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The effects of a silvicultural sludge application on small mammal populations and the potential of Giardia… Cheng, Chris 1993

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THE EFFECTS OF A SILVICULTURAL SLUDGE APPLICATION ONSMALL MAMMAL POPULATIONS ANDTHE POTENTIAL OF GIARDIA CONTAMINATION IN A FOREST ECOSYSTEMbyChris ChengB.Sc., Simon Fraser University, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Forestry)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMarch 1993© Chris Cheng, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  FoReST The University of British ColumbiaVancouver, CanadaDate  Aree,.0 2-2^1(02DE-6 (2/88)ABSTRACTThe effects of a silvicultural sludge application on resident small mammal populationswere assessed by monitoring their demographic responses before and after sewage sludgetreatment. Potential contamination of the forest ecosystem by Giardia duodenalis, a parasiticprotozoan, was also investigated. Populations of the deer mouse (Peromyscus maniculatus),Oregon vole (Microtus oregoni), Townsend chipmunk (Eutamias townsendii), shrews (Sorexspp.), and a few species caught occasionally, were sampled in control and treatment study sitesfrom May 1990 to October 1991. Sludge application occurred in November 1990. There wereno detectable differences in the abundances of deer mice, Oregon voles, and chipmunks ontreatment areas relative to controls after sludge application. This was also the case forrecruitment, survival, mean body weight, weight at sexual maturity, sex ratio, species diversityand spatial distribution. There was, however, a significant increase in the growth rate ofjuvenile deer mice on treatment sites after sludge application. This was also the only effect onsmall mammal population dynamics observed from the sludge treatment.The prevalence of Giardia spp. cysts in small mammal fecal samples on study sites didnot differ between pre-sludge and post-sludge periods. My laboratory study using wild deermice concluded that this species was capable of acting as a host to some strains of humanGiardia under specific laboratory conditions. Because of the deer mouse's apparent ability toadapt well to this environmental change, as illustrated by the lack of effect on its survival andreproduction attributes, and this species' potential ability to host human Giardia, additionalresearch and consideration are warranted prior to the implementation of large scale silviculturalsludge applications.iiTABLE OF CONTENTSABSTRACT ^ iiTABLE OF CONTENTS ^  iiiLIST OF TABLES  viLIST OF FIGURES viiiACKNOWLEDGEMENTS ^ xCHAPTER 1:General IntroductionINTRODUCTION^  1LITERATURE REVIEW: EFFECTS OF SLUDGE ON WILDLIFE ^ 4Ungulates ^  4Birds  4Small mammals  5SMALL MAMMAL STUDY OBJECTIVES ^  6CHAPTER 2:Impacts of a Silvicultural Sludge Applicationon Small Mammal PopulationsINTRODUCTION^  7MATERIALS AND METHODS ^  8Description of study areas  8Livetrapping protocol  9Population parameters ^  11iiiPopulation density and recruitment ^  12Reproduction ^  12Survival and growth  12Body weights  13Growth analysis  14Sex ratio ^  14Species diversity and richness ^  15Spatial distribution ^  16Statistical analysis  16Sludge application  17RESULTS ^  19Changes to habitat ^  19Trappability  19Total population  22Recruitment  26Reproduction ^  29Probability of survival  32Juvenile survival  32Average adult weight  32Juvenile growth rate ^  38Weight at sexual maturity  38Sex ratio ^  38Species diversity  42Spatial distribution ^  42DISCUSSION ^ 47Experimental design ^  47Density and recruitment  50Growth and survival  51Reproduction ^  51Other studies  52ivCHAPTER 3:Giardia Contamination in Small MammalsINTRODUCTION^ 55METHODS AND MATERIALS ^  561. Field Studies ^  56Establishing initial prevalence  56Giardia prevalence and sludge application ^  572. Laboratory Studies ^  58Collection of deer mice  58Metronidazole treatment  58Enzyme-linked immunosorbent assay (EIA) ^  59Source of cysts ^  59Inoculation of cysts  60Determination of infection ^  61RESULTS ^ 611. Field Studies ^  61Establishing initial prevalence ^  61Prevalence of giardiasis in small mammals ^  632. Laboratory Study ^  63DISCUSSION ^ 65OVERALL THESIS CONCLUSIONS ^  70Implications of study results for small mammal populations ^ 70Implications of Giardia transmission for sludge application  71REFERENCES ^ 73GLOSSARY OF TERMS ^  81vLIST OF TABLESTable 1. Minimum unweighted trappability estimates for the deer mouse, Oregon vole andTownsend chipmunk for three periods on all study sites ^ 20Table 2. Jolly-Seber trappability estimates for the deer mouse, Oregon vole and Townsendchipmunk for three periods on all study sites (+ 95% confidence intervals) .. 21Table 3. Proportion of male adults of three main species in breeding condition (scrotal) duringthe breeding season of each year. Sample sizes in parentheses ^ 30Table 4. Proportion of female adults of three main species in breeding condition (lactating)during the breeding season of each year. Sample sizes in parentheses ^ 31Table 5. Jolly-Seber average survival rates per period (1990-1991) for control and treatmentpopulations of deer mice (± 95% confidence intervals) ^ 33Table 6. Jolly-Seber average survival rates per period (1990-1991) for control and treatmentpopulations of Oregon voles (+ 95 % confidence intervals) ^ 34Table 7. Minimum survival rates per period for control and treatment populations of juveniledeer mice, Maple Ridge, B.C., Canada ^  36Table 8. Mean instantaneous growth rate (per day) of juvenile deer mice and ANCOVA ofgrowth rate regressed on body weight for the deer mouse. **P< 0.01 ^ 39Table 9. Sex ratio (proportion of males) for three small mammal species in summer 1990 and1991. *P< 0.05; significant difference by chi-square test. Sample sizes inparentheses   41Table 10. Shannon-Wiener indices of diversity and species richness of small mammalcommunities for 1990-1991 ^  43Table 11. Summary of demographic effects of sludge treatment on small mammal populations.Three main species of concern were P. maniculatus, M. oregoni, and E. townsendii.0 =no difference from control population, + =increased relative to control. All threespecies were analyzed unless specified with initials of species investigated . . . 48Table 12. Initial prevalence of Giardia spp. cysts near beaver dams, and on study sites prior to,and after sludge application as estimated by fecal sampling. Giardia spp. cyst contentwas tested by the formal-ether concentration method and microscopic examination ofiron-hematoxylin stained preparations   62viTable 13. Results of fecal and small intestine examinations of two groups of deer miceinoculated with different strains of and varying numbers of Giardia duodenalis^  64viiLIST OF FIGURESFigure 1. Study sites at the University of B.C. Research Forest, Maple Ridge, B.C.Stars denote study areas. Cl =control replicate one, C2 =control replicate 2,Ti =treatment replicate 1, T2 =treatment replicate 2. ^ 10Figure 2. Layout of study sites treated (Ti and T2) with sewage sludge. Blacked-out areaswere treated with sludge in November 1990 ^  18Figure 3. Population density (per ha) of deer mice on treatment and control study sites forreplicates 1 and 2. Vertical arrow indicates when sludge was applied to treatmentareas. Jolly-Seber and MNA values are both shown ^ 23Figure 4. Population density (per ha) of Oregon voles on treatment and control study sites forreplicates 1 and 2. Vertical arrow indicates when sludge was applied to treatmentareas. Jolly-Seber and MNA estimates are both shown   24Figure 5. Population density (per ha) of Townsend chipmunks on treatment and control studysites for replicates 1 and 2. Vertical arrow indicates when sludge was applied totreatment areas. Jolly-Seber and MNA estimates are both shown. Shaded areaindicates hibernation period   25Figure 6. Cumulative number of recruits (per ha) of the three major small mammal specieson all study sites during pre-sludge (unshaded) and post-sludge (shaded) periodsduring summer (1990-1991) periods, Maple Ridge, B.C., Canada   27Figure 7. Recruits of the three major small mammal species (per ha) in control (unshaded) andtreatment (shaded) populations per trapping period during summer (1990-1991)and winter (1990) periods, Maple Ridge, B.C., Canada. Arrow indicates applicationof sludge on treatment areas. ^  28Figure 8. Early juvenile survival of deer mice and Oregon voles. Index is the proportion ofjuvenile animals observed over the expected number of juvenile animals, MapleRidge, B.C., Canada., 1990-1991. Sample size (expected number of juveniles) inparentheses   35Figure 9. Mean body weights and 95% confidence limits of males and females of the threemajor small mammal species on control and treatment sites during summer andwinter periods. Sample sizes are above upper confidence limits ^ 37Figure 10. Distribution of juveniles by weight and sexual maturity for deer mice on controland treatment 1 study sites, Maple Ridge, B.C., Canada ^ 40viiiFigure 11. Species composition of study sites for summer and winter periods, Maple Ridge,B.C., Canada ^  44Figure 12. Distribution of deer mice captures on treatment sites before and after sludgetreatment. Actual area sludged was blackened on each site, Maple Ridge, B.C.,Canada ^  45a-bFigure 13. Relative number of deer mice captures in sludged areas on treatment grids(or equivalent areas sludged on control areas), as a proportion of total captures,prior to and after treatment, Maple Ridge, B.C., Canada ^ 46ixACKNOWLEDGEMENTSThere are many people who I wish to thank for helping me to complete this thesis.Firstly, I would like to thank my supervisor, Dr. Tom Sullivan, who in addition to providingmany hours of guidance and supervision, also gave me patience and understanding, especiallyduring times of need. Dr. Judy Isaac-Renton, in addition to being on my committee, spent manyhours with me organizing our project on Giardia, and offered academic and moral support.Dr. James P. Kimmins also served on my committee and I thank him for the time and valuableinformation that were given to me. Staff at the University of British Columbia Research Forestallowed me the use of their facilities and were cheerfully helpful throughout the sometime roughrunning of the study. I am also grateful for the much appreciated help of Markus Merkens inthe field. In the laboratory, I would like to thank Debra Hay, Iden Khan and Lorraine Lewinfor their expert assistance and counsel. Financial support for this project was provided by theGreater Vancouver Regional District (J. P. Kimmins and J. Isaac-Renton) and the NaturalSciences and Engineering Research Council of Canada (T. P. Sullivan).Students of the forestry-wildlife group at U.B.C. provided a supportive and stimulatingenvironment where many informal discussions generated valuable information and friendship.I owe special thanks to Markus Merkens and Todd Zimmerling for their competent assistancein dealing with computer and statistical problems, and many interesting debates.Special appreciation goes to Lois Campbell, Gabriella Matscha, Winston and Kelly Mewfor moral support.CHAPTER 1:General IntroductionINTRODUCTIONThe future disposal of sewage sludge has become a problem of major concern. Disposalof sludge into landfills is not only becoming unacceptable to the public with the recent increasein environmental awareness, but it is also prohibitively expensive in terms of transportationcosts. This problem is expected to escalate because suitable disposal sites are becoming scarceand distant from city centres. Over 54.4 dry tonnes of sewage sludge is produced daily in theGreater Vancouver region. This figure is expected to double to 108.8 dry tonnes/day within thenext decade (Kimmins et al. 1992). At the same time, existing waste water treatment plants inthe Greater Vancouver region are facing shortages in storage capacity or are requiring majorservicing of their equipment. Currently, the G.V.R.D. is reviewing six options to sewagedisposal: agricultural land application, top soil production and composting, landfill cover andgravel pit reclamation, silviculture, mine reclamation and rangeland improvement.Five constituents in sludge are considered in determining the end usage of sludge. Theseare organic content, nutrients, pathogens, trace metals and toxic organic chemicals/syntheticorganics. The organic content in sludge contains numerous essential nutrients necessary forplant growth, the most notable ones being nitrogen, phosphorus and potassium. Unfortunately,sludge also contains pathogens, a significant number of which can become concentrated in sludgeduring the wastewater treatment process. Thermophilic digestion or irradiation can destroy alarge portion of these pathogens. However, since most sludges do not contain enough of thesepathogens to warrant great concern, this process is not often considered due to its high cost.1Trace metals and inorganic ions such as