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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 ON SMALL MAMMAL POPULATIONS AND THE POTENTIAL OF GIARDIA CONTAMINATION IN A FOREST ECOSYSTEM  by Chris Cheng B.Sc., Simon Fraser University, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Forestry)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1993 © Chris Cheng, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  FoReST  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Aree,.0 2-2^1(02  ABSTRACT  The effects of a silvicultural sludge application on resident small mammal populations were assessed by monitoring their demographic responses before and after sewage sludge treatment. Potential contamination of the forest ecosystem by Giardia duodenalis, a parasitic protozoan, was also investigated. Populations of the deer mouse (Peromyscus maniculatus), Oregon vole (Microtus oregoni), Townsend chipmunk (Eutamias townsendii), shrews (Sorex spp.), and a few species caught occasionally, were sampled in control and treatment study sites from May 1990 to October 1991. Sludge application occurred in November 1990. There were no detectable differences in the abundances of deer mice, Oregon voles, and chipmunks on treatment areas relative to controls after sludge application. This was also the case for recruitment, survival, mean body weight, weight at sexual maturity, sex ratio, species diversity and spatial distribution. There was, however, a significant increase in the growth rate of juvenile deer mice on treatment sites after sludge application. This was also the only effect on small mammal population dynamics observed from the sludge treatment. The prevalence of Giardia spp. cysts in small mammal fecal samples on study sites did not differ between pre-sludge and post-sludge periods. My laboratory study using wild deer mice concluded that this species was capable of acting as a host to some strains of human Giardia under specific laboratory conditions. Because of the deer mouse's apparent ability to  adapt well to this environmental change, as illustrated by the lack of effect on its survival and reproduction attributes, and this species' potential ability to host human Giardia, additional research and consideration are warranted prior to the implementation of large scale silvicultural sludge applications. ii  TABLE OF CONTENTS  ABSTRACT ^ TABLE OF CONTENTS ^ LIST OF TABLES ^ LIST OF FIGURES ^ ACKNOWLEDGEMENTS ^  ii iii vi viii x  CHAPTER 1: General Introduction INTRODUCTION ^  1  LITERATURE REVIEW: EFFECTS OF SLUDGE ON WILDLIFE ^ 4 Ungulates ^ 4 Birds ^ 4 Small mammals ^ 5 SMALL MAMMAL STUDY OBJECTIVES ^  6  CHAPTER 2: Impacts of a Silvicultural Sludge Application on Small Mammal Populations INTRODUCTION ^  7  MATERIALS AND METHODS ^ Description of study areas ^ Livetrapping protocol ^ Population parameters ^  8 8 9 11  iii  Population density and recruitment ^ Reproduction ^ Survival and growth ^ Body weights ^ Growth analysis ^ Sex ratio ^ Species diversity and richness ^ Spatial distribution ^ Statistical analysis ^ Sludge application ^ RESULTS ^  12 12 12 13 14 14 15 16 16 17 19  Changes to habitat ^ Trappability ^ Total population ^ Recruitment ^ Reproduction ^ Probability of survival ^ Juvenile survival ^ Average adult weight ^ Juvenile growth rate ^ Weight at sexual maturity ^ Sex ratio ^ Species diversity ^ Spatial distribution ^  DISCUSSION ^  19 19 22 26 29 32 32 32 38 38 38 42 42  47  Experimental design ^ Density and recruitment ^ Growth and survival ^ Reproduction ^ Other studies ^  iv  47 50 51 51 52  CHAPTER 3:  Giardia Contamination in Small Mammals INTRODUCTION ^  55  METHODS AND MATERIALS ^  56  1. Field Studies ^ Establishing initial prevalence ^ Giardia prevalence and sludge application ^  56 56 57  2. Laboratory Studies ^ Collection of deer mice ^ Metronidazole treatment ^ Enzyme-linked immunosorbent assay (EIA) ^ Source of cysts ^ Inoculation of cysts ^ Determination of infection ^  58 58 58 59 59 60 61  RESULTS ^  61  1. Field Studies ^ Establishing initial prevalence ^ Prevalence of giardiasis in small mammals ^  61 61 63  2. Laboratory Study ^  63  DISCUSSION ^  65  OVERALL THESIS CONCLUSIONS ^  70  Implications of study results for small mammal populations ^ 70 Implications of Giardia transmission for sludge application ^ 71 REFERENCES ^  73  GLOSSARY OF TERMS ^  81  v  LIST OF TABLES  Table 1. Minimum unweighted trappability estimates for the deer mouse, Oregon vole and Townsend chipmunk for three periods on all study sites ^ 20 Table 2. Jolly-Seber trappability estimates for the deer mouse, Oregon vole and Townsend chipmunk for three periods on all study sites (+ 95% confidence intervals) .. 21 Table 3. Proportion of male adults of three main species in breeding condition (scrotal) during the breeding season of each year. Sample sizes in parentheses ^ 30 Table 4. Proportion of female adults of three main species in breeding condition (lactating) during the breeding season of each year. Sample sizes in parentheses ^ 31 Table 5. Jolly-Seber average survival rates per period (1990-1991) for control and treatment populations of deer mice (± 95% confidence intervals) ^ 33 Table 6. Jolly-Seber average survival rates per period (1990-1991) for control and treatment populations of Oregon voles (+ 95 % confidence intervals) ^ 34 Table 7. Minimum survival rates per period for control and treatment populations of juvenile deer mice, Maple Ridge, B.C., Canada ^ 36 Table 8. Mean instantaneous growth rate (per day) of juvenile deer mice and ANCOVA of growth rate regressed on body weight for the deer mouse. **P< 0.01 ^ 39 Table 9. Sex ratio (proportion of males) for three small mammal species in summer 1990 and 1991. *P< 0.05; significant difference by chi-square test. Sample sizes in parentheses 41 Table 10. Shannon-Wiener indices of diversity and species richness of small mammal communities for 1990-1991 ^ 43 Table 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 three species were analyzed unless specified with initials of species investigated . . . 48 Table 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 content was tested by the formal-ether concentration method and microscopic examination of iron-hematoxylin stained preparations 62  vi  Table 13. Results of fecal and small intestine examinations of two groups of deer mice inoculated with different strains of and varying numbers of Giardia duodenalis  ^  64  vii  LIST OF FIGURES  Figure 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. ^ 10 Figure 2. Layout of study sites treated (Ti and T2) with sewage sludge. Blacked-out areas were treated with sludge in November 1990 ^ 18 Figure 3. Population density (per ha) of deer mice on treatment and control study sites for replicates 1 and 2. Vertical arrow indicates when sludge was applied to treatment areas. Jolly-Seber and MNA values are both shown ^ 23 Figure 4. Population density (per ha) of Oregon voles on treatment and control study sites for replicates 1 and 2. Vertical arrow indicates when sludge was applied to treatment areas. Jolly-Seber and MNA estimates are both shown 24 Figure 5. Population density (per ha) of Townsend chipmunks on treatment and control study sites for replicates 1 and 2. Vertical arrow indicates when sludge was applied to treatment areas. Jolly-Seber and MNA estimates are both shown. Shaded area indicates hibernation period 25 Figure 6. Cumulative number of recruits (per ha) of the three major small mammal species on all study sites during pre-sludge (unshaded) and post-sludge (shaded) periods during summer (1990-1991) periods, Maple Ridge, B.C., Canada 27 Figure 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) periods, Maple Ridge, B.C., Canada. Arrow indicates application of sludge on treatment areas. ^ 28 Figure 8. Early juvenile survival of deer mice and Oregon voles. Index is the proportion of juvenile animals observed over the expected number of juvenile animals, Maple Ridge, B.C., Canada., 1990-1991. Sample size (expected number of juveniles) in parentheses 35 Figure 9. Mean body weights and 95% confidence limits of males and females of the three major small mammal species on control and treatment sites during summer and winter periods. Sample sizes are above upper confidence limits ^ 37 Figure 10. Distribution of juveniles by weight and sexual maturity for deer mice on control and treatment 1 study sites, Maple Ridge, B.C., Canada ^ 40  viii  Figure 11. Species composition of study sites for summer and winter periods, Maple Ridge, 44 B.C., Canada ^ Figure 12. Distribution of deer mice captures on treatment sites before and after sludge treatment. Actual area sludged was blackened on each site, Maple Ridge, B.C., 45a-b Canada ^ Figure 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 ^ 46  ix  ACKNOWLEDGEMENTS  There 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 providing many hours of guidance and supervision, also gave me patience and understanding, especially during times of need. Dr. Judy Isaac-Renton, in addition to being on my committee, spent many hours 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 valuable information that were given to me. Staff at the University of British Columbia Research Forest allowed me the use of their facilities and were cheerfully helpful throughout the sometime rough running of the study. I am also grateful for the much appreciated help of Markus Merkens in the field. In the laboratory, I would like to thank Debra Hay, Iden Khan and Lorraine Lewin for their expert assistance and counsel. Financial support for this project was provided by the Greater Vancouver Regional District (J. P. Kimmins and J. Isaac-Renton) and the Natural Sciences and Engineering Research Council of Canada (T. P. Sullivan). Students of the forestry-wildlife group at U.B.C. provided a supportive and stimulating environment where many informal discussions generated valuable information and friendship. I owe special thanks to Markus Merkens and Todd Zimmerling for their competent assistance in dealing with computer and statistical problems, and many interesting debates. Special appreciation goes to Lois Campbell, Gabriella Matscha, Winston and Kelly Mew for moral support.  CHAPTER 1: General Introduction INTRODUCTION  The future disposal of sewage sludge has become a problem of major concern. Disposal of sludge into landfills is not only becoming unacceptable to the public with the recent increase in environmental awareness, but it is also prohibitively expensive in terms of transportation costs. This problem is expected to escalate because suitable disposal sites are becoming scarce and distant from city centres. Over 54.4 dry tonnes of sewage sludge is produced daily in the Greater Vancouver region. This figure is expected to double to 108.8 dry tonnes/day within the next decade (Kimmins et al. 1992). At the same time, existing waste water treatment plants in the Greater Vancouver region are facing shortages in storage capacity or are requiring major servicing of their equipment. Currently, the G.V.R.D. is reviewing six options to sewage disposal: agricultural land application, top soil production and composting, landfill cover and gravel pit reclamation, silviculture, mine reclamation and rangeland improvement. Five constituents in sludge are considered in determining the end usage of sludge. These are organic content, nutrients, pathogens, trace metals and toxic organic chemicals/synthetic organics. The organic content in sludge contains numerous essential nutrients necessary for plant growth, the most notable ones being nitrogen, phosphorus and potassium. Unfortunately, sludge also contains pathogens, a significant number of which can become concentrated in sludge during the wastewater treatment process. Thermophilic digestion or irradiation can destroy a large portion of these pathogens. However, since most sludges do not contain enough of these pathogens to warrant great concern, this process is not often considered due to its high cost. 1  Trace 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 can serve as essential micronutrients. In high concentrations, they may be toxic to humans, plants and 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 as replenish lost nutrients in forest soils which have become nutrient deficient from a history of repeated whole-tree harvesting or poor forestry practices. Furthermore, using sludge as a silvicultural fertilizer minimizes human health hazards because timber, unlike agricultural produce, does not enter directly into the human food chain. However, this option has received mixed reviews. Major concerns include potential deleterious effects on wildlife, heavy metal contamination in both surface or ground waters, and introduction of parasites or pathogens into the ecosystem. Research from Washington State's Pack Forest in recent years has demonstrated significant responses in tree growth on some sites. In general, no 'unacceptable' environmental consequences were found (Cole et al. 1986, Henry 1989) , although the majority of past studies on the effects of sludge on wildlife considered only animal abundance and heavy metal accumulation. 