Do colobus monkeys on humanized forest edges exhibit more severe parasite infections than those on non-humanized forest edges?

Stacey A.M. Hodder Department of Anthropology McGill University, Montreal

January 2009

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Arts.

© Stacey A.M. Hodder 2009

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Table of Contents Pages

Table of Contents………………………………………………………….. i Contribution of Co-Author……………………………………………….... ii Acknowledgements………………………………………………………… iii

Abstract…………………………………………………………………….. iv Introduction………………………………………………………………… 1 Methods…………………………………………………………………...... 7 Results……………………………………………………………………… 13 Discussion………………………………………………………………….. 18 a. Nutrition……………………………………………………….... 24 b. Ranging & Behaviour…………………………………………... 26 c. Microclimate……………………………………………………. 29 d. Seasonal Effects………………………………………………… 30 e. Yearly variation…………………………………………………. 32 f. Other factors……………………………………………………... 32 Conclusions…………………………………………………………………. 33 References…………………………………………………………………... 35 Appendix……………………………………………………………………. 46

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Contribution of Co-author: Dr. Colin Chapman will be listed as a co-author on the published manuscript. He

provided funding, editorial help, and suggestions on data collection and analysis. Together we developed my initial idea. I collected the data, analyzed the data and wrote the manuscript.

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Acknowledgements: Particular thanks to my supervisor Dr. Colin Chapman for his encouragement, advice, and

guidance over the last two years. I also thank Dr. Jessica Rothman for her suggestions on drafts,

help with statistics and for helping me with parasite identification. Thank you to my committee

members, Dr. Andre Costopolous and Dr. Brian Leung, for their constructive comments and

suggestions. I thank Dr. Dwight Bowman and Dr. Ellis Greiner for help with the identification

of parasites and Tania Saj for help in cortisol collection/analysis. I am grateful to Carolyn Hall,

Mitchell Irwin, Tania Saj, and Tamaini Snaith for their comments and suggestions. I am

appreciative of all of the assistance that Dennis Twinomugisha, Patrick Omeja and Emmanuelle

Aliganyira provided me with in Kibale. For help with data collection and assistance in the lab in

Kibale, thank you (Webaale muno) to my field assistants Robert Basaija, Hillary Musinguzi,

Moses Ahebwa and Deo Twebaze as well as to Clovis Kaganzi for his help in the lab. For

assistance in the lab at McGill, I am grateful to Chesley Walsh, Paula Kaitlyn Edelson, Janet

Lee-Evoy, Irina Rozin and Cavina Bui. For performing the stress hormone analysis, thank you to

Toni Ziegler, Dan Witter and the Wisconsin National Primate Research Center Assay Services.

Thank you to Sandra Binning and Dominique Roche for translating the Abstract of my thesis.

Funding was provided by the Natural Science and Engineering Council of Canada (NSERC,

PGS-M), Primate Conservation Inc. and Sigma Xi. Thank you to Makerere Biological Field

Station and Uganda Wildlife Authority for permission for this research to be conducted. Thank

you to Cynthia Romanyk and Diane Mann for administrative support. Thank you to my mum,

dad, Brooke Hodder, Kyle Hodder, Carolyn Lue and Andrew Horvath for always encouraging

me and supporting me.

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Abstract

The aim of this research was to investigate whether gastrointestinal parasite infections in colobus

monkeys were more severe on humanized forest edges compared to non-humanized forest edges.

I examined gastrointestinal parasites and fecal cortisol in red and black-and-white colobus

(Procolobus rufomitratus; Colobus guereza) found in four habitat types in Kibale National Park,

Uganda. Three parasite types were identified: Trichuris sp., stongyles, and Strongyloides sp.

Results did not correspond to the expectation that humanized forest edges increase parasite

infection; only two measures of parasite infection in the red colobus corresponded to this

expectation. Results also did not correspond to the expectation that edge habitat causes an

increase in parasite infection. Factors that may contribute to parasite infections are discussed

and I concluded that broad classifications (e.g. “humanized”) may be too general to identify

consistent differences in infections, as factors specific to each habitat and/or group may influence

parasite infection.