arsenic, boron, cadmium, copper, lead, nickel,mercury, silver, and zinc are also constituents of sludge. In small concentrations, these canserve as essential micronutrients. In high concentrations, they may be toxic to humans, plantsand animals (Zasoski 1980, West et al. 1981).Silvicultural sludge applications are currently being considered for a number of reasons.This option has the potential to both alleviate the problem of sewage disposal, as well asreplenish lost nutrients in forest soils which have become nutrient deficient from a history ofrepeated whole-tree harvesting or poor forestry practices. Furthermore, using sludge as asilvicultural fertilizer minimizes human health hazards because timber, unlike agriculturalproduce, does not enter directly into the human food chain. However, this option has receivedmixed reviews. Major concerns include potential deleterious effects on wildlife, heavy metalcontamination in both surface or ground waters, and introduction of parasites or pathogens intothe ecosystem.Research from Washington State's Pack Forest in recent years has demonstratedsignificant responses in tree growth on some sites. In general, no 'unacceptable' environmentalconsequences were found (Cole et al. 1986, Henry 1989) , although the majority of past studieson the effects of sludge on wildlife considered only animal abundance and heavy metalaccumulation. The term 'heavy metal' does not refer specifically to high atomic weight metals.It encompasses metals with a lower atomic weight, transition metals and some truly heavy metals(such as mercury and lead). In general, heavy metals, such as copper, zinc, manganese andiron, are considered essential plant nutrients. However, they can easily exceed plantrequirements and become a problem of metal toxicity with heavy applications of sludge (Zasoski21980). Consequently, concern arises from the high metal content found in many sludges.In light of this, the Greater Vancouver Regional District (G. V.R.D.) and the Universityof British Columbia (U.B.C.) have collaborated in a large-scale research project investigatingthe potential environmental and health-related impacts of sewage sludge as a silviculturalfertilizer.Relative to other management practices, silvicultural sludge applications are not unlikethe application of inorganic fertilizers; the main differences being the amount of water that isapplied with sludge, the inability to control its exact contents and the perception of human wasteproducts as unpleasant and unsanitary. Relative to other forest management techniques, sewagesludge applications are considered to impact the ecosystem to a much smaller degree than apractice such as clearcutting, for example, which alters the microclimate of the entire site inremoving its canopy cover. Relative to other global environmental risks, such as the climatechange that is speculated to occur in the future from the increased release of greenhouse gases,it is also considered to be a very minor disturbance. More importantly, despite the large bodyof evidence supporting global warming, it is a risk that society has decided to accept. Modernrisk assessment appears to be purely subjective at times. The final decision regardingoperational silvicultural sludge applications will likely be made on a political or social ratherthan a scientific level. This study looks specifically at the potential impacts on small mammalpopulations from sludge applications and provides scientific data only to base decisions on therisks that may be taken with this group of wildlife.3LITERATURE REVIEW: EFFECTS OF SLUDGE ON WILDLIFEPast studies on the effects of sewage sludge applications on wildlife focused on five majorgroups: ungulates, small mammals (heavy metal accumulation in insectivores was alsoconsidered), birds, amphibians and reptiles. A summary of the findings on mammals isdiscussed below.UngulatesStudies on deer (Odocoileus hemionus columbianus) have found an increase in both timespent on sludged areas and fawn recruitment on a seasonal basis. Fawn survival, however, wasnot recorded. These differences may be in response to the increase in the amount of crudeprotein in key forage for deer as a result of sludge treatment (Anderson 1983, Anderson 1985,West et al. 1981). Energetically stressed deer, such as nursing does, could benefit from anincrease in forage energy. One study examining the accumulation of heavy metals in deer foundno significant accumulation in major deer repository organs (Anderson 1981).BirdsSignificant effects of sludge on bird abundance have not been reported. Some heavymetal concentrations were found to increase in some bird species as well as in certaininvertebrate species which may be prey items (Milner 1986, West and Zasoski 1986). Ingeneral, the risk of heavy metal toxicity was considered small due to the high mobility of birdsand relatively small size of sludged areas.4Small mammalsWest et al. (1981) found that of the small mammal species they studied, abundance ofinsectivores (shrews) and gramnivores did not differ between sludged and control sites, butabundance of herbivorous small mammals was significantly lower on treatment compared tocontrol sites. One study investigated the possibility of the transfer of sludge-borne parasites,specifically the nematode Toxocara canis, whose egg form is resistant to severe environmentalconditions and most likely to survive sludge applications. Small mammals did not appear to takeup eggs of this nematode parasite, although it was not clear whether the eggs of this parasitewere abundant in the sludge initially (Raedeke and West 1986).Heavy metal studies found increased accumulations of cadmium and lead in the kidneysand livers of two species of insectivores and an omnivore/gramnivore mammal'. Althoughmetal levels were too low to be considered physiologically harmful, this may become aconsideration when sludge treatments are re-applied periodically over an extended period oftime. Examination of a shrew population found no conclusive evidence of population declinedespite increased metal accumulations (Hegstrom 1986).'Heavy metals introduced into ecosystems in sludge, unlike pathogens and parasites, are notirreversibly fixed in the sludge matrix. Certain heavy metals can be taken up by plants as partof their metabolism (Taber and Zasoski 1980).5SMALL MAMMAL STUDY OBJECTIVESThere are two major objectives in this study: 1) to determine the effects of a silviculturalsludge application on the population dynamics of resident small mammal populations, and2) to evaluate the potential for Giardia contamination in a forest ecosystem. The ability of smallmammals, specifically the deer mouse (Peromyscus maniculatus), to act as a host to humanGiardia cysts, was investigated. The prevalence of Giardia spp. cysts in small mammal fecalsamples at study sites prior to and after sludge application was also monitored.6CHAPTER 2Impacts of a Silvicultural Sludge Applicationon Small Mammal PopulationsINTRODUCTIONSmall mammals were selected as the wildlife group of study for a number of reasons.Firstly, due to their abundance and ecology, they come into direct contact with sludge more thanany other group of birds or mammals. Silvicultural sludge applications are often applied at theforest floor level. Birds and arboreal mammals are further removed from contact with sludgethan small mammals which nest and feed on the forest floor directly. Small mammals, beinga numerically abundant group, can be sampled in larger numbers on a given area and aretherefore more suitable for statistical analysis. Sample sizes in studies of larger mammals, suchas beavers (Castor canadensis) or deer, are usually much smaller, given the size of study sitesand similar funding and time limitations. For these reasons, small mammals were selected asa 'sentry' wildlife group to determine the effects of silvicultural sludge applications on theirpopulation dynamics. Past small mammal studies have dealt mostly with the accumulation ofheavy metals inside livers and kidneys of specimens, but not with the short-term response ofdynamic population parameters which was a focus of this study. Consequently, live-trappingwas selected as the sampling technique so dynamic population parameters could be estimated.The second reason for studying small mammals centers around the possibility thatcontaminated sewage sludge may provide an entry path for pathogens and parasites into the7forest ecosystem and ultimately into human populations. Giardia was selected as the parasiteof interest due to its waterborne and fecal-oral modes of transmission. Giardia cysts are hardyand will survive in cold water (e.g. in streams) for up to two months (Bingham et al. 1978).The potential for giardiasis contamination through small mammals picking up cysts in their dailyactivities or beavers ingesting cysts in contaminated streams were two possibilities that neededto be addressed. The potential for small mammals to function as hosts for human Giardia is asyet undetermined, but past studies have suggested it as a possibility (Davies and Hibler 1978,Pacha et al. 1987, Roach and Wallis 1988).MATERIALS AND METHODSDescription of study areasThis livetrapping study was located at the U.B.C. Malcolm Knapp Research Forest nearMaple Ridge, B.C., Canada. The treatment study area (13 ha) was clear-cut in 1973,slashburned in the fall of 1974 and planted with Douglas-fir (Pseudotsuga menziesii) in thespring of 1975. Two 1-ha sites within this area were treated with sewage sludge. Treatment 1was located on a dry upper slope, treatment 2 at a lower, mid-slope site and spacedapproximately 250 m from treatment 1. The area was previously covered with a mature (70-90-year-old) forest dominated by western hemlock (Tsuga heterophylla), Douglas-fir and westernred cedar (Thuja plicata) (Feller 1977). Cover included well decomposed logging slash with anabundance of deciduous trees, shrubs and herbaceous vegetation. Associated with the Douglas-fir stand were western hemlock natural regeneration and shrub species such as red alder (Alnus8rubra), birch (Betula papyrifera), willow (Salix spp.), vine maple (Acer circinatum), blackraspberry (Rubus leucodermis) and salmonberry (Rubus spectabilis). Bracken fern (Pteridiumaquilinum) and fireweed (Epilobiumangustifolium) were the dominant herbaceous annual species.The control study area (15 ha) was clear-cut in 1973 and planted with Douglas-fir in1975. This area was not burned. The previous forest was also dominated by mature westernhemlock and mixed with Douglas-fir and western red cedar. The main cover was welldecomposed slash with a similar vegetation composition to the treatment site. Two control siteswere situated at similar elevations as the treatment area sites and were also 250 m apart.Treatment and control study areas were separated by approximately 500 m, which was asufficient distance to limit small mammal dispersal between the two areas. All sites wereconsidered mesic and located in the Coastal Western Hemlock biogeoclimatic zone (CWH d„,)(Meidinger and Pojar 1991) between 180 to 250 m in elevation (Fig. 1).Livetrapping protocolFrom May 1990 to October 1991, two treatment and two control grids were livetrappedat 3-week (spring, summer, and fall) and 6- to 8-week (winter) intervals with Longworth livetraps. On each grid, 49 (7 x 7) trap stations were located at 14.3-m intervals with one live trapplaced within a 2-m radius of each station. Traps were baited with oats and a slice of carrot formoisture, and coarse brown cotton for bedding. Traps were set on day 1, checked on days 2and 3, and then locked open between trapping periods.All small mammals captured, except shrews and weasels, were ear-tagged with fingerlingfish tags, sexed, reproductive condition noted, and weighed on Pesola spring balances. The9Fig. 1. Study sites at the .University of B.C. Research Forest,Maple Ridge, B.C.. Stars denote study sites. Cl =control replicateone, C2 =control replicate two, T1 =treatment replicate 1,T2 = treatment replicate two.10duration of the breeding season was noted by palpation of male testes and the condition ofmammaries of the females (Krebs et al. 1969). Small mammals were released immediately afterprocessing. All shrews (Sorex spp.) which died in the traps due to the overnight-trappingtechnique were recorded as to their station of capture, collected and frozen for later identificationof species using their dentition as the major classification criterion.Small mammal species encountered include the deer mouse, Oregon vole (Microtusoregoni), red-backed vole (Clethrionomys gapperi), Townsend chipmunk (Eutamias townsendii),Pacific jumping mouse (Zapus trinotatus), American shrew-mole (Neurotrichus gibbsii), and twospecies of shrews (Sorex vagrans, and S. monticolus), and weasels (Mustela spp.). Three majorsmall mammal species were caught consistently throughout the study: the deer mouse, Oregonvole and Townsend chipmunk.Population parametersPopulation parameters