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 and iron, are considered essential plant nutrients. However, they can easily exceed plant requirements and become a problem of metal toxicity with heavy applications of sludge (Zasoski 2  1980). 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 University of British Columbia (U.B.C.) have collaborated in a large-scale research project investigating the potential environmental and health-related impacts of sewage sludge as a silvicultural fertilizer. Relative to other management practices, silvicultural sludge applications are not unlike the application of inorganic fertilizers; the main differences being the amount of water that is applied with sludge, the inability to control its exact contents and the perception of human waste products as unpleasant and unsanitary. Relative to other forest management techniques, sewage sludge applications are considered to impact the ecosystem to a much smaller degree than a practice such as clearcutting, for example, which alters the microclimate of the entire site in removing its canopy cover. Relative to other global environmental risks, such as the climate change 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 body of evidence supporting global warming, it is a risk that society has decided to accept. Modern risk assessment appears to be purely subjective at times. The final decision regarding operational silvicultural sludge applications will likely be made on a political or social rather than a scientific level. This study looks specifically at the potential impacts on small mammal populations from sludge applications and provides scientific data only to base decisions on the risks that may be taken with this group of wildlife.  3  LITERATURE REVIEW: EFFECTS OF SLUDGE ON WILDLIFE  Past studies on the effects of sewage sludge applications on wildlife focused on five major groups: ungulates, small mammals (heavy metal accumulation in insectivores was also considered), birds, amphibians and reptiles. A summary of the findings on mammals is discussed below.  Ungulates  Studies on deer (Odocoileus hemionus columbianus) have found an increase in both time spent on sludged areas and fawn recruitment on a seasonal basis. Fawn survival, however, was not recorded. These differences may be in response to the increase in the amount of crude protein 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 an increase in forage energy. One study examining the accumulation of heavy metals in deer found no significant accumulation in major deer repository organs (Anderson 1981).  Birds  Significant effects of sludge on bird abundance have not been reported. Some heavy metal concentrations were found to increase in some bird species as well as in certain invertebrate species which may be prey items (Milner 1986, West and Zasoski 1986). In general, the risk of heavy metal toxicity was considered small due to the high mobility of birds and relatively small size of sludged areas.  4  Small mammals  West et al. (1981) found that of the small mammal species they studied, abundance of insectivores (shrews) and gramnivores did not differ between sludged and control sites, but abundance of herbivorous small mammals was significantly lower on treatment compared to control 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 environmental conditions and most likely to survive sludge applications. Small mammals did not appear to take up eggs of this nematode parasite, although it was not clear whether the eggs of this parasite were abundant in the sludge initially (Raedeke and West 1986). Heavy metal studies found increased accumulations of cadmium and lead in the kidneys and livers of two species of insectivores and an omnivore/gramnivore mammal'. Although metal levels were too low to be considered physiologically harmful, this may become a consideration when sludge treatments are re-applied periodically over an extended period of time. Examination of a shrew population found no conclusive evidence of population decline despite increased metal accumulations (Hegstrom 1986).  'Heavy metals introduced into ecosystems in sludge, unlike pathogens and parasites, are not irreversibly fixed in the sludge matrix. Certain heavy metals can be taken up by plants as part of their metabolism (Taber and Zasoski 1980). 5  SMALL MAMMAL STUDY OBJECTIVES There are two major objectives in this study: 1) to determine the effects of a silvicultural sludge application on the population dynamics of resident small mammal populations, and 2) to evaluate the potential for Giardia contamination in a forest ecosystem. The ability of small mammals, specifically the deer mouse (Peromyscus maniculatus), to act as a host to human  Giardia cysts, was investigated. The prevalence of Giardia spp. cysts in small mammal fecal samples at study sites prior to and after sludge application was also monitored.  6  CHAPTER 2 Impacts of a Silvicultural Sludge Application on Small Mammal Populations  INTRODUCTION Small 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 than any other group of birds or mammals. Silvicultural sludge applications are often applied at the forest floor level. Birds and arboreal mammals are further removed from contact with sludge than small mammals which nest and feed on the forest floor directly. Small mammals, being a numerically abundant group, can be sampled in larger numbers on a given area and are therefore more suitable for statistical analysis. Sample sizes in studies of larger mammals, such as beavers (Castor canadensis) or deer, are usually much smaller, given the size of study sites and similar funding and time limitations. For these reasons, small mammals were selected as a 'sentry' wildlife group to determine the effects of silvicultural sludge applications on their population dynamics. Past small mammal studies have dealt mostly with the accumulation of heavy metals inside livers and kidneys of specimens, but not with the short-term response of dynamic population parameters which was a focus of this study. Consequently, live-trapping was selected as the sampling technique so dynamic population parameters could be estimated. The second reason for studying small mammals centers around the possibility that contaminated sewage sludge may provide an entry path for pathogens and parasites into the 7  forest ecosystem and ultimately into human populations. Giardia was selected as the parasite of interest due to its waterborne and fecal-oral modes of transmission. Giardia cysts are hardy and 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 daily activities or beavers ingesting cysts in contaminated streams were two possibilities that needed to be addressed. The potential for small mammals to function as hosts for human Giardia is as yet undetermined, but past studies have suggested it as a possibility (Davies and Hibler 1978, Pacha et al. 1987, Roach and Wallis 1988).  MATERIALS AND METHODS Description of study areas  This livetrapping study was located at the U.B.C. Malcolm Knapp Research Forest near Maple 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 the spring of 1975. Two 1-ha sites within this area were treated with sewage sludge. Treatment 1 was located on a dry upper slope, treatment 2 at a lower, mid-slope site and spaced approximately 250 m from treatment 1. The area was previously covered with a mature (70-90year-old) forest dominated by western hemlock (Tsuga heterophylla), Douglas-fir and western red cedar (Thuja plicata) (Feller 1977). Cover included well decomposed logging slash with an abundance of deciduous trees, shrubs and herbaceous vegetation. Associated with the Douglasfir stand were western hemlock natural regeneration and shrub species such as red alder (Alnus 8  rubra), birch (Betula papyrifera), willow (Salix spp.), vine maple (Acer circinatum), black  raspberry (Rubus leucodermis) and salmonberry (Rubus spectabilis). Bracken fern (Pteridium aquilinum) 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 in 1975. This area was not burned. The previous forest was also dominated by mature western hemlock and mixed with Douglas-fir and western red cedar. The main cover was well decomposed slash with a similar vegetation composition to the treatment site. Two control sites were 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 a sufficient distance to limit small mammal dispersal between the two areas. All sites were considered 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 protocol  From May 1990 to October 1991, two treatment and two control grids were livetrapped at 3-week (spring, summer, and fall) and 6- to 8-week (winter) intervals with Longworth live traps. On each grid, 49 (7 x 7) trap stations were located at 14.3-m intervals with one live trap placed within a 2-m radius of each station. Traps were baited with oats and a slice of carrot for moisture, and coarse brown cotton for bedding. Traps were set on day 1, checked on days 2 and 3, and then locked open between trapping periods. All small mammals captured, except shrews and weasels, were ear-tagged with fingerling fish tags, sexed, reproductive condition noted, and weighed on Pesola spring balances. The 9  Fig. 1. Study sites at the .University of B.C. Research Forest, Maple Ridge, B.C.. Stars denote study sites. Cl =control replicate one, C2 =control replicate two, T1 =treatment replicate 1, T2 = treatment replicate two.  10  duration of the breeding season was noted by palpation of male testes and the condition of mammaries of the females (Krebs et al. 1969). Small mammals were released immediately after processing. All shrews (Sorex spp.) which died in the traps due to the overnight-trapping technique were recorded as to their station of capture, collected and frozen for later identification of species using their dentition as the major classification criterion. Small mammal species encountered include the deer mouse, Oregon vole (Microtus oregoni), red-backed vole (Clethrionomys gapperi), Townsend chipmunk (Eutamias townsendii),  Pacific jumping mouse (Zapus trinotatus), American shrew-mole (Neurotrichus gibbsii), and two species of shrews (Sorex vagrans, and S. monticolus), and weasels (Mustela spp.). Three major small mammal species were caught consistently throughout the study: the deer mouse, Oregon vole and Townsend chipmunk.  Population parameters  Population parameters investigated in this study were population density, recruitment, proportion of population in breeding condition, probability of survival, juvenile survival, growth parameters (including mean adult body weight, juvenile growth rate and weight at sexual maturity), sex ratio, spatial distribution and species diversity. Most of the population parameters studied were estimated for three periods: summer 1990 (May - October), winter 1990-1991 (November - April), and summer 1991 (May - October) to minimize small scale temporal variations.  