Résumé

Cette thèse a pour but d’examiner si les infections gastro-intestinales causées par des parasites

chez les singes colobus sont plus sévères chez des individus à la frontière de la forêt près

d’endroits habités ou à la frontière de la forêt loin des êtres humains. J’ai examiné des parasites

gastro-intestinaux et le taux de cortisol dans les excréments de deux espèces de singes colobus, le

colobus rouge (Procolobus rufomitratus) et le colobus noir et blanc (Colobus guereza), dans

quatre types d’habitats différents au sein du Parc National de Kibale, en Ouganda, Afrique de

l’est. Trois catégories de parasites furent identifiés: Trichuris sp., stongyles, et Strongyloides sp.

Mes résultats ne supportent pas l’hypothèse de départ que la frontière de la forêt ayant une

présence humaine plus élevée augmente le taux d’infection chez les singes colobus. En effet,

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deux mesure d’infection chez le colobus rouge confirme cette prédiction. De plus, l’hypothèse

que les singes habitant la frontière de la forêt ont un taux d’infection plus élevé que les singes

habitant l’intérieur de la forêt n’est pas supportée par cette étude. Il existe une énorme variété de

facteurs qui influence les infections parasitaires dont je discute dans cette thèse. Je conclue que

certaines classifications générales d’habitat comme « près des êtres humains » ne sont pas assez

spécifiques afin de pouvoir identifier des différences constantes du taux d’infection parce que

des facteurs spécifiques à chaque habitat ou groupe d’habitats peuvent influencer les infections

parasitaires.

Introduction

Anthropogenic disturbance has played a major role in animal-to-human disease transmission

(Greger 2007) and concern over the potential for increased zoonotic disease transmission has

grown with increasing human encroachment and increasing human population size (Chomel et

al. 2007; Desjeux 2004; Desjeux 2001; Ferber 2000; Muriuki et al. 1998; Wolfe et al. 1998).

Approximately 60% of infectious diseases that affect humans are zoonotic (Hopkins & Nunn

2007; Taylor et al. 2001) and some zoonotic diseases such as avian influenza, West Nile virus,

and Ebola are among those that can be fatal to humans and often to their animal hosts (Kramer et

al. 2008; Capua & Alexander 2007; Sadek et al. 1999). In fact, avian influenzas have shown to

be fatal in approximately 43% of human cases (Capua & Alexander 2007) and the Ebola virus

has been fatal in 70% of human cases (Groseth et al. 2007). Aside from threats to human health,

other consequences of such zoonotic disease transmission include the cost of treating disease

outbreaks (Greger 2007; Desjeux 2004) and the possibility of disease transmission from wild

animals to agriculturally important animals, such as cattle and poultry (Menzano et al. 2007;

Gortazar 2007; Webster et al. 2006; Delahay et al. 2001).

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Similarly, the potential consequences of disease transmission to wildlife populations,

especially endangered or threatened species, can be severe. For example, the scabies parasite

(Sarcoptes scabiei) was responsible for a 47 - 77% decline in some chamois (Rupicapra

rupicapra) populations in Italy (Rossi et al. 2007). It thus raises great concern when a species

such as the mountain gorilla (Gorilla beringei) which has a population estimated to be only 650

individuals, was found to be infected with scabies that could have been transmitted from

domesticated animals or people, since it was common in villages in the area surrounding their

natural habitat (Kalema-Zikusoka et al. 2002).

Another zoonotic disease, the Ebola virus, was speculated to be responsible for

population declines in gorillas (56%), chimpanzees (89%) and duikers (53%) in Central Africa

(Leroy et al. 2004). This virus is also thought to have contributed to a 99% decline in the gorilla

(Gorilla gorilla) population in the Minkébé forest region of Gabon over one decade (Walsh et al.