investigated in this study were population density, recruitment,proportion of population in breeding condition, probability of survival, juvenile survival, growthparameters (including mean adult body weight, juvenile growth rate and weight at sexualmaturity), sex ratio, spatial distribution and species diversity. Most of the population parametersstudied were estimated for three periods: summer 1990 (May - October), winter 1990-1991(November - April), and summer 1991 (May - October) to minimize small scale temporalvariations.11Population density and recruitmentDue to variation in trappability of small mammal species, population densities wereestimated by minimum number alive (MNA) (Krebs 1966), as well as the Jolly-Seber (J/S)method (Seber 1982). Enumeration by MNA is an accurate measure when trappabilities are high( > 80%) (Hilborn et al. 1976), whereas J/S estimates are more accurate when trappabilities arelow (Jolly and Dickson 1983). Density values were estimated for each trapping period for thethree numerically dominant small mammal species; the deer mouse, Oregon vole and Townsendchipmunk.Recruitment, the number of new animals joining the population, was calculated fromobserved numbers as a cumulative value for two main periods: pre-sludge (summer 1990) andpost-sludge (summer 1991). Recruitment per trapping period (J/S estimates) on all study sitesprior to (summer 1990) and after (summer 1991) sludge application was also compared.The winter of 1990-91 was omitted from this analysis as no equivalent period in 1991 wasavailable for comparison.ReproductionNumbers of males and females reproductively active (i.e., scrotal or lactating) wereobtained from field data with each animal being tallied at each capture. These values were thensummed and calculated as a proportion of the total number of animals caught during that period.Survival and growthAverage survival rates (J/S) for males and females in control and treatment populations12of both the deer mouse and Oregon vole were calculated. Survival values for chipmunksexhibited large variation in J/S values, and therefore were not used in this analysis. All survivalvalues were calculated for three periods (summer 1990, winter 1990-91, summer 1991) with anindividual animal being tallied at each capture.Juvenile survival was calculated by two methods. Early juvenile survival, defined as thepercentage of observed juveniles/expected number of juveniles, was calculated for the deermouse and Oregon vole (low sample size for Townsend chipmunk). Expected number ofjuveniles was the product of the number of successful pregnancies (based on consecutive capturesof lactating females) and the average number of juveniles per litter {as recorded in literature:4.5/litter for the deer mouse (Sheppe 1963, Sadleir 1974): 3.2/litter for the Oregon vole(Gashwiler 1972): 4.0/litter for the Townsend chipmunk (Tevis 1955, Gashwiler 1976)}. Theobserved number of juveniles was obtained from field data using weight as the definingparameter (deer mouse: < 17 g; Oregon vole: < 20 g; Townsend chipmunk < 75 g) (Sullivan1990b, Sullivan et al. 1983).Minimum juvenile survival values were summed over each period with an individualanimal being tallied each time it was captured. Only the deer mice had enough juveniles for thisanalysis.Body weightsAverage adult weights for the three major species were calculated for the three periods.Adults were separated from juveniles by their weight and reproductive condition(deer mouse: .^  17 g; Oregon vole: > 20 g; Townsend chipmunk: ^ 75 g) (Sullivan 1990b,13Sullivan et al. 1983). Only males were used during the summer seasons because female datawere complicated by undetected pregnancies.Growth analysisGrowth rates were considered as an indicator of condition in small mammals.Instantaneous growth rates (per day) were calculated for all juvenile animals (males and females)for periods 1 and 3 when young animals were actively growing. Juveniles from treatment andcontrol areas showed similar growth rates to each other (i.e., treatment 1 and 2, control 1 and2) prior to and after sludge treatment and justified the pooling of juvenile growth rates for thisanalysis. As growth rate is dependent on body weight, an ANCOVA of growth rate regressedon body weight was done for juvenile deer mice. Temporal (pre vs. post-sludge periods) andspatial (treatment vs. control sites) comparisons were then made.Only deer mice had a sufficient sample size for this analysis. Smaller sample sizes ofOregon voles and Townsend chipmunks are not suited to this analysis due to the variability indata which would have masked any significant differences.Weight at sexual maturity of deer mice was also graphed for all study sites, pre- andpost-sludge treatment. Juveniles from 11-16 grams were categorized as to their breedingcondition (scrotal or abdominal, lactating or non-lactating) for all study areas for the summerperiods. Minimum weights at sexual maturity were then compared spatially and temporally.Sex ratioSex ratio (proportion of males to total population) was calculated using MNA values.14Individual animals were tallied at each capture and summed over each period. For reasons thatare discussed in the statistical analysis section, values were compared using the chi-square test.Species diversity and richnessThe Shannon-Wiener index of diversity (Pielou 1966) was used in this study to considerboth evenness and species richness in small mammal communities. The availability of a t-test(Magurran 1988) allowed community indices to be compared. This index is also sensitive tochanges in rare species (Peet 1974). The assumptions of this index are that individuals arerandomly sampled from an infinite population (i.e., an open versus a closed population) and allsmall mammal species were captured in the sampling; which holds true for this study.Values for the calculation of Shannon-Wiener indices were obtained from J/S estimatesfor the three main species. Actual observed numbers from field data were used for lessabundant species due to a lack of J/S estimates. Rarer species included shrews (Sorex spp.),weasels (Mustela spp.), red-backed voles, jumping mice and shrew-moles. As only dead shrewswere identified, these samples were used in the calculation (the percentage of live shrews rangedfrom 33-50% of actual number of shrews caught). This method was considered acceptablebecause the proportion of live shrews caught was relatively similar on all study sites.Both spatial (treatment vs. control) and temporal (pre- vs. post-sludge) comparisons weremade between indices. Species richness was measured by the total number of species sampled(Krebs 1989).One limitation of Shannon-Wiener diversity indices is that subtle changes in speciescomposition may occur even though overall diversity does not change (i.e., the relative15abundances of species in a community may change, but if the overall ratio of species remain thesame, these changes will not be detected in the diversity index). To reinforce this diversityanalysis, the relative cumulative proportions of species captured were also estimated for all sitesbefore and after sludge application.Spatial distributionThe effect of sludge application on the spatial distribution of deer mice was studied bycomparing the relative proportion of deer mice captures in the sludged area inside each grid tothat of the total number of captures on the study grid. These values were then comparedbetween pre-sludge and post-sludge periods. However, duration of the periods used forcomparison varied between replicates. In treatment 1, a total of 8 trapping periods prior to andafter sludge applications was considered in the calculation. In treatment 2, only a total of 4trapping periods was used. This was due to bear disturbance on study sites which resulted ina reduced number of operable live traps on certain sites at various times. The total number ofoperable traps was kept constant for the comparisons, hence the variation in temporal duration.Statistical analysisStandard statistical analysis was inappropriate for many comparisons because the analysesinvolved samples of the same animals captured in consecutive trapping periods. The chi-squaretest, based on the null hypothesis of independence, was used to compare frequencies (Zar 1984)as samples were not strictly independent (Hurlbert 1984). This test was used in place of ananalysis of variance because it is a more robust test when deviations from the basic assumptions16occur. Consequently, test results were only an indication of the degree of difference betweensets of data and may not be statistically valid. This was the case for proportion of populationbreeding, survival, average adult weight, weight at sexual maturity and sex ratio. The one-tailedFisher Exact test was used for the analysis of the proportion of population reproducing in theTownsend chipmunk due to low sample size (Zar 1984).Sludge applicationAnaerobically-digested sludge was obtained from the Annacis Island Sewage TreatmentPlant in Delta, B.C.. Truckloads of sludge were transported to the Research Forest and storedin a waterproof Smithrite container located as close to the actual application site as possible.Sludge was protected from precipitation dilution by a plastic cover. Prior to application, atractor fitted with an agitator (propeller on a long extension) mixed the sludge to a uniformconsistency. This 'slurry' was then pumped into trash hoses (10 cm diameter) and onto the site.The total area treated was 3.58 hectares (Fig. 2). Sludge application only partially coveredtreatment grids. The actual areas sludged are marked in Figure 12 as part of the spatialdistribution analysis. The total amount of sludge applied was 173,823 kg (22% solids) of whichthe average nitrogen content was 3.9%. Variation between applications was monitored byperiodic collection of sludge samples and analysis for nitrogen content. An estimated averageof approximately 500 kg N/ha was applied to sites (Kimmins et al. 1992). Sludge applicationbegan on November 25 and was completed on November 30, 1990.17Fig. 2. Layout of study sites treated (T1 and T2)with sewage sludge. Blacked-out areas weretreated with sludged in November 1990.18RESULTSChanges to habitatEffects of sludge application on vegetation were studied on the treatment study areasconcurrently with this study. Changes in parameters such as percent cover, height, light, andfoliage nitrogen and phosphorus content were monitored. Significant increases in nitrogen andphosphorus content were found in salal leaves (Gaultheria shallon) on treatment areas comparedto control areas. In addition, relative percent cover decreased for salal but increased for brackenfern in the post-sludge year (L. Coward, pers. comm.) 2 .TrappabilityThe enumeration of small mammals is based on the assumption that most individuals arecaptured in a given population. Estimates of trappability used in this study were the minimumunweighted and J/S calculations (Krebs and Boonstra 1984). Minimum unweighted trappability(MUT) eliminates first and last captures, and hence all animals caught only once or twice.Jolly-Seber trappability (JST) was also calculated and most parameters in this study were basedon these estimates. Both trappabilities were generally high for deer mice (MUT:73-82%,JST:71-91%) but variable for Oregon voles (MUT:40-79% , JST:57-77%) and chipmunks(MUT:10-75%, JST:33-57%) (Tables 1 and 2). No difference in trappability was noted betweenfemale and male deer mice. Oregon vole and chipmunk values were too variable for patternsto be discerned.2Laura Coward, M. Sc. candidate in Forest Sciences. Thesis topic was on the effects onvegetation from a silvicultural sludge application, Department of Forest Sciences, University ofBritish Columbia, Vancouver, B.C. Canada.19Table 1. Minimum unweighted trappability estimates for the deer mouse, Oregon vole and Townsend chipmunk for threeperiods on all study sites. Sample sizes in parentheses. Minimum unweighted trappability eliminates first and last captures andprovides only one value for each individual regardless of how long it lives.Control 1 Control 2 Treatment 1 Treatment 2P. maniculatus Males Females Males Females Males Females Males FemalesSummer 1990 0.79 (22) 0.74 (27) 0.83 (9) 0.74 (10) 0.86 (17) 0.88 (20) 0.78 (19) 0.65 (20)Winter 1990-91 0.96 (11) 0.91 (13) 0.89 (6) 1.00 (3) 0.64 (12) 0.80 (8) 0.70 (10) 0.85 (10)Summer 1991 0.78 (3) 0.76 (7) 1.00 (3) 0.92 (2) 0.68 (8) 0.61 (8) 0.78 (9) 0.86 (6)M. oregoniSummer 1990 0.59 (7) 0.46 (13) 0.80 (12) 0.78 (18) 0.37 (3) 0.47 (3) 0.50 (5) 0.76 (5)Winter 1990-91 0.81 (1) 0.33 (2) 0.69 (5) 0.27 (5) 0.80 (1) 0.00 (1) 0.80 (2) 0.33 (2)Summer 1991 0.50 (1) 0.83 (2) 0.50 (2) 0.63 (6) 0.67 (1) 0.20 (1) 1.00 (1) 0.50 (1)E. townsendiiSummer 1990 0.24 (4) 0.20 (2) 0.38 (4) 0.48 (3) 0.57 (8) 0.50 (4) 0.78 (5) 0.67 (5)Winter 1990-91 0.13 (3) 0.00 (1) 0.11 (3) 0.40 (1) 0.10 (6) 0.00 (2) 0.20 (3) 0.08 (5)Summer 1991 0.33 (4) 1.00 (1) 0.50 (1) 1.00 (1) 0.00 (0) 0.12 (3) 0.19 (3) 0.35 (6)Table 2. Jolly-Seber trappability estimates for the deer mouse (Peromyscus maniculatus), Oregon vole (Microtus oregoni) and Townsendchipmunk (Eutamias townsendii) for three periods on all study sites ( ± 95% confidence intervals).PERIOD Control 1 Control 2 Treatment 1 Treatment 2P. maniculatus Males Females Males Females Males Females Males FemalesSummer 1990 0.69 0.71 0.83 0.83 0.78 0.82 0.69 0.75(0.52-0.86) (0.61-0.81) (0.73-0.93) (0.71-0.96) (0.64-0.91) (0.69-0.95) (0.53-0.84) (0.64-.86)Winter 1990-91 0.89 0.84 0.84 1.00 0.79 0.82 0.73 0.78(0.63-1.00) (0.65-1.00) (0.56-0.00) (1.00-0.00) (0.58-1.00) (0.62-1.00) (0.44-1.00) (0.61-0.96)Summer 1991 0.68 0.81 0.86 0.86 0.73 0.78 0.83 0.81(0.45-0.92) (0.64-0.99) (0.51-1.00) (0.36-1.00) (0.36-1.00) (0.45-1.00) (0.69-0.96) (0.62-1.00)M. oregoniSummer 1990 0.80 0.61 0.90 0.75 0.26 0.56 0.59 0.80(0.59-1.00) (0.44-0.77) (0.80-1.00) (0.54-0.96) (0.05-0.47) (0.15-0.96) (0.34-0.84) (0.60-1.00)t)0--Winter 1990-91 0.80(0.24-1.00)0.41(0.00-0.93)0.73(0.34-1.00)0.50(0.00-1.00)0.80(0.24-1.00)0.25(0.00-0.79)0.72(0.23-1.00)0.74(0.30-1.00)Summer 1991 0.64 0.86 0.78 0.78 0.71 0.43 0.43 0.61(0.20-1.00) (0.63-1.00) (0.44-1.00) (0.56-1.00) (0.26-1.00) (0.00-0.92) (0.00-0.92) (0.15-1.00))E. townsendiiSummer 1990 0.38 0.56 0.47 0.59 0.56 0.48 0.80 0.65(0.13-0.62) (0.15-0.96) (0.31-0.64) (0.26-0.92) (0.31-0.81) (0.27-0.69) (0.57-1.00) (0.36-0.95)Winter 1990-91 0.15 0.20 0.47 0.40 0.15 0.00 0.33 0.15(0.00-0.51) (0.00-0.76) (0.00-1.00) (0.00-1.00) (0.00-0.37) (0.00-0.00)) (0.00-0.70) (0.00-0.42))Summer-1991 0.29 0.43 0.57 0.43 0.29 0.33 0.41 0.44(0.00-0.64) (0.00-0.93) (0.07-1.00) (0.00-0.93) (0.00-0.74) (0.00-0.68) (0.07-0.75) (0.27-0.61))Total populationPre-sludge application period (Summer 1990)Population density estimates calculated by MNA and Jolly-Seber exhibited comparablefluctuations over time for all three species (Figs. 3-5). For this reason, these estimates areassumed to be reasonably accurate in reflecting the numerical responses to treatment for thesethree small mammal species.Population density fluctuations of deer mice on all study sites were similar for thesummer of 1990 (May-August). All populations had an initial density of approximately 15 deermice/ha (J/S). With the onset of breeding, populations experienced a significant increase. InSeptember 1990, however, the control population in replicate 1 increased but the replicate 2control population declined (Fig. 3).Oregon vole populations increased steadily through the late summer and fall of 1990 to22/ha on control site 1 and 28/ha on control site 2 (J/S) (Fig. 4). Both treatment volepopulations appeared to decrease marginally in September from initial densities of approximately4-8 voles/ha (J/S).Chipmunk densities increased gradually due to juvenile recruitment over the summer(Fig. 5). Treatment chipmunk populations sustained a higher population (11-15/ha) than controlpopulations (7-8/ha) (J/S), particularly in the late summer-fall period (September-November1990) (Fig. 5).22T^T^I^I^I-^-I-1990M S 0Time 19910Perom yscus manicula tus7060501:1'10 40s:1O300 20C:1410I^1^■ 1^I^,^I^I^,^1M^3^3^S^0^D^M M^3^J^1990 1991TimeFig. 3. Population density (per ha) of deer mice on treatment (—) andcontrol (-) study sites for replicates 1 and 2. Vertical arrow indicateswhen sludge was applied to treatment areas. Jolly-Seber (q) and MNA(+) values are both shown.S23STAIIVII^1111D M M J^JTime^ 19910M^J^J^S1990r0IIIIIIIII1 -111-^I-IMicro tus oregoniM^J^J^S^0 D M M J^J S^0^1990 Time^ 1991Fig. 4. Population density (per ha) of Oregon voles on treatment (—) andcontrol (-) study sites for replicates 1 and 2. Vertical arrow indicateswhen sludge was applied to treatment areas. Jolly-Seber (a) and MNA(+) estimates are both shown.24Eutamias townsenciiiM^J^J^S^0^D M M^J^J^S^01990 Time^1991M^J^J^S^0^D^M^M^J^J^S^0^1990 Time 1991Fig. 5. Population density (per ha) of Townsend chipmunks on treatment (—)and control (-) study sites for replicates 1 and 2. Vertical arrow indicates whensludge was applied to treatment areas. Jolly-Seber (m) and MNA (+) estimatesare both shown. Shaded area indicates hibernation period.25Sludge application period (Winter 1990-1991)Deer mouse and Oregon vole populations did not undergo any changes in abundanceimmediately after sludge treatment (arrow in Figs. 3 and 4). Effects on the Townsend chipmunkwere not detected during their hibernation period. An decrease in abundance in December forthese three species was experienced on all study sites and continued throughout the winterperiod.Post-sludge application period (Summer 1991)A consistent pattern of reduction in abundance in the post-treatment year was found inthe deer mouse and Oregon vole populations on all study sites independent of sludge treatment.Population densities remained low throughout this period. This was illustrated by similarchanges expressed by both estimates of abundance used. Increases in abundance as observedin July 1990 were not repeated. Deer mouse and Oregon vole populations on control site 2decreased to almost zero in July in the post-sludge period and increased marginally over the restof the period. Chipmunk densities in the second year were slightly lower than first yeardensities on all study sites.RecruitmentAll study sites experienced a large decrease (minimum 50%) in both cumulative {totalnumber of recruits/period (field data)} (Fig. 6) and weekly number of recruits in the post-sludgeperiod for all three species (J/S estimates) (Fig. 7). No consistent effects from sludge treatmentwere found. Cumulative recruitment of voles was higher on control than treatment sites in the26TMT 1 CON 1 TMT 2 CON 2Cumlotive number of recruitsCON 1 TMT 2 CON 2Cumulative number of recruits60201011.11TMT 1Peromyscus maniculatusCD Pre-sludge^MI Post-sludgeMicrotus oregoniPre-sludge^Post-sludgeEutamias townsendiiCumulative number of recruitsPre-sludge^Mg Post-sludgeFig. 6. Cumulative number of recruits (per ha) ofthe three major small mammal species on all studysites during pre-sludge (unshaded) and post-sludge(shaded) periods during summer (1990-1991)periods, Maple Ridge, B.C., Canada.27TrIssa.11^C:3 6.0.11CD cO.rvai2520 -151 01190 19911 J1110At^J1111Eutamias townsendii3020 -1 0Ip11, 11 P,NU„S 0Pig. 7. Recruits of the three major small mammal species (per ha) in control (unshaded) and treatment (shaded)populations per trapping period during summer (1990-1991) and winter (1990-91) periods, Maple Ridge, B.C.,Canada. Arrow indicates application of sludge on treatment areas.20IS1 01110S^0 D M M J^J^SO1991Peromyscus maniculatusTreaomeol I^CDMicrotus oregoni11111 Truamew I^C.1 C..0.11 I1M^.1^J^S1990(^111 M^I^11111M 5^0PEI^3^JM^J^JISSOJS^0^D^14MJISO106201 0first year. A notable decrease occurred for the Oregon vole on control sites from the pre-sludgeto post-sludge period. Chipmunk recruitment followed a similar pattern to the other two speciesin having a generally higher recruitment in the first year than the second. However, smallersample sizes reflected a small degree of difference (2-4 chipmunks/ha) (Fig. 6).Deer mouse recruitment on a trapping week basis was variable. Two periods of highrecruitment (J/S estimates) prior to sludge application were found: on control site 1 andtreatment site 2. This increase was not repeated on any other site in the post-treatment year.Periods of high recruitment were also observed in the Oregon vole population, occurring duringperiods of low deer mouse recruitment (Fig. 7) in this period. Contrary to the other twospecies, periods of high recruitment in the Townsend chipmunk were repeated in the secondyear, although to varying degrees on the study sites.ReproductionNo significant differences were found for the Oregon vole in spatial (treatment vs.control) and temporal (pre- vs. post-sludge) comparisons in the proportion of animals in breedingcondition. However, this parameter was found to have increased significantly (P< 0.01) ontreatment site 1 for both male deer mice and Townsend chipmunks (low sample size) betweenpre- and post-sludge periods. This increase was not observed on control sites (Table 3). Dueto the small sample size, however, it was concluded to be attributed solely to the sludgetreatment.Significant increases in the proportion of females in breeding condition (i.e. lactating)were found for deer mice in post-sludge periods for all four populations (Table 4).29Table 3. Proportion of male adults of three main species in breeding condition (scrotal) during thebreeding season of each year. Sample sizes in parentheses. Replicate 1^ Replicate 2Control 1^Treatment 1^Control 2^Treatment 2P. maniculatus1990^0.41 (104)^0.33 (95)a^0.36 (69)^0.35 (92)1991 0.42 (31) 0.60 (45)a 0.40 (25) 0.33 (51)M. oregoni1990^0.30 (37)^0.67 (9)^0.34 (59)^0.67 (27)oL.)^1991 0.27 (11) 0.83 (6) 0.35 (23) 1.00 (4)E. townsendii1990^0.12 (17)^0.15 (34)b^0.37 (19)^0.07 (27)1991 0.00 (10) 0.75 (4) b 0.00 (7) 0.13 (8)a-a, P< 0.01; values are significantly different by chi-square.b-b, P< 0.01; values are significantly different by the one-tailed Fisher Exact test.1990=pre-sludge period, 1991=post-sludge period.Table 4. Proportion of female adults of three main species in breeding condition (lactating) during thebreeding season of each year. Sample sizes in parentheses.Replicate 1^ Replicate 2Control 1^Treatment 1^Control 2^Treatment 2P. maniculatus1990 0.33 (49)a 0.27 (52)b 0.15 (39)c 0.29 (48)d1991 0.92 (26)a 0.65 (31)b 0.60 (10)c 0.77 (22)dM. oregoni1990 0.53 (30) 0.67 (6) 0.32 (19) 0.40 (15)1991 0.67 (3) 0.33 (3) 0.86 (14) 0.60 (5)E. townsendii1990 0.00 (9) 0.00 (7) 0.46 (13) 0.05 (21)1991 0.40 (5) 0.33 (3) 1.00 (2) 0.29 (14)a-a, b-b, c-c, d-d, P< 0.01; values followed by the same letter are significantly different by chi-square.1990 =pre-sludge period, 1991 =post-sludge period.Probability of survivalNo significant changes in spatial and temporal comparisons for either sex of deer miceor Oregon voles (± 95% CI, Tables 5 and 6) were found. Estimates of Townsend chipmunksurvival values were too variable for analysis.Juvenile survivalConsistent with other results, percentage juvenile survival for deer mice decreasedsignificantly in the second year on all study sites (Fig. 8). Total number of expected juveniles,however, increased for all sites. Juvenile survival for the Oregon vole showed an increase ontreatment site 1, but a decrease on treatment site 2 (Fig. 8). Both control sites experienced adecrease. Sample sizes for the Townsend chipmunk were too low for evaluation of thisparameter.Mean minimum juvenile survival rates were also calculated for deer mice (sample sizetoo low for Oregon vole and Townsend chipmunk) (Table 7). Juvenile survival decreased onall sites in general agreement with previous percentage juvenile survival values (Fig. 8) excepton control site 2 where it increased. This may be an artifact of small sample size. Noconsistent effects could be found attributable to sludge treatment.Average adult weightAverage male and female adult weights were found to be not significantly different(based on 95 % confidence intervals) between control and treatment populations in temporal andspatial comparisons for the deer mouse, Oregon vole, and Townsend chipmunk (Fig. 9).32Table 5. Jolly-Seber average survival rates per period (1990-1991) for control and treatment populationsof deer mice (± 95% confidence intervals).Season Females MalesControl Treatment Control TreatmentReplicate 1Summer 1990 0.86 (0.77-0.94) 0.87 (0.79-0.95) 0.75 (0.66-0.85) 0.78 (0.73-0.83)Winter 1990-1991 0.73 (0.62-0.83) 0.73 (0.66-0.88) 0.53 (0.38-0.69) 0.64 (0.73-0.87)Summer 1991 0.77 (0.52-1.00) 0.61 (0.37-1.00) 0.75 (0.52-0.99) 0.56 (0.23-1.00)Replicate 2Summer 1990 0.79 (0.69-0.90) 0.85 (0.75-0.95) 0.77 (0.66-0.89) 0.78 (0.66-0.90)Winter 1990-1991 0.80 (0.64-0.96) 0.77 (0.63-0.84) 0.77 (0.62-0.93) 0.80 (0.51-0.76)Summer 1991 0.83 (0.30-1.00) 0.83 (0.41-0.81) 0.49 (0.13-0.86) 0.56 (0.56-0.93)Table 6. Jolly-Seber average survival rates per period (1990-1991) for control and treatment populationsof Oregon voles (± 95% confidence intervals).Season^Females^ MalesControl^Treatment^Control^TreatmentReplicate 1Summer 1990^0.72 (0.52-0.92)^0.70 (0.23-1.00)^0.45 (0.23-0.68)^0.94 (0.08-0.83)(,)4=•^Winter 1990-1991^0.76 (0.44-1.00)^0.79 (0.66-1.00)^0.77 (0.45-1.00)^0.69 (0.36-1.00)Summer 1991^0.83 (0.52-1.00)^1.00 (0.00-1.00)^1.00 (0.26-1.00)^1.00 (0.26-1.00)Replicate 2Summer 1990^0.78 (0.71-0.90) 0.77 (0.48-1.00) 0.70 (0.23-0.68) 0.70 (0.23-1.00)^Winter 1990-1991 0.68 (0.00-1.00)^0.61 (0.00-1.00) 0.65 (0.45-1.00) 0.66 (0.00-1.00)Summer 1991^0.65 (0.20-1.00)^1.00 (0.13-1.00)^0.52 (0.26-1.00)^1.00 (0.00-1.00)100158.5)(27)131.51(131TMT 2^CON 2140.51CON 180 -14.1.5)TMT160 -40 -20 -% Juvenile SurvivalPeromyscus maniculatus% Juvenile SurvivalMicro tus oregoniKW132)122.011.4119.1)112.1) (32)112.11TMT1^CON 1^TMT 2^CON 2PRE-SLUDGE^al POST-SLUDGEFig 8. Early juvenile survival of deer mice and Oregon voles. Index is theproportion of juvenile