11  Population density and recruitment  Due to variation in trappability of small mammal species, population densities were estimated 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 are low (Jolly and Dickson 1983). Density values were estimated for each trapping period for the three numerically dominant small mammal species; the deer mouse, Oregon vole and Townsend chipmunk. Recruitment, the number of new animals joining the population, was calculated from observed numbers as a cumulative value for two main periods: pre-sludge (summer 1990) and post-sludge (summer 1991). Recruitment per trapping period (J/S estimates) on all study sites prior 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 was available for comparison.  Reproduction  Numbers of males and females reproductively active (i.e., scrotal or lactating) were obtained from field data with each animal being tallied at each capture. These values were then summed and calculated as a proportion of the total number of animals caught during that period.  Survival and growth  Average survival rates (J/S) for males and females in control and treatment populations 12  of both the deer mouse and Oregon vole were calculated. Survival values for chipmunks exhibited large variation in J/S values, and therefore were not used in this analysis. All survival values were calculated for three periods (summer 1990, winter 1990-91, summer 1991) with an individual animal being tallied at each capture. Juvenile survival was calculated by two methods. Early juvenile survival, defined as the percentage of observed juveniles/expected number of juveniles, was calculated for the deer mouse and Oregon vole (low sample size for Townsend chipmunk). Expected number of juveniles was the product of the number of successful pregnancies (based on consecutive captures of 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)}. The observed number of juveniles was obtained from field data using weight as the defining parameter (deer mouse: < 17 g; Oregon vole: < 20 g; Townsend chipmunk < 75 g) (Sullivan 1990b, Sullivan et al. 1983). Minimum juvenile survival values were summed over each period with an individual animal being tallied each time it was captured. Only the deer mice had enough juveniles for this analysis.  Body weights  Average 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,  13  Sullivan et al. 1983). Only males were used during the summer seasons because female data were complicated by undetected pregnancies.  Growth analysis  Growth 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 and control areas showed similar growth rates to each other (i.e., treatment 1 and 2, control 1 and 2) prior to and after sludge treatment and justified the pooling of juvenile growth rates for this analysis. As growth rate is dependent on body weight, an ANCOVA of growth rate regressed on body weight was done for juvenile deer mice. Temporal (pre vs. post-sludge periods) and spatial (treatment vs. control sites) comparisons were then made. Only deer mice had a sufficient sample size for this analysis. Smaller sample sizes of Oregon voles and Townsend chipmunks are not suited to this analysis due to the variability in data which would have masked any significant differences. Weight at sexual maturity of deer mice was also graphed for all study sites, pre- and post-sludge treatment. Juveniles from 11-16 grams were categorized as to their breeding condition (scrotal or abdominal, lactating or non-lactating) for all study areas for the summer periods. Minimum weights at sexual maturity were then compared spatially and temporally.  Sex ratio Sex ratio (proportion of males to total population) was calculated using MNA values. 14  Individual animals were tallied at each capture and summed over each period. For reasons that are discussed in the statistical analysis section, values were compared using the chi-square test.  Species diversity and richness  The Shannon-Wiener index of diversity (Pielou 1966) was used in this study to consider both 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 to changes in rare species (Peet 1974). The assumptions of this index are that individuals are randomly sampled from an infinite population (i.e., an open versus a closed population) and all small 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 estimates for the three main species. Actual observed numbers from field data were used for less abundant 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 shrews were identified, these samples were used in the calculation (the percentage of live shrews ranged from 33-50% of actual number of shrews caught). This method was considered acceptable because 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 were made 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 species composition may occur even though overall diversity does not change (i.e., the relative 15  abundances of species in a community may change, but if the overall ratio of species remain the same, these changes will not be detected in the diversity index). To reinforce this diversity analysis, the relative cumulative proportions of species captured were also estimated for all sites before and after sludge application.  Spatial distribution  The effect of sludge application on the spatial distribution of deer mice was studied by comparing the relative proportion of deer mice captures in the sludged area inside each grid to that of the total number of captures on the study grid. These values were then compared between pre-sludge and post-sludge periods. However, duration of the periods used for comparison varied between replicates. In treatment 1, a total of 8 trapping periods prior to and after sludge applications was considered in the calculation. In treatment 2, only a total of 4 trapping periods was used. This was due to bear disturbance on study sites which resulted in a reduced number of operable live traps on certain sites at various times. The total number of operable traps was kept constant for the comparisons, hence the variation in temporal duration.  Statistical analysis  Standard statistical analysis was inappropriate for many comparisons because the analyses involved samples of the same animals captured in consecutive trapping periods. The chi-square test, 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 an analysis of variance because it is a more robust test when deviations from the basic assumptions 16  occur. Consequently, test results were only an indication of the degree of difference between sets of data and may not be statistically valid. This was the case for proportion of population breeding, survival, average adult weight, weight at sexual maturity and sex ratio. The one-tailed Fisher Exact test was used for the analysis of the proportion of population reproducing in the Townsend chipmunk due to low sample size (Zar 1984).  Sludge application  Anaerobically-digested sludge was obtained from the Annacis Island Sewage Treatment Plant in Delta, B.C.. Truckloads of sludge were transported to the Research Forest and stored in 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, a tractor fitted with an agitator (propeller on a long extension) mixed the sludge to a uniform consistency. 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 covered treatment grids. The actual areas sludged are marked in Figure 12 as part of the spatial distribution analysis. The total amount of sludge applied was 173,823 kg (22% solids) of which the average nitrogen content was 3.9%. Variation between applications was monitored by periodic collection of sludge samples and analysis for nitrogen content. An estimated average of approximately 500 kg N/ha was applied to sites (Kimmins et al. 1992). Sludge application began on November 25 and was completed on November 30, 1990.  17  Fig. 2. Layout of study sites treated (T1 and T2) with sewage sludge. Blacked-out areas were treated with sludged in November 1990.  18  RESULTS Changes to habitat  Effects of sludge application on vegetation were studied on the treatment study areas concurrently with this study. Changes in parameters such as percent cover, height, light, and foliage nitrogen and phosphorus content were monitored. Significant increases in nitrogen and phosphorus content were found in salal leaves (Gaultheria shallon) on treatment areas compared to control areas. In addition, relative percent cover decreased for salal but increased for bracken fern in the post-sludge year (L. Coward, pers. comm.) 2 .  Trappability  The enumeration of small mammals is based on the assumption that most individuals are captured in a given population. Estimates of trappability used in this study were the minimum unweighted 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 based on 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 between female and male deer mice. Oregon vole and chipmunk values were too variable for patterns to be discerned.  Laura Coward, M. Sc. candidate in Forest Sciences. Thesis topic was on the effects on vegetation from a silvicultural sludge application, Department of Forest Sciences, University of British Columbia, Vancouver, B.C. Canada. 2  19  Table 1. Minimum unweighted trappability estimates for the deer mouse, Oregon vole and Townsend chipmunk for three periods on all study sites. Sample sizes in parentheses. Minimum unweighted trappability eliminates first and last captures and provides only one value for each individual regardless of how long it lives.  Control 1  Control 2  Treatment 1  Treatment 2  P. maniculatus  Males  Females  Males  Females  Males  Females  Males  Females  Summer 1990 Winter 1990-91 Summer 1991  0.79 (22) 0.96 (11) 0.78 (3)  0.74 (27) 0.91 (13) 0.76 (7)  0.83 (9) 0.89 (6) 1.00 (3)  0.74 (10) 1.00 (3) 0.92 (2)  0.86 (17) 0.64 (12) 0.68 (8)  0.88 (20) 0.80 (8) 0.61 (8)  0.78 (19) 0.70 (10) 0.78 (9)  0.65 (20) 0.85 (10) 0.86 (6)  0.59 (7) 0.81 (1) 0.50 (1)  0.46 (13) 0.33 (2) 0.83 (2)  0.80 (12) 0.69 (5) 0.50 (2)  0.78 (18) 0.27 (5) 0.63 (6)  0.37 (3) 0.80 (1) 0.67 (1)  0.47 (3) 0.00 (1) 0.20 (1)  0.50 (5) 0.80 (2) 1.00 (1)  0.76 (5) 0.33 (2) 0.50 (1)  0.24 (4) 0.13 (3) 0.33 (4)  0.20 (2) 0.00 (1) 1.00 (1)  0.38 (4) 0.11 (3) 0.50 (1)  0.48 (3) 0.40 (1) 1.00 (1)  0.57 (8) 0.10 (6) 0.00 (0)  0.50 (4) 0.00 (2) 0.12 (3)  0.78 (5) 0.20 (3) 0.19 (3)  0.67 (5) 0.08 (5) 0.35 (6)  M. oregoni  Summer 1990 Winter 1990-91 Summer 1991 E. townsendii  Summer 1990 Winter 1990-91 Summer 1991  Table 2. Jolly-Seber trappability estimates for the deer mouse (Peromyscus maniculatus), Oregon vole (Microtus oregoni) and Townsend chipmunk (Eutamias townsendii) for three periods on all study sites ( ± 95% confidence intervals). PERIOD  P. maniculatus Summer 1990 Winter 1990-91 Summer 1991  M. oregoni Summer 1990 t) 0--  Winter 1990-91 Summer 1991  E. townsendii Summer 1990 Winter 1990-91 Summer-1991  Control 1  Control 2  Treatment 1  Treatment 2  Males  Females  Males  Females  Males  Females  Males  Females  0.69 (0.52-0.86) 0.89 (0.63-1.00) 0.68 (0.45-0.92)  0.71 (0.61-0.81) 0.84 (0.65-1.00) 0.81 (0.64-0.99)  0.83 (0.73-0.93) 0.84 (0.56-0.00) 0.86 (0.51-1.00)  0.83 (0.71-0.96) 1.00 (1.00-0.00) 0.86 (0.36-1.00)  0.78 (0.64-0.91) 0.79 (0.58-1.00) 0.73 (0.36-1.00)  0.82 (0.69-0.95) 0.82 (0.62-1.00) 0.78 (0.45-1.00)  0.69 (0.53-0.84) 0.73 (0.44-1.00) 0.83 (0.69-0.96)  0.75 (0.64-.86) 0.78 (0.61-0.96) 0.81 (0.62-1.00)  0.80 (0.59-1.00) 0.80 (0.24-1.00) 0.64 (0.20-1.00)  0.61 (0.44-0.77) 0.41 (0.00-0.93) 0.86 (0.63-1.00)  0.90 (0.80-1.00) 0.73 (0.34-1.00) 0.78 (0.44-1.00)  0.75 (0.54-0.96) 0.50 (0.00-1.00) 0.78 (0.56-1.00)  0.26 (0.05-0.47) 0.80 (0.24-1.00) 0.71 (0.26-1.00)  0.56 (0.15-0.96) 0.25 (0.00-0.79) 0.43 (0.00-0.92)  0.59 (0.34-0.84) 0.72 (0.23-1.00) 0.43 (0.00-0.92)  0.80 (0.60-1.00) 0.74 (0.30-1.00) 0.61 (0.15-1.00))  0.38 (0.13-0.62) 0.15 (0.00-0.51) 0.29 (0.00-0.64)  0.56 (0.15-0.96) 0.20 (0.00-0.76) 0.43 (0.00-0.93)  0.47 (0.31-0.64) 0.47 (0.00-1.00) 0.57 (0.07-1.00)  0.59 (0.26-0.92) 0.40 (0.00-1.00) 0.43 (0.00-0.93)  0.56 (0.31-0.81) 0.15 (0.00-0.37) 0.29 (0.00-0.74)  0.48 (0.27-0.69) 0.00 (0.00-0.00)) 0.33 (0.00-0.68)  0.80 (0.57-1.00) 0.33 (0.00-0.70) 0.41 (0.07-0.75)  0.65 (0.36-0.95) 0.15 (0.00-0.42)) 0.44 (0.27-0.61))  Total population  Pre-sludge application period (Summer 1990)  Population density estimates calculated by MNA and Jolly-Seber exhibited comparable fluctuations over time for all three species (Figs. 3-5). For this reason, these estimates are assumed to be reasonably accurate in reflecting the numerical responses to treatment for these three small mammal species. Population density fluctuations of deer mice on all study sites were similar for the summer of 1990 (May-August). All populations had an initial density of approximately 15 deer mice/ha (J/S). With the onset of breeding, populations experienced a significant increase. In September 1990, however, the control population in replicate 1 increased but the replicate 2 control population declined (Fig. 3). Oregon vole populations increased steadily through the late summer and fall of 1990 to 22/ha on control site 1 and 28/ha on control site 2 (J/S) (Fig. 4). Both treatment vole populations appeared to decrease marginally in September from initial densities of approximately 4-8 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 control populations (7-8/ha) (J/S), particularly in the late summer-fall period (September-November 1990) (Fig. 5).  