 3 years, for antibodies against four rodent-borne, potentially zoonotic viruses or virus groups-hantaviruses, lymphocytic choriomeningitis virus (LCMV), Ljungan virus (LV), and orthopoxviruses (OPV). Antibodies against all four viruses were detected. However, seroprevalence of hantaviruses, LV, and LCMV was low. OPV antibodies (most likely cowpox) were more common but restricted geographically to southeastern Finland. Within these sites, antibody prevalence showed delayed density dependence in spring and direct density dependence in fall. Higher seroprevalence was found in spring than fall. These results substantially increase knowledge of the presence and distribution of viruses of field voles in Finland, as well as CPXV infection dynamics.

Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument

In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods.

Statistical comparison of excystation methods in Cryptosporidium parvum oocysts

Excystation of sporozoites of Cryptosporidium parvum from oocysts is essential for successful in vitro assays. It has also been traditionally used as a measure for oocyst viability and infectivity. Laboratories use various excystation protocols so there is a need to clarify which method is the best. In this study, six different protocols for in vitro excystation of C. parvum oocysts were compared to find the most efficient excystation method (expressed as percentage excystation). Tested protocols differed in chemical pre-incubation steps, excystation media or time of incubation. There were significant differences in percentage of excysted oocysts among groups excysted by different methods. There were also significant differences in percentage of excysted oocysts between methods using pre-incubation with sodium hypochlorite and those without. The other variables examined; the presence of trypsin, kind of excystation medium and the incubation time, did not show statistical differences in percentage excystation among groups. Pre-incubation steps which included sodium hypochlorite, enhancing the permeability of the oocysts were found to increase the excystation ratio and methods using this step were the most effective.

Parasitic nematodes of the genus Syphacia Seurat, 1916 infecting Muridae in the British Isles, and the peculiar case of Syphacia frederici.

Syphacia stroma (von Linstow, 1884) Morgan, 1932 and Syphacia frederici Roman, 1945 are oxyurid nematodes that parasitize two murid rodents, Apodemus sylvaticus and Apodemus flavicollis, on the European mainland. Only S. stroma has been recorded previously in Apodemus spp. from the British Isles. Despite the paucity of earlier reports, we identified S. frederici in four disparate British sites, two in Nottinghamshire, one each in Berkshire and Anglesey, Wales. Identification was based on their site in the host (caecum and not small intestine), on key morphological criteria that differentiate this species from S. stroma (in particular the tail of female worms) and by sequencing two genetic loci (cytochrome C oxidase 1 gene and a section of ribosomal DNA). Sequences derived from both genetic loci of putative British S. frederici isolates formed a tight clade with sequences from continental worms known to be S. frederici, clearly distinguishing these isolates from S. stroma which formed a tight clade of its own, distinct from clades representative of Syphacia obvelata from Mus and S. muris from Rattus. The data in this paper therefore constitute the first record of S. frederici from British wood mice, and confirm the status of this species as distinct from both S. obvelata and S. stroma.