animals observed over the expected number of juvenileanimals, Maple Ridge, B. C., Canada, 1990-91. Sample size (expected numberof juveniles) in parentheses.10080604020035Table 7. Mean minimum survival rates per period for control and treatment populations of juvenile deermice, Maple Ridge, B. C., Canada.Period^Control^Treatment^Control^TreatmentFemales^ MalesReplicate 1 Summer 1990^0.86^(42)^0.85^(34)^0.64^(50)^0.87^(3 1)Winter 1990-91^0.67^(52)^0.62^(21)^0.46^(41)^0.34^(29)a■w^ Summer 1991 0.00^(6)^0.62^(13)^0.20^(10)^0.36^(11)Replicate 2 Summer 1990^0.65^(20)^0.84^(31)^0.70^(23)^0.86^(37)Winter 1990-91^0.50^(12)^0.60^(40)^0.75^(20)^0.56^(32)Summer 1991 0.83^(6)^0.53^(15)^0.36^(11)^0.72^(18)1171I27■^ Control^Trrattnent(51117 1iaal16911211125 )^ Control^• Treatment110151 191139/^ Control^• Treatment^lel^ 151(23119)262524U)Eco 23CI2221209085coEco 8007570Peromyscus maniculatusSummer^Winter^ Summer1990 1990-1991 1991Microtus oregoniSummer^Winter^ Summer1990 1990-1991 1991Eutamias to wnsendiiSummer^Winter^Summer1990 1990-1991 1991Fig. 9. Mean body weight and 95% confidence intervals of malesand females of the three major small mammal species on control(1) and treatment (■) sites during summer and winter periods.Sample sizes are above upper confidence limits.2 120181737Juvenile growth rateA significant difference was found in growth rate of juvenile deer mice in treatmentpopulations after sludge application. Mean instantaneous growth rate (per day) for the post-sludge period was almost three times that of the pre-sludge period (F value s , 181) =11.43,P< 0.01). All other temporal and spatial comparisons were found to be not statisticallysignificant (Table 8).Weight at sexual maturityOnly one small change in minimum weight at sexual maturity was found for all spatialand temporal comparisons for deer mice. Juvenile males on control site 1 in summer 1990began breeding (i.e. scrotal) at 14 grams, whereas breeding males were found at 16 grams insummer 1991 (Fig. 10). All other comparisons found weight at sexual maturity to be similar.Females rarely became sexually mature before 17 grams.Sex RatioNo consistent differences were found in temporal or spatial comparisons for both the deermouse and Oregon vole (chi-square analysis). The pre-sludge sex ratio value for the Townsendchipmunk, however, was found to be significantly higher than the post-sludge value (P< 0.05,Table 9).38Table 8. Mean instantaneous growth rate (per day) of juvenile deer mice perperiod and ANCOVA of growth rate regressed on body weight for the deermouse. **P<0.01.Comparison Mean instantaneousgrowth rate (per day)F-value Probability(X 10)Pre-control vs. 2.7 0.32 0.75Post-control 3.7Pre-treatment vs. 2.3 0.40 0.59Pre-control 2.7Post-treatment vs. 6.1 1.26 0.27Post-control 3.5Pre-treatment vs. 2.3 11.43 0.0009**Post-treatment 6.1200dz50252007c• IS• 1020z0Control 1 NI Abdominal^Scrotal161511^13^14Weight (grams)15 1611^12^13^14Weight (grams)Juveniled deer micePeriod 3Juvenile a deer micePeriod 3202520=d 10zS015012.100dzSJuveniled deer mice^ Juvenile (5 deer micePeriod 1^Period 111^12^13^14^IS^16^ 1 1^12^13^14^15^16Weight (grams) Weight (grams)Fig. 10. Distribution of juveniles by weight and sexual maturity for deer mice on control andtreatment 1 study sites, Maple Ridge, British Columbia, Canada.Table 9. Sex ratio (proportion of males) for three small mammalspecies in summer 1990 and 1991. *P< 0.05; significant difference bychi-square test. Sample sizes in parentheses.Pre-sludge Post-sludgeP. maniculatusTreatment 1 0.43 (220) 0.41 (109)2 0.41 (221) 0.50 (102)Control 1 0.56 (257) 0.55 (78)2 0.45 (155) 0.56 (45)M. oregoniTreatment 1 0.73 (37) 0.67 (18)2 0.38 (71) 0.20 (20)Control 1 0.48 (130) 0.61 (26)2 0.49 (158) 0.52 (56)E. townsendiiTreatment 1 0.82 (74)* 0.59 (29)*2 0.64 (72) 0.62 (55)Control 1 0.80 (46) 0.76 (34)2 0.75 (51) 0.77 (13)Species DiversityDiversity indices of small mammals (calculated for pooled treatment and controlpopulations: i.e. replicates 1 and 2 combined) between treatment and control sites prior to sludgetreatment were similar. Temporal and spatial comparisons found no significant differencesbetween diversity indices on all sites (t-test). Species richness values also were similar betweenpopulations (Table 10).The proportion of each species captured relative to the total number of captures wasrepresented and compared between periods. This analysis was carried out to detect changes inspecies composition. No difference was found attributable to sludge treatment (Fig. 11).Spatial distributionOverlays of actual areas sludged before and after treatment failed to reveal any obviouschanges in spatial distribution of deer mice as indicated by the number of captures at each station(Fig. 12). In both replicates, no notable difference was observed between the relative proportionof deer mice captures in the sludged areas to the entire study area before and after sludgeapplication (Fig. 13). This analysis was not possible for the Oregon vole and the Townsendchipmunk, due to their low sample sizes.42Table 10. Shannon-Wiener indices of diversity and species richness of study sites for1990-1991.Period Shannon-WienerIndicesSite No. ofspeciesControl Pre-sludge 1.57 1 62 7Treatment Pre-sludge 1.58 1 62 9Control Post-sludge 1.76 1 52 7Treatment Post-sludge 1.48 1 62 643Fig. 11. Species composition of study sites for summer and winter periods, Maple Ridge, B.C.Canada.Oregon vole^=1 Chipmunk^EEM St Inn. I0 Jump mouse MI Red-bodied yobINN Deer mouse1.1 Shrew 2Cumulative Proportions: Control 1/NW AMP%,,,,,,,,,,,,,,,,,,,,,,,,I 00806040200Summer 1990 Summer 1991Winter 1990.91Per 10./14^EIEM Orate. vole^=1 Chipmuck^FEE S►re. IShrma 1 Jump. mouse 11111 Red-backed voleCumulative Proportions: Treatment 1Cumulative Proportions: Control 2Summer 1991Winter 1990.91Summer 1990Cumulative Proportions: Treatment 2Summer 1990 Winter 1990-91 Summer 1991 Summer 1990 Winter 1990-91 Summer 1991:a4;;O/4.4:4;i100so40200MN Deer mouse Oregon vole =1 Chipmunk Shrew 1 Door moos WO Oregon yak Ea Chipmunk k'1,X'^Wow IShrew 2 C.= Jump. moos IBM Weasel iM:1^Red -backed vole MN Shrew 2 =1 Jump. mouse 111111 Weasel Etg^Red-tacked role10080604020MIL,ILL7,11008060402002^3^4^5^6^720 -15 -10 -125PRE-SLUDGE TREATMENT 1Peromyscus maniculatusPRE-SLUDGE TREATMENT 2Peromyscus maniculatus2520 -15 -10 -1 2 3 4 5 6 7To compare spatial distribution of deer mice captures before and after sludgeapplication, superimpose this figure onto Figure 12.45aPOST-SLUDGE TREATMENT 1Perom yscus maniculatus1^2^3^4^6^7POST-SLUDGE TREATMENT 2Perbmyscus maniculatus1 2 3 4 5 - 6 7Fig. 12. Distribution of deer mice captures on treatment sites before f] andafter 11 sludge treatment. Actual area sludged was blackened on each site,Maple Ridge, B.C., Canada.45b%%/SLUDGED UNSLUDGEDUNSLUDGEDIIM SLUDGEDPre-sludge^Post-sludgeCONTROL 1.771Post-sludgeCONTROL 20EQUIVALENT AREASPre-sludge^Post-sludgeTREATMENT 1Pre-sludge^Post-sludgeTREATMENT 2Fig. 13. Relative number of deer mice captures in sludgedareas on treatment grids (or equivalent areas sludged oncontrol areas), as a proportion of total captures, prior toand after treatment, Maple Ridge, B.C., Canada.46DISCUSSIONThe application of sewage sludge could potentially have significant effects on thepopulation dynamics of small mammals in a forest environment. However, from data gatheredhere, it would appear that there was no major effect on population parameters attributable tosludge treatment one year after application. These parameters included abundance, reproduction,growth, survival, sex ratio, species diversity and spatial distribution. Juvenile growth rates ofdeer mice did increase after sludge treatment. No other population parameters appeared to beaffected (Table 11).Experimental designImprovements in the experimental design suggested for future studies include increasingthe number of replicates, as well as in the scale, timing and duration of application. Only twosites were treated with sludge in this study as part of the overall project design. Moreover,sludge was only applied to parts of the treatment grids. A minimum of three replicates isrecommended in future studies. This would have also helped to clarify site-specific resultswhich could not be attributed entirely to the sludge treatment. Population densities of theOregon vole varied between treatment and control populations. Control populations occurredat higher densities than treatment populations in the pre-sludge year. A possible cause for thiscould be the concurrent occurrence of weasels on treatment grids. Weasels are efficientpredators and females have been recorded to prefer voles as a prey species (Microtuspennsylvanicus) (Raymond et al. 1990).A similar difference in population density was also found for the Townsend chipmunk.47Table 11. Summary of demographic effects of sludge treatment on small mammalpopulations. Three main species of concern were P. maniculatus, M. oregoni, andE. townsendii. 0 =no difference from control population, + =increased relative to control.All three species were analyzed unless specified with initials of species investigated.Total population (MNA and J/S)per trapping sessionRecruitmentcumulative no. of recruits/periodweekly number of recruitsProportion of animals breeding/periodmales scrotalfemales lactatingProbability of survival (J/S) {pm,mo}summer 1990 vs. summer 1991Juvenile survival^{pm,mo}percentage juvenile survivalminimum survival rates (per 21 days)Mean body weightper periodAge at sexual maturity^{pm}per periodJuvenile growth^{pm}per periodSex ratio (MNA)per periodSpecies diversityShannon-Wiener indicesspecies compositionSpatial distribution^{pm}relative no. of deer mouse captures(per period)distribution of captures(per period)000+ (treatment 1 only)Pm000000+ pm00000pm: P. maniculatusmo: M. oregoni48Treatment population densities were consistently higher than control densities in the pre-sludgeyear. This, in addition to the difference observed in Oregon vole populations, suggested thatthere may be the existence of 'metapopulations', or isolated populations acting independently ofother surrounding populations. It is difficult to determine what are the direct causes of thispattern without data from additional replicates. However, an overview of my results withrespect to population parameters (Table 11) showed a consistent lack of effect from the sludgeapplication.Application on a larger scale (e.g. 5-10 ha) is also suggested. Sludge applications in thesize range of this study may be too small to significantly impact mobile small mammals such asthe deer mouse and chipmunk. These generalist species may be able to modify their behaviorand territories to adjust to the sludge treatment relatively easily when the area treated is small.Operational applications are likely to be of much larger scale. Other wildlife groups suitablefor small scale studies such as this include soil invertebrates, insects, and amphibians. It isimportant to note that this study concentrated on only one wildlife group: small mammals. Otherresident fauna could potentially be affected by the sludge treatment and need to be consideredas well.Future studies should apply sludge in the middle of the summer season when vegetationand small mammals are actively growing. Parameters such as reproduction, growth andrecruitment are best studied at this time. The application of sludge in this study took place inlate November and this timing may have contributed to the limited effects observed. Lastly,increasing the duration of this study to two or three years post-treatment may reveal long-termeffects on population dynamics not exhibited in a short-term study.49Density and recruitmentAnnual and multi-annual fluctuations in abundance of small mammal populations are notunexpected and have been well documented in the literature (P. maniculatus: Sadleir 1965,Healey 1967; M. oregoni: Gashwiler 1972, Petticrew and Sadleir 1974, Hooven and Black 1976)and were evident on all study areas. The low abundance of control populations in 1991 suggeststhat small mammal populations were at a low point in their cycle rather than their low populationnumbers being a result of sludge treatment. Decreases in Oregon vole abundance on all siteswere attributed to the characteristic cyclic fluctuations of this species (Gashwiler 1972, Hoovenand Black 1976, Sullivan and Krebs 1981b), with the 1991 data covering a presumed decline invole abundance.The observed increase in Oregon vole recruitment during periods of reduced deer mouserecruitment is likely related to competitive interactions between the two species. Data collectedfrom treatment site 2 and control site 2 during a previous study supports this conclusion(Sullivan 1990a). Interspecific competition has also been recorded previously for these twospecies in a young plantation (Petticrew and Sadleir 1974). Moreover, Oregon voles have beenshown to competitively exclude deer mice from grassland areas (Grant 1971, Redfield et al.1977).Data from this study