22  Perom yscus manicula tus  T^T^I^I^I-^-I-  M  S Time  1990  0  1991  70 60 50 1:1 '10 40  s:1  O  30 0 20 C:14 10  1^I^,^I^I^,^1  I^1^■^  M^3^3^S^0^D^M M^3^J ^1990^  Time  1991  S  Fig. 3. Population density (per ha) of deer mice on treatment (—) and control (-) study sites for replicates 1 and 2. Vertical arrow indicates when sludge was applied to treatment areas. Jolly-Seber (q) and MNA (+) values are both shown.  23  0  Micro tus oregoni  r  M^J^J^S  0  TAIIVII^1111  D M M J^J Time^  1990  IIIIIIIII1  -  Time^  0  1991  111-^I-I  M^J^J^S^0 D M M J^J ^1990^  S  ^ ^ 0 S  1991  Fig. 4. Population density (per ha) of Oregon voles on treatment (—) and control (-) study sites for replicates 1 and 2. Vertical arrow indicates when sludge was applied to treatment areas. Jolly-Seber (a) and MNA (+) estimates are both shown. 24  Eutamias townsenciii  M^J^J^S  ^  ^ ^ ^ 0^D M M 0 S J^J ^ 1991 Time  1990^  M  ^  J^J  ^1990  ^  S^0^D^M  ^  Time  ^ ^ ^ ^ 0 S M J^J  ^1991  Fig. 5. Population density (per ha) of Townsend chipmunks on treatment (—) and control (-) study sites for replicates 1 and 2. Vertical arrow indicates when sludge was applied to treatment areas. Jolly-Seber (m) and MNA (+) estimates are both shown. Shaded area indicates hibernation period. 25  Sludge application period (Winter 1990-1991)  Deer mouse and Oregon vole populations did not undergo any changes in abundance immediately after sludge treatment (arrow in Figs. 3 and 4). Effects on the Townsend chipmunk were not detected during their hibernation period. An decrease in abundance in December for these three species was experienced on all study sites and continued throughout the winter period.  Post sludge application period (Summer 1991) -  A consistent pattern of reduction in abundance in the post-treatment year was found in the 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 similar changes expressed by both estimates of abundance used. Increases in abundance as observed in July 1990 were not repeated. Deer mouse and Oregon vole populations on control site 2 decreased to almost zero in July in the post-sludge period and increased marginally over the rest of the period. Chipmunk densities in the second year were slightly lower than first year densities on all study sites.  Recruitment  All study sites experienced a large decrease (minimum 50%) in both cumulative {total number of recruits/period (field data)} (Fig. 6) and weekly number of recruits in the post-sludge period for all three species (J/S estimates) (Fig. 7). No consistent effects from sludge treatment were found. Cumulative recruitment of voles was higher on control than treatment sites in the 26  Peromyscus maniculatus Cumlotive number of recruits  TMT 1  CON 1  TMT 2  CD Pre-sludge^  MI  CON 2  Post-sludge  Microtus oregoni 60  Cumulative number of recruits  20  10  11.11 TMT 1  CON 1  TMT 2  CON 2  Pre-sludge^Post-sludge  Eutamias townsendii Cumulative number of recruits  Pre-sludge^  Mg  Post-sludge  Fig. 6. Cumulative number of recruits (per ha) of the three major small mammal species on all study sites during pre-sludge (unshaded) and post-sludge (shaded) periods during summer (1990-1991) periods, Maple Ridge, B.C., Canada. 27  Eutamias townsendii  Microtus oregoni  Peromyscus maniculatus  10 Treaomeol I^  11111 Truamew I^C.1 C..0.11 I  CD  25  20  20 -  6  IS 15  10 10 2  1 M^.1^J^S 1990  M  (^111  M^I^1 1111  0  S^0 D M M J^J^SO  5^0 1110  1110  J  1  At^J  1111  1991  10  30 TrIssa.11^C:3 6.0.11  CD cO.rvai 20  PEI^3^J  -  10  S  0  Ip11, 1 P,NU„  JS^0^D^14MJISO 1190  M^J^J ISSO  1991  Pig. 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.  first year. A notable decrease occurred for the Oregon vole on control sites from the pre-sludge to post-sludge period. Chipmunk recruitment followed a similar pattern to the other two species in having a generally higher recruitment in the first year than the second. However, smaller sample 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 high recruitment (J/S estimates) prior to sludge application were found: on control site 1 and treatment 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 during periods of low deer mouse recruitment (Fig. 7) in this period. Contrary to the other two species, periods of high recruitment in the Townsend chipmunk were repeated in the second year, although to varying degrees on the study sites.  Reproduction  No 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 breeding condition. However, this parameter was found to have increased significantly (P< 0.01) on treatment site 1 for both male deer mice and Townsend chipmunks (low sample size) between pre- and post-sludge periods. This increase was not observed on control sites (Table 3). Due to the small sample size, however, it was concluded to be attributed solely to the sludge treatment. 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). 29  Table 3. Proportion of male adults of three main species in breeding condition (scrotal) during the breeding season of each year. Sample sizes in parentheses. Replicate 2 Replicate 1^ ^ Control 2^Treatment 2 Control 1^Treatment 1  P. maniculatus  1990^0.41 (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. oregoni  1990^0.30 (37)^0.67 (9)^0.34 (59)^0.67 (27) L.)^ 1991^0.27 (11)^0.83 (6)^0.35 (23)^1.00 (4) o E. townsendii 1990^0.12 (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 the breeding season of each year. Sample sizes in parentheses. Replicate 2 Replicate 1^ ^ Control 2^Treatment 2 Control 1^Treatment 1 P. maniculatus  1990 1991  0.33 (49)a 0.92 (26)a  0.27 (52)b 0.65 (31)b  0.15 (39)c 0.60 (10)c  0.29 (48)d 0.77 (22)d  0.53 (30) 0.67 (3)  0.67 (6) 0.33 (3)  0.32 (19) 0.86 (14)  0.40 (15) 0.60 (5)  0.00 (9) 0.40 (5)  0.00 (7) 0.33 (3)  0.46 (13) 1.00 (2)  0.05 (21) 0.29 (14)  M. oregoni  1990 1991 E. townsendii  1990 1991  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 survival  No significant changes in spatial and temporal comparisons for either sex of deer mice or Oregon voles (± 95% CI, Tables 5 and 6) were found. Estimates of Townsend chipmunk survival values were too variable for analysis.  Juvenile survival  Consistent with other results, percentage juvenile survival for deer mice decreased significantly 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 on treatment site 1, but a decrease on treatment site 2 (Fig. 8). Both control sites experienced a decrease. Sample sizes for the Townsend chipmunk were too low for evaluation of this parameter. Mean minimum juvenile survival rates were also calculated for deer mice (sample size too low for Oregon vole and Townsend chipmunk) (Table 7). Juvenile survival decreased on all sites in general agreement with previous percentage juvenile survival values (Fig. 8) except on control site 2 where it increased. This may be an artifact of small sample size. No consistent effects could be found attributable to sludge treatment.  Average adult weight  Average male and female adult weights were found to be not significantly different (based on 95 % confidence intervals) between control and treatment populations in temporal and spatial comparisons for the deer mouse, Oregon vole, and Townsend chipmunk (Fig. 9). 32  Table 5. Jolly-Seber average survival rates per period (1990-1991) for control and treatment populations of deer mice (± 95% confidence intervals). Season Replicate 1 Summer 1990 Winter 1990-1991 Summer 1991  Females  Males  Treatment  Treatment  Control  0.86 (0.77-0.94) 0.73 (0.62-0.83) 0.77 (0.52-1.00)  0.87 (0.79-0.95) 0.73 (0.66-0.88) 0.61 (0.37-1.00)  0.75 (0.66-0.85) 0.53 (0.38-0.69) 0.75 (0.52-0.99)  0.78 (0.73-0.83) 0.64 (0.73-0.87) 0.56 (0.23-1.00)  0.79 (0.69-0.90) 0.80 (0.64-0.96) 0.83 (0.30-1.00)  0.85 (0.75-0.95) 0.77 (0.63-0.84) 0.83 (0.41-0.81)  0.77 (0.66-0.89) 0.77 (0.62-0.93) 0.49 (0.13-0.86)  0.78 (0.66-0.90) 0.80 (0.51-0.76) 0.56 (0.56-0.93)  Control  Replicate 2  Summer 1990 Winter 1990-1991 Summer 1991  ^  Table 6. Jolly-Seber average survival rates per period (1990-1991) for control and treatment populations of Oregon voles (± 95% confidence intervals). ^ Males Season Females^ Control^Treatment^Control^Treatment Replicate 1 Summer 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 2 Summer 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)  % Juvenile Survival Peromyscus maniculatus 100 140.51  158.5) (27)  80 -  14.1.5)  60 -  131.51  (131  40 -  20 -  TMT 2^CON 2  CON 1  TMT1  % Juvenile Survival Micro tus oregoni 100  132) KW  80 122.0 11.41  60  19.1) (32)  112.1)  40  112.11  20  0 TMT1  ^  CON 1  ^  TMT 2  ^  CON 2  PRE-SLUDGE^al POST-SLUDGE Fig  8.  Early juvenile survival of deer mice and Oregon voles. Index is the  proportion of juvenile animals observed over the expected number of juvenile animals, Maple Ridge, B. C., Canada, 1990-91. Sample size (expected number of juveniles) in parentheses. 35  Table 7. Mean minimum survival rates per period for control and treatment populations of juvenile deer mice, Maple Ridge, B. C., Canada. ^ Period Control^Treatment^Control^Treatment Females^ Males  Replicate 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) w^ Summer 1991^0.00^(6)^0.62^(13)^0.20^(10)^0.36^(11) a■ 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)  ^lel  Peromyscus maniculatus 21 I27■ ^  Control^Trrattnent  20 125 ) 17 1i  a  (511  1691  al  1211  1171  18  17  ^ ^ Summer Winter Summer ^ ^ 1990 1990-1991 1991  Microtus oregoni 26 139/  ^  Control^• Treatment  25  191  24  U)  E  co CI  23  110  151  22  21  20  ^ ^ Summer Winter Summer ^ ^ 1991 1990 1990-1991  Eutamias to wnsendii  90 ^  Control^• Treatment  19)  85  co  E  co  (231  ^  151  80  0  75  70  ^ ^ Summer Winter Summer ^ ^ 1991 1990 1990-1991  Fig. 9. Mean body weight and 95% confidence intervals of males and 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. 37  Juvenile growth rate A significant difference was found in growth rate of juvenile deer mice in treatment populations after sludge application. Mean instantaneous growth rate (per day) for the postsludge 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 statistically significant (Table 8).  Weight at sexual maturity Only one small change in minimum weight at sexual maturity was found for all spatial and temporal comparisons for deer mice. Juvenile males on control site 1 in summer 1990 began breeding (i.e. scrotal) at 14 grams, whereas breeding males were found at 16 grams in summer 1991 (Fig. 10). All other comparisons found weight at sexual maturity to be similar. Females rarely became sexually mature before 17 grams.  Sex Ratio No consistent differences were found in temporal or spatial comparisons for both the deer mouse and Oregon vole (chi-square analysis). The pre-sludge sex ratio value for the Townsend chipmunk, however, was found to be significantly higher than the post-sludge value (P< 0.05, Table 9).  38  Table 8. Mean instantaneous growth rate (per day) of juvenile deer mice per period and ANCOVA of growth rate regressed on body weight for the deer mouse. **P<0.01. Mean instantaneous growth rate (per day) (X 10)  F-value  Probability  Pre-control vs. Post-control  2.7 3.7  0.32  0.75  Pre-treatment vs. Pre-control  2.3 2.7  0.40  0.59  Post-treatment vs. Post-control  6.1 3.5  1.26  0.27  Pre-treatment vs. Post-treatment  2.3 6.1  11.43  0.0009**  Comparison  •  ^ Juveniled deer^ mice Period 1  20  Juvenile (5 deer mice Period 1  25  20 07  c IS 2  0  •  0  d  10  z  z 5  0 11  ^  0  ^ ^ ^ IS 16 ^ Weight (grams)  12^13^14  11  ^  12^13^14  Weight (grams)  Juveniled deer mice Period 3 20  Control 1  ^ ^ 16 15  Juvenile a deer mice Period 3  25 NI Abdominal^Scrotal 20  15 0  =  12.10  0  d  d 10  z  z  S S  11  ^  13^14 Weight (grams)  15  16  0 11  ^  12^13^14  15  Weight (grams)  Fig. 10. Distribution of juveniles by weight and sexual maturity for deer mice on control and treatment 1 study sites, Maple Ridge, British Columbia, Canada.  16  Table 9. Sex ratio (proportion of males) for three small mammal species in summer 1990 and 1991. *P< 0.05; significant difference by chi-square test. Sample sizes in parentheses. Pre sludge -  P. maniculatus Treatment 1 2 Control 1 2  Post sludge -  0.43 0.41 0.56 0.45  (220) (221) (257) (155)  0.41 0.50 0.55 0.56  (109) (102) (78) (45)  0.73 0.38 0.48 0.49  (37) (71) (130) (158)  0.67 0.20 0.61 0.52  (18) (20) (26) (56)  0.82 0.64 0.80 0.75  (74)* (72) (46) (51)  0.59 0.62 0.76 0.77  (29)* (55) (34) (13)  M. oregoni  Treatment 1 2 Control 1 2 E. townsendii  Treatment 1 2 Control 1 2  Species Diversity  Diversity indices of small mammals (calculated for pooled treatment and control populations: i.e. replicates 1 and 2 combined) between treatment and control sites prior to sludge treatment were similar. Temporal and spatial comparisons found no significant differences between diversity indices on all sites (t-test). Species richness values also were similar between populations (Table 10). The proportion of each species captured relative to the total number of captures was represented and compared between periods. This analysis was carried out to detect changes in species composition. No difference was found attributable to sludge treatment (Fig. 11).  Spatial distribution  Overlays of actual areas sludged before and after treatment failed to reveal any obvious changes 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 proportion of deer mice captures in the sludged areas to the entire study area before and after sludge application (Fig. 13). This analysis was not possible for the Oregon vole and the Townsend chipmunk, due to their low sample sizes.  42  Table 10. Shannon-Wiener indices of diversity and species richness of study sites for 1990-1991. Period  Shannon-Wiener Indices  Site  No. of species  Control  Pre-sludge  1.57  1 2  6 7  Treatment  Pre-sludge  1.58  1 2  6 9  Control  Post-sludge  1.76  1 2  5 7  Treatment  Post-sludge  1.48  1 2  6 6  43  Cumulative Proportions: Control 2  Cumulative Proportions: Control 1  /NW AMP%  I 00  ,,,,,,,,,,,,,,,,,,,,,,,,  100  80  80  60  60  40  40  20  20  0  0 Summer 1990  Per 10./14^  Winter 1990.91  EIEM Orate. vole^=1 Chipmuck^FEE  Shrma 1^Jump. mouse  Winter 1990.91  Summer 1990  Summer 1991  INN  S►re. I  11111 Red-backed vole  Oregon vole^=1 Chipmunk^EEM St Inn. I  Deer mouse  1.1 Shrew 2  Summer 1991  0  Jump mouse  MI  Red-bodied yob  Cumulative Proportions: Treatment 2  Cumulative Proportions: Treatment 1 100  100 80 60  MIL,ILL7,1  so  40  40  20  20 0  Summer 1990  MN Deer mouse Shrew 2  Winter 1990-91  =1 Chipmunk Oregon vole C.= Jump. moos IBM Weasel  Summer 1991 Shrew 1 iM:1^Red -backed vole  :a4;;O/4.4:4;i  Summer 1990 Door moos  MN Shrew 2  Winter 1990-91 WO Oregon yak  =1 Jump. mouse  Ea  Chipmunk  111111  Weasel  Summer 1991 k'1,X'^Wow I  Etg^Red-tacked role  Fig. 11. Species composition of study sites for summer and winter periods, Maple Ridge, B.C. Canada.  PRE-SLUDGE TREATMENT 1 Peromyscus maniculatus  25 20 15 10 -  1  2^3^4^5^6^7  PRE-SLUDGE TREATMENT 2 Peromyscus maniculatus  25 20 15 10 -  1  2  3  4  5  6  7  To compare spatial distribution of deer mice captures before and after sludge application, superimpose this figure onto Figure 12. 45a  POST-SLUDGE TREATMENT 1 Perom yscus maniculatus  1  ^ ^ ^ ^ ^7 6 4 2 3  POST-SLUDGE TREATMENT 2 Perbmyscus maniculatus  1  2  3  4  5 - 6 7  f]  Fig. 12. Distribution of deer mice captures on treatment sites before and after sludge treatment. Actual area sludged was blackened on each site, Maple Ridge, B.C., Canada.  11  45b  EQUIVALENT AREAS SLUDGED  / %%  UNSLUDGED  .77  1 Post-sludge  0 Pre-sludge^Post-sludge  CONTROL 1  CONTROL 2  IIM SLUDGED  UNSLUDGED  Pre-sludge^Post-sludge  Pre-sludge^Post-sludge  TREATMENT 2  TREATMENT 1  Fig. 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.  46  DISCUSSION The application of sewage sludge could potentially have significant effects on the population dynamics of small mammals in a forest environment. However, from data gathered here, it would appear that there was no major effect on population parameters attributable to sludge treatment one year after application. These parameters included abundance, reproduction, growth, survival, sex ratio, species diversity and spatial distribution. Juvenile growth rates of deer mice did increase after sludge treatment. No other population parameters appeared to be affected (Table 11).  Experimental design  Improvements in the experimental design suggested for future studies include increasing the number of replicates, as well as in the scale, timing and duration of application. Only two sites 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 is recommended in future studies. This would have also helped to clarify site-specific results which could not be attributed entirely to the sludge treatment. Population densities of the Oregon vole varied between treatment and control populations. Control populations occurred at higher densities than treatment populations in the pre-sludge year. A possible cause for this could be the concurrent occurrence of weasels on treatment grids. Weasels are efficient predators and females have been recorded to prefer voles as a prey species (Microtus pennsylvanicus) (Raymond et al. 1990).  A similar difference in population density was also found for the Townsend chipmunk. 47  Table 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 three species were analyzed unless specified with initials of species investigated. 0  Total population (MNA and J/S)  per trapping session  Recruitment  0 0  cumulative no. of recruits/period weekly number of recruits  Proportion of animals breeding/period  + (treatment 1 only)Pm 0  males scrotal females lactating  Probability of survival (J/S) {pm,mo}  summer 1990 vs. summer 1991  0  Juvenile survival^{pm,mo}  0 0  percentage juvenile survival minimum survival rates (per 21 days)  Mean body weight  per period  Age at sexual maturity  ^  per period  0 {pm}  0  Juvenile growth^{pm}  per period  + pm  Sex ratio (MNA) per period  0  Species diversity  0 0  Shannon-Wiener indices species composition  Spatial distribution^{pm}  0  relative no. of deer mouse captures (per period) distribution of captures (per period)  0  pm: P. maniculatus mo: M. oregoni  48  Treatment population densities were consistently higher than control densities in the pre-sludge year. This, in addition to the difference observed in Oregon vole populations, suggested that there may be the existence of 'metapopulations', or isolated populations acting independently of other surrounding populations. It is difficult to determine what are the direct causes of this pattern without data from additional replicates. However, an overview of my results with respect to population parameters (Table 11) showed a consistent lack of effect from the sludge application. Application on a larger scale (e.g. 5-10 ha) is also suggested. Sludge applications in the size range of this study may be too small to significantly impact mobile small mammals such as the deer mouse and chipmunk. These generalist species may be able to modify their behavior and 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 suitable for small scale studies such as this include soil invertebrates, insects, and amphibians. It is important to note that this study concentrated on only one wildlife group: small mammals. Other resident fauna could potentially be affected by the sludge treatment and need to be considered as well. Future studies should apply sludge in the middle of the summer season when vegetation and small mammals are actively growing. Parameters such as reproduction, growth and recruitment are best studied at this time. The application of sludge in this study took place in late 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-term effects on population dynamics not exhibited in a short-term study. 49  Density and recruitment  Annual and multi-annual fluctuations in abundance of small mammal populations are not unexpected 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 suggests that small mammal populations were at a low point in their cycle rather than their low population numbers being a result of sludge treatment. Decreases in Oregon vole abundance on all sites were attributed to the characteristic cyclic fluctuations of this species (Gashwiler 1972, Hooven and Black 1976, Sullivan and Krebs 1981b), with the 1991 data covering a presumed decline in vole abundance. The observed increase in Oregon vole recruitment during periods of reduced deer mouse recruitment is likely related to competitive interactions between the two species. Data collected from 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 two species in a young plantation (Petticrew and Sadleir 1974). Moreover, Oregon voles have been shown 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 sludge treatment as illustrated by the lack of effect on its survival and reproductive attributes. This is reinforced by the work of Petticrew and Sadleir (1974) and Sullivan (1979) which concluded that there was little variation in demographic parameters of deer mouse populations in different habitats of coastal forests. 50  Growth and survival  The increase in growth rate of deer mice on treatment sites suggests that fertilization effects from the sludge may have caused an increase in growth of juvenile deer mice by increasing the nutrient content in the vegetation. Increases in nitrogen and phosphorus were also found 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 in 1991 did not affect this species' abundance, survival, recruitment or average adult weight. This may be due to short-term effects of the sludge application. The timing of the application may have also contributed to a limited effect. Sludge was applied in late November when deciduous vegetation was in senescence. More extensive effects may have occurred if sludge was applied in the midst of summer when both small mammals and ground vegetation were actively growing.  Reproduction  A site-specific increase in the proportion of breeding male deer mice in the post-treatment year was found on treatment site 1. This may be related to the increase in the growth rate of juvenile deer mice. Some distinguishing feature on treatment site 1 may have increased sensitivity of deer mice to sludge treatment. However, this site-specific response cannot be solely attributed to sludge application. Additional replicates would have been needed to identify any treatment effects. The proportion of breeding female deer mice increased in the second year on all four experimental sites. This may have been due to concurrent population decreases observed on 51  these sites, which in turn relaxed competitive pressures for resources. This gender-specific response may stem from different behavioral responses between sexes during various seasons of the year. Agonistic behaviour between male deer mice during the breeding season has been suggested 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 same extent (Petticrew and Sadleir 1974). Juvenile male deer mice, despite a decrease in overall abundance, are still faced with agonistic behaviour from established adult males.  