indicate that the deer mouse appeared to adapt well to the sludgetreatment as illustrated by the lack of effect on its survival and reproductive attributes. This isreinforced by the work of Petticrew and Sadleir (1974) and Sullivan (1979) which concluded thatthere was little variation in demographic parameters of deer mouse populations in differenthabitats of coastal forests.50Growth and survivalThe increase in growth rate of deer mice on treatment sites suggests that fertilizationeffects from the sludge may have caused an increase in growth of juvenile deer mice byincreasing the nutrient content in the vegetation. Increases in nitrogen and phosphorus were alsofound in salal foliage in the post-treatment year (L. Coward, pers. comm.).The increase in the mean growth rate of juvenile deer mice found on treatment sites in1991 did not affect this species' abundance, survival, recruitment or average adult weight. Thismay be due to short-term effects of the sludge application. The timing of the application mayhave also contributed to a limited effect. Sludge was applied in late November when deciduousvegetation was in senescence. More extensive effects may have occurred if sludge was appliedin the midst of summer when both small mammals and ground vegetation were actively growing.ReproductionA site-specific increase in the proportion of breeding male deer mice in the post-treatmentyear was found on treatment site 1. This may be related to the increase in the growth rate ofjuvenile deer mice. Some distinguishing feature on treatment site 1 may have increasedsensitivity of deer mice to sludge treatment. However, this site-specific response cannot besolely attributed to sludge application. Additional replicates would have been needed to identifyany treatment effects.The proportion of breeding female deer mice increased in the second year on all fourexperimental sites. This may have been due to concurrent population decreases observed on51these sites, which in turn relaxed competitive pressures for resources. This gender-specificresponse may stem from different behavioral responses between sexes during various seasonsof the year. Agonistic behaviour between male deer mice during the breeding season has beensuggested as a mechanism for regulating the number of breeding adult males (Sadleir 1965).Subadult and immigrant females are tolerated, so their numbers are not regulated to the sameextent (Petticrew and Sadleir 1974). Juvenile male deer mice, despite a decrease in overallabundance, are still faced with agonistic behaviour from established adult males.Other studiesOther studies investigating the effects of sewage sludge on small mammals have revealedvariable results. Hegstrom's (1986) study on shrews found no response in abundance to sludgetreatment, although insectivores had higher levels of cadmium in their livers and kidneys aftertreatment. Conversely, West et al. (1981) found that treatment populations of herbivorousrodents (M. townsendii, M. oregoni, C. gapperi) were significantly lower in density than thoseon control sites, presumably due to changes in plant species which acted as food and cover forthis group. Their study, however, was based on data from one trapping period of four days induration and lacked temporal controls. No other studies were found that investigated theinfluence of sludge on the population dynamics of small mammals.Other habitat alteration studies have found that some rodents have a strong negativereaction to disturbance or loss of their habitat (as cited in Sullivan and Sullivan 1982). This isespecially pronounced in 'intolerant' or specialist species such as the Oregon vole. Birney etal. (1976) proposed that a minimal level of vegetative cover was necessary to permit Microtus52spp. to increase in abundance during their 3-4 year cycle. This reliance on cover was attributedto the need for visual cover from diurnal predators. Unfortunately, the Microtus species in mystudy underwent a population decline in the post-treatment year which made analysis difficultdue to a small sample size. Based on the data from this study, however, M. oregoni was notobserved to be affected by the treatment.Studies on the effects of forest herbicide use on small mammals found that, in general,no significant effects were observed on their population parameters (Sullivan and Sullivan 1981,1982, Sullivan 1990b). Parameters investigated in these studies included abundance,reproductive performance (measured in terms of the proportion of breeding animals and numberof successful pregnancies), growth and survival. This lack of effect was not observed forclearcut logging or burning. Sullivan and Krebs (1981a) found an 'irruption' in the number ofdeer mice in an old-field habitat adjacent to a forest immediately after the forest was harvestedby clearcutting. This increase in recruitment was not recorded in the study site in the loggedarea. In another study, a significant decrease in deer mice abundance was found shortly aftera prescribed burn on a clearcut site as compared to a control site. This reduction in abundance,however, was short-lived and returned to a level comparable to the control area within a fewmonths (T. Sullivan, pers. comm.) 3 .Relative to other habitat alteration practices, silvicultural sludge applications are expectedto have less impact on small mammal populations than logging or burning. In sludgeapplications, deciduous vegetation is not altered as intensely as a herbicide application or a3Dr. T. P. Sullivan, professor of forests and wildlife, Department of Forest Sciences,University of British Columbia, Vancouver, B.C., Canada.53clearcut logging operation. In addition, fertilization effects of the sludge may enhance somevegetation after treatment. Consequently, the impacts on small mammals which utilize thisvegetation as food and habitat are expected to be not as severe. This prediction is supported bymy findings in this study.Other wildlife groups, however, may not be as adaptable. Insectivores (such as shrews),ground nesting birds, or amphibians, are a few species that may be more sensitive to metalaccumulation or the sludge application itself. Furthermore, future sludge applications areunlikely to be of such small scale or short duration as in this pilot study. In light of this, it issuggested that additional research be conducted on small mammals and other wildlife groups.54CHAPTER 3:Giardia contamination in Small MammalsINTRODUCTIONOne of the primary health issues surrounding sludge applications as silvicultural fertilizersis the introduction of pathogens and parasites into the forest ecosystem, a potentially irreversibleprocess. The parasite group of concern in this study is Giardia duodenalis. Giardia spp. is aparasitic protozoan which emerges from its dormant cyst form upon ingestion by an appropriatehost, and develops into a motile form capable of multiplying known as the trophozoite.Morphological criteria, based on the shape of a microtubular structure of unknown function, themedian body, have been used to divide this genus into three morphological groups:G. duodenalis (includes G. lamblia) which infects a variety of mammals including humans,G. muris which infects rodents, and G. agilis which infects amphibians. Symptoms of giardiasisare many and varied, from mild flu-like symptoms to severe diarrhea and vomiting. Symptomslast from weeks to months depending on the individual and perhaps the parasite strain.Fortunately, there is medical treatment available for this condition. Despite this, it is the mostprevalent gastrointestinal condition in the United States (Warhurst and Smith 1992) and the mostcommon protozoan identified in human stool specimens worldwide (Isaac-Renton 1987).The condition caused by this organism, giardiasis, commonly known as beaver fever,may be acquired by drinking contaminated water. The beaver is considered a potential reservoirof infection of human strains of Giardia. In addition to potentially contaminating water sources,the beaver has also been considered as an amplification host. Other mammal hosts to Giardia55spp. include smaller rodents such as deer mice and voles (Microtus longicaudus, M.pennsylvanicus, M. richardsoni) (Grant and Woo 1978, Pacha et al. 1987) and domestic animalssuch as dogs and cats (Baker et al. 1987, Collins et al. 1987). There have also been giardiasiscases found in budgerigars (Box 1981), a heron (Georgi et al. 1986), sheep and even llamas(Kiorpes et al. 1987). Small mammals were selected as the subject of this study due to theirnumerical abundance and mobility,Infectivity of G. duodenalis to other mammal species is uncertain. One major difficultylies in the uncertainty of species classification based on cyst morphology. For accurate speciesidentification, trophozoites, available only from the intestinal lining of a live host, are required.Most studies, to date, have dealt mainly with cysts found in fecal samples.This part of the study examined: 1) the initial prevalence of small mammal giardiasis inthe Research Forest and prevalence of small mammal giardiasis before and after sludgetreatment; and 2) the ability of a small wild rodent, the deer mouse, to host strains of humantype G. duodenalis in the laboratory.METHODS AND MATERIALS1. Field StudiesEstablishing initial prevalenceA baseline estimate of the prevalence of Giardia spp. in field populations of smallmammals was determined prior to sludge application from analysis of fecal samples collectedfrom two main groups of wildlife: beavers and small mammals. Samples of the former were56collected near active or abandoned beaver dams. Information regarding locations was obtainedfrom Research Forest staff and reconnaissance surveys were then conducted. Twenty-fivesamples of small mammal feces were obtained from animals captured in three small mammallive-trap lines set up near active or abandoned beaver dams. Fecal samples from aquaticmammals included only the beaver due to its close association with the spread of giardiasis. Atotal of 13 samples was collected from three different sites (distributed throughout the southernhalf of the forest). As beavers are known to show high site fidelity, this was assumed to be atleast three different animals. Samples were transported to the Vancouver General HospitalGiardia laboratory and analyzed by the formol-ether concentration method and microscopicexamination of iron-hematoxylin stained preparations (Garcia and Bruckner 1988), a highlysensitive detection method for fecal Giardia cyst content.Giardia prevalence and sludge applicationOne hundred fresh fecal samples from small mammals were obtained from the live-trapping study sites (Chapter 1) prior to and after sludge application. Samples were collectedfrom 100 individual specimens (25/study site) which were caught in live-traps overnight. Thesesamples were placed in parasite preservative (sodium acetate, acetic acid, formalin) and testedfor the presence of Giardia spp. cysts in the laboratory. This part of the analysis was conductedunder the direction of Dr. J. Isaac-Renton.572. Laboratory StudiesCollection of deer miceTwenty-six deer mice were live-trapped at the Malcolm Knapp Research Forest insouthwestern British Columbia in western Canada at a sufficient distance away from sludgedareas to prevent any dispersal effects. All deer mice were captured in Longworth live traps thatwere baited with oats and supplied with cotton bedding and a slice of carrot for moisture. Trapsset in various locations in the forest were baited and checked twice daily over a two to three dayperiod. In total, six different sites were sampled. All animals trapped were tagged withfingerling fish tags and then transported in traps to the parasitology laboratory at the VancouverGeneral Hospital. Only males were kept for use in these experiments. Animals were kept in thelaboratory in cages (47 x 26 x 12.7 cm) in pairs and provided with laboratory rodent chow (PMIFeeds Inc. Richmond, B.C.) and water ad libitum. Small plastic water bottles were used asnesting chambers. All animals were acclimatized for seven days after being trapped and fecalexaminations were then carried out. For optimal laboratory examination, fecal samples werecollected from each animal and placed in SAF (sodium acetate, acetic acid and formalin) parasitepreservative for processing. Each SAF-preserved sample was tested by microscopic examinationfollowing the formol-ether concentration method and microscopic examination of iron-hematoxylin stained preparations (Garcia and Bruckner 1988).Metronidazole treatmentNo natural infections with Giardia were detected in the trapped deer mice, but to ensurethat none of the test or control animals were infected, they were all treated with metronidazole.58Animals were treated orally using a gavage needle (gauge #22) with three consecutive dailydoses of 7.5 mg of metronidazole for each deer mouse. Each mouse was treated whilst mildlyanaesthetized with Metofane (methoxyfluorane). Following a