Other studies  Other studies investigating the effects of sewage sludge on small mammals have revealed variable results. Hegstrom's (1986) study on shrews found no response in abundance to sludge treatment, although insectivores had higher levels of cadmium in their livers and kidneys after treatment. Conversely, West et al. (1981) found that treatment populations of herbivorous rodents (M. townsendii, M. oregoni, C. gapperi) were significantly lower in density than those on control sites, presumably due to changes in plant species which acted as food and cover for this group. Their study, however, was based on data from one trapping period of four days in duration and lacked temporal controls. No other studies were found that investigated the influence of sludge on the population dynamics of small mammals. Other habitat alteration studies have found that some rodents have a strong negative reaction to disturbance or loss of their habitat (as cited in Sullivan and Sullivan 1982). This is especially pronounced in 'intolerant' or specialist species such as the Oregon vole. Birney et al. (1976) proposed that a minimal level of vegetative cover was necessary to permit Microtus  52  spp. to increase in abundance during their 3-4 year cycle. This reliance on cover was attributed to the need for visual cover from diurnal predators. Unfortunately, the Microtus species in my study underwent a population decline in the post-treatment year which made analysis difficult due to a small sample size. Based on the data from this study, however, M. oregoni was not observed 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 number of successful pregnancies), growth and survival. This lack of effect was not observed for clearcut logging or burning. Sullivan and Krebs (1981a) found an 'irruption' in the number of deer mice in an old-field habitat adjacent to a forest immediately after the forest was harvested by clearcutting. This increase in recruitment was not recorded in the study site in the logged area. In another study, a significant decrease in deer mice abundance was found shortly after a 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 few months (T. Sullivan, pers. comm.) 3 . Relative to other habitat alteration practices, silvicultural sludge applications are expected to have less impact on small mammal populations than logging or burning. In sludge applications, deciduous vegetation is not altered as intensely as a herbicide application or a  Dr. T. P. Sullivan, professor of forests and wildlife, Department of Forest Sciences, University of British Columbia, Vancouver, B.C., Canada. 3  53  clearcut logging operation. In addition, fertilization effects of the sludge may enhance some vegetation after treatment. Consequently, the impacts on small mammals which utilize this vegetation as food and habitat are expected to be not as severe. This prediction is supported by my 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 metal accumulation or the sludge application itself. Furthermore, future sludge applications are unlikely to be of such small scale or short duration as in this pilot study. In light of this, it is suggested that additional research be conducted on small mammals and other wildlife groups.  54  CHAPTER 3:  Giardia contamination in Small Mammals  INTRODUCTION  One of the primary health issues surrounding sludge applications as silvicultural fertilizers is the introduction of pathogens and parasites into the forest ecosystem, a potentially irreversible process. The parasite group of concern in this study is Giardia duodenalis. Giardia spp. is a parasitic protozoan which emerges from its dormant cyst form upon ingestion by an appropriate host, 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, the median 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 giardiasis  are many and varied, from mild flu-like symptoms to severe diarrhea and vomiting. Symptoms last 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 most prevalent gastrointestinal condition in the United States (Warhurst and Smith 1992) and the most common 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 reservoir of 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 Giardia 55  spp. 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 animals  such as dogs and cats (Baker et al. 1987, Collins et al. 1987). There have also been giardiasis cases 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 their numerical abundance and mobility, Infectivity of G. duodenalis to other mammal species is uncertain. One major difficulty lies in the uncertainty of species classification based on cyst morphology. For accurate species identification, 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 in the Research Forest and prevalence of small mammal giardiasis before and after sludge treatment; and 2) the ability of a small wild rodent, the deer mouse, to host strains of human type G. duodenalis in the laboratory.  METHODS AND MATERIALS 1. Field Studies Establishing initial prevalence  A baseline estimate of the prevalence of Giardia spp. in field populations of small mammals was determined prior to sludge application from analysis of fecal samples collected from two main groups of wildlife: beavers and small mammals. Samples of the former were 56  collected near active or abandoned beaver dams. Information regarding locations was obtained from Research Forest staff and reconnaissance surveys were then conducted. Twenty-five samples of small mammal feces were obtained from animals captured in three small mammal live-trap lines set up near active or abandoned beaver dams. Fecal samples from aquatic mammals included only the beaver due to its close association with the spread of giardiasis. A total of 13 samples was collected from three different sites (distributed throughout the southern half of the forest). As beavers are known to show high site fidelity, this was assumed to be at least three different animals. Samples were transported to the Vancouver General Hospital Giardia laboratory and analyzed by the formol-ether concentration method and microscopic  examination of iron-hematoxylin stained preparations (Garcia and Bruckner 1988), a highly sensitive detection method for fecal Giardia cyst content.  Giardia prevalence and sludge application  One hundred fresh fecal samples from small mammals were obtained from the livetrapping study sites (Chapter 1) prior to and after sludge application. Samples were collected from 100 individual specimens (25/study site) which were caught in live-traps overnight. These samples were placed in parasite preservative (sodium acetate, acetic acid, formalin) and tested for the presence of Giardia spp. cysts in the laboratory. This part of the analysis was conducted under the direction of Dr. J. Isaac-Renton.  57  2. Laboratory Studies Collection of deer mice  Twenty-six deer mice were live-trapped at the Malcolm Knapp Research Forest in southwestern British Columbia in western Canada at a sufficient distance away from sludged areas to prevent any dispersal effects. All deer mice were captured in Longworth live traps that were baited with oats and supplied with cotton bedding and a slice of carrot for moisture. Traps set in various locations in the forest were baited and checked twice daily over a two to three day period. In total, six different sites were sampled. All animals trapped were tagged with fingerling fish tags and then transported in traps to the parasitology laboratory at the Vancouver General Hospital. Only males were kept for use in these experiments. Animals were kept in the laboratory in cages (47 x 26 x 12.7 cm) in pairs and provided with laboratory rodent chow (PMI Feeds Inc. Richmond, B.C.) and water ad libitum. Small plastic water bottles were used as nesting chambers. All animals were acclimatized for seven days after being trapped and fecal examinations were then carried out. For optimal laboratory examination, fecal samples were collected from each animal and placed in SAF (sodium acetate, acetic acid and formalin) parasite preservative for processing. Each SAF-preserved sample was tested by microscopic examination following the formol-ether concentration method and microscopic examination of ironhematoxylin stained preparations (Garcia and Bruckner 1988).  Metronidazole treatment  No natural infections with Giardia were detected in the trapped deer mice, but to ensure that none of the test or control animals were infected, they were all treated with metronidazole. 58  Animals were treated orally using a gavage needle (gauge #22) with three consecutive daily doses of 7.5 mg of metronidazole for each deer mouse. Each mouse was treated whilst mildly anaesthetized with Metofane (methoxyfluorane). Following a seven day interval of rest after metronidazole therapy, six deer mice were randomly selected and further fecal samples were examined. Deer mice were then assigned into two groups of 13 each; 10 as test animals and 3 as controls.  Enzyme linked immunosorbent assay (EIA) -  An enzyme-linked immunosorbent assay of sera collected prior to inoculation was used to 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 mice previously inoculated with Giardia antigen and producing high anti-Giardia immunoglobulin G were used as positive EIA controls. Preimmune sera, also collected from Balb/c mice, were used as negative EIA controls. Sera from the deer mice were collected by tail-bleeding and stored at -20°C until testing.  Source of cysts  Cysts for inoculation of deer mice, obtained from previously infected laboratory gerbils (Meriones unguiculatus), were collected from feces using a sucrose gradient concentration  method (Roberts-Thompson et al. 1976). One-day old cysts were used in all experiments to eliminate variation of infectivity based on cyst-age. One Giardia strain was obtained from a human-source (symptomatic child) and a second strain was collected from a beaver determined 59  to 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 to human source strains of Giardia retrieved during the outbreak. Trophozoites from both strains were also confirmed as being in the G. duodenalis morphological group by Giemsa staining and microscopic examination for median body morphology (Filice 1952).  Inoculation of cysts  All test and control mice were inoculated on the same day. Cysts were quantified using a counting chamber (Neubauer) and appropriate concentrations (1X10 25) prepared in distilled water. 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 serve as controls, four mice were inoculated with 0.1 mL distilled water containing 1X10 5 cysts, two deer mice were inoculated with 0.1 mL containing 1X10 4 cysts, two were inoculated with 1X10 3 and two with 1X10 2 cysts. A higher proportion of mice were inoculated with the highest cyst concentration to provide a larger sample size with optimal conditions for infection. Animals were anaesthetized when gavage treated with the designated suspension of cysts. The experiment was 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 the  inoculation of the deer mice to test the viability of the infecting cysts. Fecal samples from gerbils were analyzed using the same methods as the test and control deer mice.  60  Determination of infection  Starting one week post-inoculation, three fecal samples were collected at weekly intervals for each test and control animal. Fecal samples were collected and placed in SAF parasite preservative and then examined by the two methods described above (microscopy after formolether concentration and examination of iron-hematoxylin stained preparations) for the presence of Giardia cysts or trophozoites. All animals were sacrificed at the end of three weeks and their small intestines removed. The anterior half of the small intestine was removed and impression smears prepared from sections near the pylorus of the stomach. A wet preparation was also prepared from the intestinal contents. Both preparations were cover-slipped and examined microscopically (Nikon, Labophot X 200) for Giardia trophozoites. Trophozoites were identified on the basis of pear-shaped body, motility and size (10-12 um in length).  