seven day interval of rest aftermetronidazole therapy, six deer mice were randomly selected and further fecal samples wereexamined. Deer mice were then assigned into two groups of 13 each; 10 as test animals and 3as controls.Enzyme-linked immunosorbent assay (EIA)An enzyme-linked immunosorbent assay of sera collected prior to inoculation was usedto determine whether any of the deer mice had evidence of previous infection with Giardia.The EIA was carried out as described by Birkhead et al. (1989). Sera from Balb/c micepreviously inoculated with Giardia antigen and producing high anti-Giardia immunoglobulin Gwere used as positive EIA controls. Preimmune sera, also collected from Balb/c mice, wereused as negative EIA controls. Sera from the deer mice were collected by tail-bleeding andstored at -20°C until testing.Source of cystsCysts for inoculation of deer mice, obtained from previously infected laboratory gerbils(Meriones unguiculatus), were collected from feces using a sucrose gradient concentrationmethod (Roberts-Thompson et al. 1976). One-day old cysts were used in all experiments toeliminate variation of infectivity based on cyst-age. One Giardia strain was obtained from ahuman-source (symptomatic child) and a second strain was collected from a beaver determined59to be the source of a large waterborne outbreak in humans (Isaac-Renton et al. 1992).Chromosomal banding patterns on DNA karyotyping and isoenzyme analysis were similar tohuman source strains of Giardia retrieved during the outbreak. Trophozoites from both strainswere also confirmed as being in the G. duodenalis morphological group by Giemsa staining andmicroscopic examination for median body morphology (Filice 1952).Inoculation of cystsAll test and control mice were inoculated on the same day. Cysts were quantified usinga counting chamber (Neubauer) and appropriate concentrations (1X1025) prepared in distilledwater. Deer mice treated and tested as above were inoculated with different cyst concentrations.In each group, three deer mice were inoculated with 0.10 mL of distilled water alone to serveas controls, four mice were inoculated with 0.1 mL distilled water containing 1X10 5 cysts, twodeer mice were inoculated with 0.1 mL containing 1X10 4 cysts, two were inoculated with 1X10 3and two with 1X102 cysts. A higher proportion of mice were inoculated with the highest cystconcentration to provide a larger sample size with optimal conditions for infection. Animalswere anaesthetized when gavage treated with the designated suspension of cysts. The experimentwas then repeated for the second duodenalis isolate in the second group of mice. Gerbils(Meriones unguiculatus) were also inoculated in both experiments on the same day as theinoculation of the deer mice to test the viability of the infecting cysts. Fecal samples fromgerbils were analyzed using the same methods as the test and control deer mice.60Determination of infectionStarting one week post-inoculation, three fecal samples were collected at weekly intervalsfor each test and control animal. Fecal samples were collected and placed in SAF parasitepreservative and then examined by the two methods described above (microscopy after formol-ether concentration and examination of iron-hematoxylin stained preparations) for the presenceof Giardia cysts or trophozoites. All animals were sacrificed at the end of three weeks and theirsmall intestines removed. The anterior half of the small intestine was removed and impressionsmears prepared from sections near the pylorus of the stomach. A wet preparation was alsoprepared from the intestinal contents. Both preparations were cover-slipped and examinedmicroscopically (Nikon, Labophot X 200) for Giardia trophozoites. Trophozoites wereidentified on the basis of pear-shaped body, motility and size (10-12 um in length).RESULTS1. Field StudiesEstablishing initial prevalenceLaboratory results from fecal sample analysis found very low (small mammals: 8%) tono (beavers: 0%) Giardia contamination prior to sludge application. Furthermore, Giardia cystswere not identified to the species (a limitation of cyst identification). Endemic giardiasis inrodents (G. muris) has been found to be prevalent and is not considered a human health concern(Grant and Woo 1978, Kirkpatrick and Benson 1987, Pacha et al. 1987). These results suggestthat initial prevalence of Giardia at the Research Forest was low (Table 12).61Table 12. Initial prevalence of Giardia spp. cysts near beaver dams and onstudy sites prior to, and after sludge application as estimated by fecalsampling. Giardia spp. cyst content was tested by the formol-etherconcentration method and microscopic examination of iron-hematoxylinstained preparations. ^No.^Source^Time of^Giardia spp. cystSamples Sampling positive (%) Small mammals:25^(near beaver dams)^Pre-sludge^2 (8.0%)Beavers:c■N^ 13^(near beaver dams)^Pre-sludge^0 (0.0%)Small mammals:100^(study sites)^Pre-sludge^11 (11.0%)Small mammals:98^(study sites)^Post-sludge^4 (4.1 %)Prevalence of giardiasis in small mammalsPrevalence of Giardia spp. cysts in study sites estimated from small mammal fecalanalysis were found to be slightly lower after sludge application. Due to the small degree indifference in these estimates, it was concluded that prevalence of Giardia did not appear to beaffected by sludge treatment in this case (Table 12). However, it was unknown whether or notthe sludge utilized for this study was contaminated by Giardia previous to application. Nooutbreaks of giardiasis were reported in the areas where the sludge was collected. Samplingsludge for Giardia spp. cyst content was not considered because past attempts have found thisprocedure to be both inefficient and prohibitively expensive in terms of labour and analysiscosts.2. Laboratory StudyNone of the trapped deer mice showed evidence of endogenous infection prior toinoculation as determined by multiple fecal examinations. None of the deer mice showedevidence of past infection as demonstrated by the absence of anti-Giardia IgG by EIA. Hightitres of specific antibody were, however, obtained from the Balb/c mice used as EIA positiveserum controls. The group of deer mice used as negative controls remained Giardia-negativethroughout both sets of experiments and were uninfected at necropsy indicating that cross-contamination did not occur between cages. Cysts from both Giardia strains used in theseexperiments were infective in gerbils and therefore were considered viable at time of inoculation.Results of deer mouse inoculations using the different isolates and different cyst concentrationsare seen in Table 13. None of the animals inoculated with the human-source G. duodenalis63Table 13. Results of fecal and small intestine examinations of two groups of deer miceinoculated with different strains of and varying numbers of G. duodenalis.Giardia Strain Concentration^Number of^% Positive Testsof cysts^deer miceinoculated inoculatedper deer mouseFecals1^2 3GutExams0 0 0 00 0 0 00 0 0 00 0 0 025 50 50 750 0 0 00 0 0 00 0 0 0Symptomatic Child^1 x 105^n = 41 x 104^n = 21 x 103^n = 21 x 102^n = 2Beaver source^1 x 105^n = 4causing 1 x 104^n = 2human outbreak^1 x 103^n = 21 x 102^n = 264became infected even when a dose of 1 X 10 5 cysts was given. Two of the four animalsinoculated with 1 X 10 5 cysts of the beaver-source isolate associated with the human outbreak,however, became infected. One Giardia-infected deer mouse was found to be positive on fecalexamination during the first week post-inoculation and a second animal was found to be positiveduring the second week post-inoculation. Both of these animals remained Giardia positive untilnecropsy. Giardia trophozoites were found on examination of small intestinal contents on a thirdinfected mouse which had been consistently cyst-negative on fecal examinations. Thus, threeof the four mice in the group inoculated with 1 X 10 5 cysts were infected with the outbreak-associated Giardia isolate.DISCUSSIONThese experiments confirm the observation that deer mice may become infected withsome strains of the G. duodenalis morphological group which are associated with infections inhumans. The strain shown to be infective in these small rodents in the present experimentsrequired a large number of cysts for infection to be produced. I also note that three of the fourdeer mice infected with this strain were young animals from the same litter. As noted by others,it is therefore possible that these infections demonstrate that age-associated immunity plays arole in resistance to infection (Woo and Paterson 1986). Other factors that determine resistanceto infection include acquired immunity due to previous infections. Results of serological testingin these experiments indicated that none of the deer mice had been previously exposed toGiardia, and therefore were unlikely to be resistant to infection due to acquired immunity.65Evidence to suggest the transmission of animal-source Giardia to humans so far has beencircumstantial, yet the frequency with which human infections have been traced to a potentialanimal source suggests that humans and animals may be susceptible to infection with some ofthe same strains of G. duodenalis. The beaver (Castor canadensis) has been the animal mostcommonly implicated in zoonotic infection (Frost et al. 1980, Isaac-Renton 1987, Erlandsen andBemrick 1988). This water-dwelling member of the rodent family may maintain infection in acolony by vertical transmission and may also act as amplification hosts (Thompson et al. 1990).Beavers have also been associated, both epidemiologically and most recently by laboratoryevidence, with waterborne epidemics (Dykes et al. 1980, Healy 1990). Biotyping of human,beaver, and drinking water source isolates (Isaac-Renton et al. 1992) from a waterborneoutbreak, in which a beaver contaminated the drinking water source, is highly suggestive of arole for this animal in some human infections. There is evidence that both beavers and muskrats(Ondatra zibethica) may act as hosts to some strains of G. duodenalis (Davies and Hibler 1978,Bemrick et al. 1984). Other aquatic mammals such as the muskrat have also been clearly shownto harbour Giardia infections (Pacha et al. 1985, Isaac-Renton et al. 1987, Kirkpatrick andBenson 1987). Giardia cysts from some human sources have also shown to be capable ofinfecting muskrats (Erlandsen et al. 1988).Cross-infection studies have shown that cysts from some human source strains are capableof establishing successful infections in a range of wild mammals other than these rodents.Davies and Hibler (1978) reported that human source Giardia cysts were infective to wildanimals such as rats (Rattus norvegicus), beavers, raccoons (Procyon lotor), and bighorn Xmouflon sheep (Ovis canadensis X 0. musimon); and domestic animals such as gerbils (Gerbillus66gerbillus), guinea pigs (Cavia porcellus) and dogs (Canis familiaris). Other animals that mayact as potential reservoirs for G. duodenalis include cats (Healy 1990) and cattle (Buret et al.1990). Erlandsen and Bemrick (1988) found Giardia trophozoites in wading birds (green heronsand egrets, species not given) and noted that they possessed the typical duodenalis-shaped,clawhammer-like, median body (as cited in Erlandsen and Bemrick 1988). In a recent study,these bird-source isolates (G. psittaci and G. ardeae) were not infective to the mammalian hoststested (Erlandsen et al. 1991).Small mammals such as deer mice, voles (Microtus spp.) and chipmunks (Eutamias spp.)are found in high population densities and often traverse large areas. Grant and Woo (1978)found that Giardia infections were common in wild small mammal populations in Ontario. Intheir latter study, 98% of live-trapped voles (Microtus pennsylvanicus) and 97.9 % of live-trappeddeer mice (Peromyscus maniculatus) were found to harbour Giardia spp. The morphologicalgroup based on Filice's classification of Giardia found in these animals, however, was notdetermined. Other reports (Kirkpatrick and Benson 1987, Pacha et al. 1987, Hay 1990) havenoted that the prevalence of giardiasis in many wild rodent populations can be as high as 90%.Pacha et al. (1987) examined the cell morphology of both cysts and trophozoites fromsmall mammals and found that organisms from the microtines of the genus Microtus (voles)resembled the G. duodenalis type. The present experiments also confirm the findings of Roachand Wallis (1988) in which three cultured, but not morphologically defined, human-sourceisolates were used to inoculate deer mice. In those experiments, one of the three isolates usedinfected deer mice. The minimum number of cysts required to produce infection was not given.These data and those from the present study are consistent with the concept that some strains of67G. duodenalis are not host-specific whereas others are rigidly so (Erlandsen and Bemrick 1988).Generalization of the results of cross-transmission studies is difficult when otherbiological and biochemical characteristics