RESULTS 1. Field Studies Establishing initial prevalence  Laboratory results from fecal sample analysis found very low (small mammals: 8%) to no (beavers: 0%) Giardia contamination prior to sludge application. Furthermore, Giardia cysts were not identified to the species (a limitation of cyst identification). Endemic giardiasis in rodents (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 suggest that initial prevalence of Giardia at the Research Forest was low (Table 12). 61  ^  Table 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 content was tested by the formol-ether concentration method and microscopic examination of iron-hematoxylin stained preparations. No.^Source^Time of^Giardia spp. cyst Samples^ 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 mammals  Prevalence of Giardia spp. cysts in study sites estimated from small mammal fecal analysis were found to be slightly lower after sludge application. Due to the small degree in difference in these estimates, it was concluded that prevalence of Giardia did not appear to be affected by sludge treatment in this case (Table 12). However, it was unknown whether or not the sludge utilized for this study was contaminated by Giardia previous to application. No outbreaks of giardiasis were reported in the areas where the sludge was collected. Sampling sludge for Giardia spp. cyst content was not considered because past attempts have found this procedure to be both inefficient and prohibitively expensive in terms of labour and analysis costs.  2. Laboratory Study  None of the trapped deer mice showed evidence of endogenous infection prior to inoculation as determined by multiple fecal examinations. None of the deer mice showed evidence of past infection as demonstrated by the absence of anti-Giardia IgG by EIA. High titres of specific antibody were, however, obtained from the Balb/c mice used as EIA positive serum controls. The group of deer mice used as negative controls remained Giardia-negative throughout both sets of experiments and were uninfected at necropsy indicating that crosscontamination did not occur between cages. Cysts from both Giardia strains used in these experiments 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 concentrations are seen in Table 13. None of the animals inoculated with the human-source G. duodenalis 63  Table 13. Results of fecal and small intestine examinations of two groups of deer mice inoculated with different strains of and varying numbers of G. duodenalis.  Giardia Strain  Concentration^Number of^% Positive Tests of cysts^deer mice inoculated^inoculated Fecals Gut per deer mouse 1^2 3 Exams  Symptomatic Child^1 x 105^n 1 x 104^n 1 x 103^n 1 x 102^n  = = = =  4 2 2 2  0 0 0 0  0 0 0 0  0 0 0 0  0 0 0 0  Beaver source^1 x 105^n causing^1 x 104^n human outbreak^1 x 103^n 1 x 102^n  = = = =  4 2 2 2  25 0 0 0  50 0 0 0  50 0 0 0  75 0 0 0  64  became infected even when a dose of 1 X 10 5 cysts was given. Two of the four animals inoculated 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 fecal examination during the first week post-inoculation and a second animal was found to be positive during the second week post-inoculation. Both of these animals remained Giardia positive until necropsy. Giardia trophozoites were found on examination of small intestinal contents on a third infected mouse which had been consistently cyst-negative on fecal examinations. Thus, three of the four mice in the group inoculated with 1 X 10 5 cysts were infected with the outbreakassociated Giardia isolate.  DISCUSSION  These experiments confirm the observation that deer mice may become infected with some strains of the G. duodenalis morphological group which are associated with infections in humans. The strain shown to be infective in these small rodents in the present experiments required a large number of cysts for infection to be produced. I also note that three of the four deer 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 a role in resistance to infection (Woo and Paterson 1986). Other factors that determine resistance to infection include acquired immunity due to previous infections. Results of serological testing in these experiments indicated that none of the deer mice had been previously exposed to Giardia, and therefore were unlikely to be resistant to infection due to acquired immunity.  65  Evidence to suggest the transmission of animal-source Giardia to humans so far has been circumstantial, yet the frequency with which human infections have been traced to a potential animal source suggests that humans and animals may be susceptible to infection with some of the same strains of G. duodenalis. The beaver (Castor canadensis) has been the animal most commonly implicated in zoonotic infection (Frost et al. 1980, Isaac-Renton 1987, Erlandsen and Bemrick 1988). This water-dwelling member of the rodent family may maintain infection in a colony 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 laboratory evidence, 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 waterborne outbreak, in which a beaver contaminated the drinking water source, is highly suggestive of a role 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 shown to harbour Giardia infections (Pacha et al. 1985, Isaac-Renton et al. 1987, Kirkpatrick and Benson 1987). Giardia cysts from some human sources have also shown to be capable of infecting muskrats (Erlandsen et al. 1988). Cross-infection studies have shown that cysts from some human source strains are capable of 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 wild animals such as rats (Rattus norvegicus), beavers, raccoons (Procyon lotor), and bighorn X mouflon sheep (Ovis canadensis X 0. musimon); and domestic animals such as gerbils (Gerbillus 66  gerbillus), guinea pigs (Cavia porcellus) and dogs (Canis familiaris). Other animals that may  act 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 herons and 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 hosts tested (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. In their latter study, 98% of live-trapped voles (Microtus pennsylvanicus) and 97.9 % of live-trapped deer mice (Peromyscus maniculatus) were found to harbour Giardia spp. The morphological group based on Filice's classification of Giardia found in these animals, however, was not determined. Other reports (Kirkpatrick and Benson 1987, Pacha et al. 1987, Hay 1990) have noted 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 from small 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 Roach and Wallis (1988) in which three cultured, but not morphologically defined, human-source isolates were used to inoculate deer mice. In those experiments, one of the three isolates used infected 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 of 67  G. 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 other biological and biochemical characteristics of parasite isolates are poorly defined. The two isolates used in the present experiments have been characterized by DNA karyotyping; one has been shown to be capable of infecting both beaver and humans (Isaac-Renton et al. 1992), the second isolate, recovered from a human, has not been as epidemiologically well-characterized as the epidemic-associated isolate. It appears that different isolates within the Giardia duodenalis morphological group have differing ranges of host specificities as well as different  biochemical (Nash et al. 1985, Meloni et al. 1989) and biological behaviours. The differences in infectivity in deer mice shown by the two isolates in these experiments may reflect further differences 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 infecting humans and small rodents. The importance of the role of large water-dwelling rodents such as beaver and muskrats in transmission of giardiasis to humans is based on their potential for excretion 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 of other 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 in terms of host-specificities, interspecies biochemical relationships and biological behaviours. Since cross-infection of such common small rodents as Peromyscus has been demonstrated (albeit 68  using large inocula with the strains used), precautions should be taken to keep sewage for silvicultural or agricultural application away from water sources that may be contaminated by run-off containing cysts or from areas in which domestic pets may become infected following a predatorial incident.  69  OVERALL THESIS CONCLUSIONS  Implications of study results for small mammal populations  In this study, the only effect on population parameters attributable to sludge treatment was found in the deer mouse. Fortunately, of the three small mammal species caught consistently in this study, the deer mouse was the best measure of habitat suitability due to its annual cycle of abundance (Gashwiler 1972, Petticrew and Sadleir 1974, Hooven and Black 1976). Oregon voles are known for their characteristic 3-4 year cycles in abundance and may require a longer study period for effects to be detected. The Townsend chipmunk was trapped in 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 posttreatment period. Potential impacts on other species from this effect is unknown. In times of limited resources, increased interspecific competition with the Oregon vole or other more specialist species may cause consequent declines of these species. Their respective predators may also be negatively affected. These two potential impacts reflect a change in the balance of the ecosystem from an artificially implemented situation, and warrant more research prior to the broad application of sewage sludge on a field scale. The proportion of male deer mice in breeding condition also experienced an increase in the post-sludge year but on treatment site 1 only. This effect, however, cannot be attributed solely to the sludge treatment due to its site specificity. In general, the three major small mammal species that were investigated in this study, the deer mouse, Oregon vole and Townsend chipmunk, did not appear to be significantly affected by the sludge application. 70  More subtle changes in the ecology or population dynamics of small mammal populations might have been revealed had the study been continued for longer, with more replicates, over a larger area of application, with more frequent applications of sludge, or with a wider diversity of study animals than what was available in this study. Furthermore, small mammals are only one of many groups of wildlife that may be affected. Research is needed to determine the magnitude of the effects of sludge applications if operational scale applications ( > 10 ha) are undertaken. Additional in-depth studies may also pinpoint areas of modification in application which might help minimize effects.  