of parasite isolates are poorly defined. The twoisolates used in the present experiments have been characterized by DNA karyotyping; one hasbeen shown to be capable of infecting both beaver and humans (Isaac-Renton et al. 1992), thesecond isolate, recovered from a human, has not been as epidemiologically well-characterizedas the epidemic-associated isolate. It appears that different isolates within the Giardiaduodenalis morphological group have differing ranges of host specificities as well as differentbiochemical (Nash et al. 1985, Meloni et al. 1989) and biological behaviours. The differencesin infectivity in deer mice shown by the two isolates in these experiments may reflect furtherdifferences in host-specificity demonstrated by others (Meloni et al. 1989, Nash et al. 1987).Virulence markers have not yet been described in Giardia.This study demonstrated that some strains of G. duodenalis are capable of infectinghumans and small rodents. The importance of the role of large water-dwelling rodents such asbeaver and muskrats in transmission of giardiasis to humans is based on their potential forexcretion of large numbers of cysts directly into surface water supplies. On the other hand,population densities of small rodents such as deer mice are higher than beaver or muskrats.While it is possible that their role in transmission to humans may be related to the infection ofother potential animal reservoirs such as dogs or cats, this concept requires further study.Before the role of animal reservoirs is fully understood, Giardia isolates need to be defined interms of host-specificities, interspecies biochemical relationships and biological behaviours.Since cross-infection of such common small rodents as Peromyscus has been demonstrated (albeit68using large inocula with the strains used), precautions should be taken to keep sewage forsilvicultural or agricultural application away from water sources that may be contaminated byrun-off containing cysts or from areas in which domestic pets may become infected followinga predatorial incident.69OVERALL THESIS CONCLUSIONSImplications of study results for small mammal populationsIn this study, the only effect on population parameters attributable to sludge treatmentwas found in the deer mouse. Fortunately, of the three small mammal species caughtconsistently in this study, the deer mouse was the best measure of habitat suitability due to itsannual cycle of abundance (Gashwiler 1972, Petticrew and Sadleir 1974, Hooven and Black1976). Oregon voles are known for their characteristic 3-4 year cycles in abundance and mayrequire a longer study period for effects to be detected. The Townsend chipmunk was trappedin low numbers only and the data were not suitable for statistical analysis.The growth rate of juvenile deer mice was found to be significantly increased in the post-treatment period. Potential impacts on other species from this effect is unknown. In times oflimited resources, increased interspecific competition with the Oregon vole or other morespecialist species may cause consequent declines of these species. Their respective predatorsmay also be negatively affected. These two potential impacts reflect a change in the balance ofthe ecosystem from an artificially implemented situation, and warrant more research prior to thebroad application of sewage sludge on a field scale. The proportion of male deer mice inbreeding condition also experienced an increase in the post-sludge year but on treatment site 1only. This effect, however, cannot be attributed solely to the sludge treatment due to its sitespecificity. In general, the three major small mammal species that were investigated in thisstudy, the deer mouse, Oregon vole and Townsend chipmunk, did not appear to be significantlyaffected by the sludge application.70More subtle changes in the ecology or population dynamics of small mammal populationsmight have been revealed had the study been continued for longer, with more replicates, overa larger area of application, with more frequent applications of sludge, or with a wider diversityof study animals than what was available in this study. Furthermore, small mammals are onlyone of many groups of wildlife that may be affected. Research is needed to determine themagnitude of the effects of sludge applications if operational scale applications ( > 10 ha) areundertaken. Additional in-depth studies may also pinpoint areas of modification in applicationwhich might help minimize effects.Implications of Giardia transmission for sludge applicationsIn terms of public health considerations, potential for Giardia contamination wasdemonstrated from the findings of this study. P. maniculatus has been shown to be capable ofacting as a host to some strains of human Giardia under specific laboratory conditions. Thissmall mammal species also did not appear to be negatively affected from sludge treatment asreflected by the study of its population parameters. During human giardiasis outbreaks, sewagesludge could become a source of contamination whereby Giardia duodenalis can enter theecosystem through deer mice acting as a host.The risk of exposure to Giardia cysts in field applications, however, is considered smalldue to the poor survival of Giardia cysts under conditions of desiccation (Dr. J. Isaac-Renton,71pers. comm.)4 , the large inoculum dosage required to cause infection, and the chance of havingthis high level of cyst density in sewage sludge. From this information, it may be beneficial forapplications to take place in the summer when desiccation conditions would be most likelyencountered. Alternately, a summer sludge application may also have the most adverse effecton small mammal populations. Presently, field applications are ideally undertaken in earlyspring (January-March) when winter rains can effectively wash sludge into the soil. In addition,fertilization effects of the sludge at this time would benefit trees more than a late fall or winterapplication (M. van Ham, pers. comm.)5 . Without further studies, an alternative time forapplication cannot be suggested. Currently, the main precaution taken with silvicultural sludgeapplications is to ensure they are located at a sufficient distance away from streams or creeksin order to prevent potential run-off of sewage into water sources. My findings reinforce thisprecautionary measure.Of particular concern is the intent to undertake broad scale silvicultural sludgeapplications. Due to the short duration of fertilization effects, it has been considered that sludgeapplications be repeated periodically (perhaps once every 10 to 20 years) over an extendedperiod of time. In field application, areas treated would be considerably larger than those in thisexperiment. A larger study conducted for a longer interval of time is therefore recommended.'Dr. J. L. Isaac-Renton, Department of Pathology, Division of Medical Microbiology,University of British Columbia, and British Columbia Centres for Disease Control, Ministry ofHealth, Vancouver, Canada.5M. van Ham. Ph. D. candidate in Forest Sciences. Thesis topic on the effects ofsilvicultural sludge applications on forest ecosystems, Department of Forest Sciences, Universityof British Columbia, Vancouver, B.C. Canada.72REFERENCESAnderson, D.A. 1981. Response of the Columbian black-tailed deer to fertilization of Douglas-fir forests with municipal sewage sludge. Ph.D. Diss., Univ. Washington. Seattle. 176pp(as cited in Anderson 1983).Anderson, D.A. 1983. Reproductive success of columbian Black-tailed deer in a sewage-fertilized forest in western Washington. J. Wildl. Manage. 47(1): 243-247.Anderson, D.A. 1985. Influence of sewage sludge fertilization on food habits of deer inWestern Washington. J. Wildl. Manage. 49(1): 91-95.Ash, L.R., and Orihel, T.C. 1987. 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Micro. 27:1707-1710.Birney, E.C., Grant, W.E. and Baird, D.D. 1976. Importance of vegetative cover to cyclesof Microtus populations. Ecology 57: 1043-1051.Box, E.D. 1981. Observations on Giardia of budgerigars. J. Protozool. 28(4): 491-494.Buret, A., denHollander, N., Wallis, P.M., Befus, D., and Olson, M.E. 1990. Zoonoticpotential of giardiasis in domestic ruminants. J. Infect. Dis. 163: 231-237.Cole, D.W., Henry, C.I. and Nutter, W.L. 1986. The forest alternative for treatment andutilization of municipal and industrial wastes. Univ. of Wash. Press, Seattle (as cited inKimmins et al. 1992).73Collins, G.H., Pope, S.E., Griffin, D.L., Walker, J., and Connor, G. 1987. Diagnosis andprevalence of Giardia spp. in dogs and cats. Australian Veterinary Journal. 64(3): 89-90.Davies, R.B., and Hibler, C.P. 1978. Animal reservoirs and cross-species transmission ofGiardia. In Waterborne transmission of giardiasis. Edited by W. Jakubowski and J.C.Hoff. Proceedings of Symposium, Cincinnati, Ohio. Sept. 18-20. pp. 104-126.Dykes, A.C., Juranek, D.D., Lorenz, R.A., Sinclair, S., Jakubowski, W., and Davies, R.1980. Municipal waterborne giardiasis: an epidemiologic investigation. Ann. Int. Med.92 (Part I): 165-170.Erlandsen, S.L., and Bemrick, W.J. 1988. Waterborne Giardiasis: sources of Giardia cysts andevidence pertaining to their implication in human infection. In Advances in Giardiaresearch. Edited by P.M. Wallis and B.R.U. Hammond. Calgary Press, Calgary. pp.227-236.Erlandsen, S.L., Sherlock, L.A., Januschka, M., Schupp, D.G., Schaefer III, F.W.,Jakubowski, W. and Bemrick, W.J. 1988. Cross-species transmission of Giardia spp.:inoculation of beavers and muskrats with cysts of human, beaver, mouse and muskratorigin. Appl. Environ. Microbiol. 54: 2777-2785.Erlandsen, S.L., Bemrick, W.J., and Jukubowski, W. 1991. 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Fauna on sludge-treated and untreated study sites. InNutritional and toxic effects of sewage sludge in forest ecosystems. Edited by S.D. Westand R.J. Zasoski. College of Forest Resources, University of Washington. Seattle.(abstract).Woo, P.T.K. 1984. Evidence for animal reservoirs and transmission species. In Giardia andGiardiasis. Edited by S.L. Erlandsen and E.A. Meyer. Plenum Press, New York. pp.341-364.Woo, P.T.K., and Paterson, W.B. 1986. Giardia lamblia in children in day-care centres insouthern Ontario, Canada, and susceptibility of animals to G. lamblia. Trans. Roy. Soc.Trop. Med. Hyg. 80: 56-59.79Zar, J.H. 1984. Biostatistical analysis. Prentice-Hall, Inc., Inglewood Cliffs, New Jersey.718 p.Zasoski, R.J. 1980. Heavy metal mobility in sludge amended soils. In Sludge applicationsin Pacific Northwest Forest Lands. Edited by C.S. Bledsoe. Coll. of Forest Resources.U. of W. Seattle, Wash. pp. 67-72.80GLOSSARY OF TERMSBalb/c mouse:a special type of mouse used for laboratory experiments.Biotyping:using biological characteristics to differentiate between different isolates.Enzyme-linked immunosorbent assay (EIA):a sensitive serological method of detecting antibodies to infective organisms. May detectevidence of recent OR past infection.Formol-ether concentration method:an efficient method of detecting the presence of parasites including Giardia cysts in fecalsamples. It uses different chemicals to separate organisms from fecal fat and debris.Although it is quicker and more sensitive than the sucrose-gradient method, cysts arekilled in the preservative used.Gavage:inoculation directly into the stomach using a tube passed through the flares, pharynx, ormouth and through the esophagus.Giemsa stain:one of the Romanosky group of combination stains often used for staining parasites ofblood. Used in these experiments to stain median bodies in trophozoites formorphological identification of groups.Incidence:frequency of a occurrence over time.Intestinal analysis:in this experiment, this referred to the dissection and removal of the small intestine,making intestinal scrapings of the contents and microscopically scanning contents forGiardia trophozoites.Iron-hematoxylin stained preparations:preparations made from feces and stained with the named reagent to demonstrate parasitemorphology. Recommended for the most sensitive method of detecting intestinalparasites when combined with examination of results of a concentration method.Isolate:one strain of parasite from one particular source.81Karyotyping:arranging patterns of chromosomes based on size.Metronidazole:anti-Giardial agent. Used routinely in the treatment of human and animal infections.Necropsy:autopsy.Prevalence:the number of cases of a disease present in a specifiedpopulation at a given time.Serological:of or relating to serum.Sucrose-gradient method:method as described by Roberts-Thomson et al for separation of Giardia cyst from fecaldebris. Cysts maintain viability when this method is used.Virulence markers:signs or characteristics that indicate a particularly virulent strain when comparingdifferent strains, eg. a specific enzyme.Zoonosis:infections that may be transmitted between vertebrate animals and humans.82


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