Implications of Giardia transmission for sludge applications  In terms of public health considerations, potential for Giardia contamination was demonstrated from the findings of this study. P. maniculatus has been shown to be capable of acting as a host to some strains of human Giardia under specific laboratory conditions. This small mammal species also did not appear to be negatively affected from sludge treatment as reflected by the study of its population parameters. During human giardiasis outbreaks, sewage sludge could become a source of contamination whereby Giardia duodenalis can enter the ecosystem through deer mice acting as a host. The risk of exposure to Giardia cysts in field applications, however, is considered small due to the poor survival of Giardia cysts under conditions of desiccation (Dr. J. Isaac-Renton,  71  pers. comm.)4 , the large inoculum dosage required to cause infection, and the chance of having this high level of cyst density in sewage sludge. From this information, it may be beneficial for applications to take place in the summer when desiccation conditions would be most likely encountered. Alternately, a summer sludge application may also have the most adverse effect on small mammal populations. Presently, field applications are ideally undertaken in early spring (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 winter application (M. van Ham, pers. comm.) 5 . Without further studies, an alternative time for application cannot be suggested. Currently, the main precaution taken with silvicultural sludge applications is to ensure they are located at a sufficient distance away from streams or creeks in order to prevent potential run-off of sewage into water sources. My findings reinforce this precautionary measure. Of particular concern is the intent to undertake broad scale silvicultural sludge applications. Due to the short duration of fertilization effects, it has been considered that sludge applications be repeated periodically (perhaps once every 10 to 20 years) over an extended period of time. In field application, areas treated would be considerably larger than those in this experiment. 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 of Health, Vancouver, Canada. M. van Ham. Ph. D. candidate in Forest Sciences. Thesis topic on the effects of silvicultural sludge applications on forest ecosystems, Department of Forest Sciences, University of British Columbia, Vancouver, B.C. Canada. 5  72  REFERENCES Anderson, D.A. 1981. Response of the Columbian black-tailed deer to fertilization of Douglasfir 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 sewagefertilized 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 in Western Washington. J. Wildl. Manage. 49(1): 91-95. Ash, L.R., and Orihel, T.C. 1987. Preparation of permanent-stained smears. In: Parasites: a guide to laboratory procedures and identification. Chicago, ASCP Press, 37. Baker, D.G., Strombeck, D.R., and Gershwin, L.J. 1987. Laboratory diagnosis of Giardia duodenalis infection in dogs. 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Journ. 29(6): 341-344. Isaac-Renton, J.L., Moricz, M., and Proctor, E.M. 1987. A Giardia survey of fur-bearing water mammals in British Columbia, Canada. J. Environ. Hlth. 50: 80-82. 75  Isaac-Renton, J.L., Cordeiro, C., Sarafis, K., and Shahriahi, H. 1992. Characterization of Giardia duodenalis isolates from a waterborne outbreak. J. Infect. Dis. In press. Jolly, G.M. and Dickson, J.M. 1983. The problem of unequal catchability in mark-recapture estimation of small mammal populations. Can. J. Zool. 61: 922-927. Kimmins J. P., Noon, T., van Ham, M., Prescott, C., Tsze, K.M., Peddie, C.C., and Lee, K.W.. 1992. Sewage sludge as a slow release organic fertilizer, December 1991 Report to Ministry of the Environment, Environment Protection Division. Section II: Discussion of interim results Phase I, II. Kiorpes, A.L., Kirkpatrick C.E., and Bowman, D.D. 1987. Isolation of Giardia from a llama and from sheep. Can. J. Vet. Res. 51: 277-280. Kirkpatrick, C.E., and Benson, C.E. 1987. Presence of Giardia spp. and absence of Salmonella spp. in New Jersey muskrats (Ondatra zibethicus). App. Environ. Micro. 53: 1790-1792. Klinka, K. 1976. Ecosystem units, their classification, interpretation, and mapping in the University of British Columbia Research Forest. Ph.D. Thesis, Faculty of Forestry, University of British Columbia, Vancouver. Krebs, C.J. 1966. Demographic changes in fluctuating populations of Microtus califomicus. Ecol. Monogr. 36: 239-273. Krebs, C.J. 1989. Ecological Methodology. Harper and Row Publishers, New York. 654 p. Krebs, C. J., and Boonstra, R. 1984. Trappability estimates for mark-recapture data. Can. J. Zool. 62: 2440-2444. Krebs, C. J., Keller, B.L., and Tamarin, R.H. 1969. Microtus population biology:demographic changes in fluctuating populations of M. ochrogaster and M. pennsylvanicus in Southern Indiana. Ecology. 50(4): 587-607. Magurran, A.E. 1988. Ecological diversity and its measurement. Princeton University Press, Princeton, New Jersey. 179 pp. Meidinger, D., and Pojar, J. 1991. Ecosystems of British Columbia, Research Branch, Ministry of Forests. Edited by Research Branch of the Ministry of Forests, Victoria, B.C. 330 pp. Meloni, B.D., and Thompson, R.C.A. 1987. Comparative studies on the axenic in vitro cultivation of Giardia of human and canine origin: evidence for intraspecific variation. Trans. Roy. Soc. Trop. Med. Hyg. 81: 637-640. 76  Meloni, B.P., Lymbery, A.J., and Thompson, R.C.A. 1989. Characterization of Giardia isolates using a non-radiolabelled DNA probe and correlation with the results of isoenzyme analysis. Am. J. Trop. Med. Hyg. 406: 629-637. Milner, R.L. 1986. Response of birds to potential habitat alteration and heavy metal accumulation following sewage application in Douglas-fir forests. In Nutritional and toxic effects of sewage sludge in forest ecosystems. Edited by S.D. West, and R.J. Zasoski. College of Forest Resources, University of Washington. Seattle. (abstract). Nash, T.E., McCutchan, T., Keister, D., Dame, J. B., Conrad, J. D., and Gillin, F. D. 1985. Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J. Infect. Dis. 152: 64-73. Nash, T.E., Herrington, D.A., Losonsky, G.A., and Levine, M.M. 1987. Experimental human infections with Giardia lamblia. J. Inf. Dis. 156(6): 974-984. Pacha, R.E., Clark, G.W., and Williams, E.A. 1985. Occurrence of Campylacter jejuni and Giardia species in muskrat (Ondatra zibethica). Appl. Env. Micro. 50(1): 177-178. Pacha, R.E., Clark, G.W., Williams, E.A., Carter, A.M., Scheffelmaier, J.J. and Debusschere, P. 1987. Small rodents and other mammals associated with mountain meadows as reservoirs of Giardia spp. and Campylobacter spp. Appl. Env. Microbi. 53(7): 15741579. Peet, R.K. 1974. The measurement of species diversity. Annu. Rev. Ecol. Syst. 5: 285-307. Petticrew, B.G. and Sadleir, R.M.F.S. 1974. The ecology of the deer mouse, Peromyscus maniculatus, in a coastal coniferous forest. I. Population dynamics. Can. J. Zool. 52: 107-118. Pielou, E.C. 1966. The measurement of diversity in different types of biological collections. J. Theor. Biol. 13: 131-144. Raedeke, K.J., and West, S. 1986. Transmission of sludge-borne parasites to free-living mammals. In Nutritional and toxic effects of sewage sludge in forest ecosystems. Edited by S.D. West, and R.J. Zasoski. College of Forest Resources, University of Washington. Seattle. (abstract). Raymond, M., Robitaille, J.F. , Lauzon, P. and Vaudry, R. 1990. Prey-dependent profitability of foraging behavior of male and female ermine, Mustela erminea. Oikos 58: 323-328. Redfield, J. A., Krebs, C.J., and Taitt, M.J. 1977. Competition between Peromyscus maniculatus and Microtus townsendii in grasslands of coastal British Columbia. J. Anim. Ecol. 46: 607-616. 77  Roach, P.D., and Wallis, P.M. 1988. Transmission of Giardia duodenalis from human and animal sources in wild mice. In Advances in Giardia research. Edited by P.M. Wallis and B.R. Hammond. U. of Calgary Press, Calgary. pp.79-82. Roberts-Thomson, I.C., Stevens, D.P., Mahmoud, A.A.F., and Warren, K.S. 1976. Giardiasis in the mouse: an animal model. Gastroenterol. 71: 57-61. Sadleir, R.M.F.S. 1965. The relationship between agonistic behavior and population changes in the deer mouse Peromyscus maniculatus Wagner. J. Anim. Ecol. 34: 331-352. Sadleir, R.M.F.S. 1974. The ecology of the deer mouse Peromyscus maniculatus in a coastal coniferous forest II. Reproduction. Can. J. Zool. 42: 119-131. Seber, G.A.F. 1982. The estimation of animal abundance and related parameters. 2nd ed. Charles Griffin & Co. Ltd., London. Sheppe, W. 1963. Population structure of the deer mouse, Peromyscus, in the Pacific Northwest. J. Mammal. 44(2): 180-185. Sullivan, T.P. 1977. 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Microtus population biology: demography of M. oregoni in southwestern British Columbia. Can. J. Zool. 59(11): 2092-2102.  78  Sullivan, T.P., and Sullivan, D.S. 1981. Responses of a deer mouse population to a forest herbicide application: reproduction, growth, and survival. Can J. Zool. 59: 1148-1154. Sullivan, T.P., and Sullivan, D.S. 1982. Responses of small mammal populations to a forest herbicide application in a 20-year-old conifer plantation. J. Appl. Ecol. 19:95-106. Sullivan, T.P., Sullivan D.S., and Krebs, C.J. 1983. Demographic responses of a chipmunk (Eutamias townsendii) population with supplemental food. J. Anim. Ecol. 52: 743-755. Taber, R.D., and Zasoski, R.J. 1980. Heavy metal transfer to plants and rodents. In Use of dewatered sludge as an amendment for forest growth. Vol III. Edited by R.L. Edmonds, and D.W. Cole. Center for ecosystem studies, College of Forest Resources, U. of W., Seattle. Abstract. Tevis, L. 1955. Observations on chipmunks and mantled squirrels in northeastern California. Am. Midl. Nat. 53: 71-78. Thompson, R.C.A., Lymbery, A.J., and Meloni, B.P. 1990. Genetic variation in Giardia, Kunstler, 1882: taxonomic and epidemiological significance. Protozool. Abst. 14: 1-28. University of Washington College of forest resources pack forest sludge research. 1972-1989. Draft abstracts of scientific papers and theses. Compiled by Charles L. Henry and B.F. Jensen. Warhurst, D.C., and Smith, H. 1992. Getting to the guts of the problem. Parasitol. Today 8: 292-93. West, S.D., Taber, R.D., and Anderson, D.A. 1981. Wildlife in sludge-treated plantations. In Sludge applications in Pacific Northwest Forest Lands. Edited by C.S. Bledsoe. Coll. of Forest Resources. U. of W. Seattle, Wash. pp. 115-121. West, S.D., and Zasoski, R.J. 1986. Fauna on sludge-treated and untreated study sites. In Nutritional and toxic effects of sewage sludge in forest ecosystems. Edited by S.D. West and 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 and Giardiasis. 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 in southern Ontario, Canada, and susceptibility of animals to G. lamblia. Trans. Roy. Soc. Trop. Med. Hyg. 80: 56-59. 79  Zar, 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 applications in Pacific Northwest Forest Lands. Edited by C.S. Bledsoe. Coll. of Forest Resources. U. of W. Seattle, Wash. pp. 67-72.  80  GLOSSARY OF TERMS  Balb/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 detect evidence of recent OR past infection. Formol-ether concentration method: an efficient method of detecting the presence of parasites including Giardia cysts in fecal samples. 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 are killed in the preservative used. Gavage: inoculation directly into the stomach using a tube passed through the flares, pharynx, or mouth and through the esophagus. Giemsa stain: one of the Romanosky group of combination stains often used for staining parasites of blood. Used in these experiments to stain median bodies in trophozoites for morphological 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 for Giardia trophozoites. Iron-hematoxylin stained preparations: preparations made from feces and stained with the named reagent to demonstrate parasite morphology. Recommended for the most sensitive method of detecting intestinal parasites when combined with examination of results of a concentration method. Isolate:  one strain of parasite from one particular source.  81  Karyotyping: 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 specified population 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 fecal debris. Cysts maintain viability when this method is used. Virulence markers: signs or characteristics that indicate a particularly virulent strain when comparing different strains, eg. a specific enzyme. Zoonosis: infections that may be transmitted between vertebrate animals and humans.  82  

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