2003). However, Leroy et al. (2004) note that the Ebola outbreaks in the apes were observed

prior to the outbreaks in the local human populations and that humans likely became infected by

coming into contact with infected ape carcasses. While this suggests that the disease likely did

not spread from humans to apes, the above examples raise concern for the health of both humans

and non-human primates where the two populations live in close proximity and these examples

are also worrisome from a primate conservation perspective (Hopkins and Nunn 2007; Bermejo

et al. 2006; Travis et al. 2006).

Zoonotic disease transmission is of particular concern for primates because of the

relatedness of humans and non-human primates and thus their similarities in morphology,

physiology, and behaviour (Muriuki et al. 1998; Travis et al. 2006; Wallis & Lee 1999; Wolfe et

al. 1998). In fact, concerns about the spread of diseases between humans and non-human

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primates have been noted by several researchers (Goldberg et al. 2007; Chapman et al. 2006a;

Phillips et al. 2004; Legesse & Erko 2004; Hahn et al. 2003; Wallis & Lee 1999; Muriuki et al.

1998; Wolfe et al. 1998).

The forested edges of primate habitat, especially those that border humanized landscapes

(e.g., villages, crop fields), provide opportunities for humans and non-human primates to come

into close proximity (Chapman et al. 2006a) and research seems to indicate that primates

inhabiting areas close to humans do experience poorer health. For example, Ekanayake et al.

(2006) investigated the gastrointestinal parasite infections of toque macaques (Macaca sinica),

gray langurs (Semnopithecus priam), and purple-faced langurs (Trachypithecus vetulus) in areas

used by humans and in areas that were not used by humans. Human uses included tourism,

religious purposes, grazing livestock, picnicking, and defecation. In contrast, only livestock were

present in the areas considered unused by humans (Ekanayake et al. 2006). Interestingly, the

prevalence of Cryptosporidium sp. infections in the macaques and purple-faced langurs that

ranged into the areas used by humans was found to be greater than the prevalence of

Cryptosporidium infections in the monkeys that did not range into the areas used by humans.

Furthermore, a greater prevalence of Enterobius sp., Strongyloides sp., Trichuris sp., strongyle

type eggs, Entamoeba coli, and E. hystolytica/dispar infections were found in the macaques that

ranged in areas used by humans. However for other intestinal parasites, differences in

prevalence were not found between macaques in the different areas. For example, prevalence of

a spiruroid type parasite, Iodamoeba sp., Balintidium coli, Chilomastix sp., and E. hartmanni did

not differ between macaques from either area (Ekanayake et al. 2006).

Similarly, research by Chapman et al. (2006a) investigated the gastrointestinal parasite

infection of colobus monkeys (red colobus – Procolobus rufomitratus, black-and-white colobus -

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Colobus guereza) inhabiting forest edges and interior forest in Kibale National Park, Uganda.

Overall, a greater number of colobus monkeys with more than one parasite infection were

documented in groups inhabiting forest edges. These researchers also found one type of parasite,

a strongyle, to be more common in the monkeys that inhabited forest edges. Contact with

humans along the forest edges was one of the reasons that Chapman et al. speculated could

explain the increased parasitism in these colobines. Unfortunately, forest edges far from any

human activity were not included in the study so it is not known whether the results found for the

colobus monkeys were a consequence of their proximity to edge habitat or a consequence of

their proximity to human activity.

It remains unclear what role forest edges play with respect to host/parasite dynamics

(Chapman et al. 2006a). The microclimate along forest edges should be less conducive to

parasite transmission because forest edges may receive increased wind, increased solar radiation,

and may be drier than interior forest environments (Matlack 1993; Murcia 1995) and dry

conditions would be expected to desiccate parasite infective stages, making survival more

difficult (Gillespie 2001; Larsen & Roepstorff 1999; Appleton & Brain 1993). For example, in

an experimental study Larsen and Roepstorff (1999) demonstrated a reduction in the number of

pig parasite eggs recovered in hot, dry months compared to wetter months. Dehydration and

high temperatures were considered as causes of the observed pattern. The results of the study by

Chapman et al. (2006a) are intriguing because they are the opposite pattern of what would be

expected based on the possibility that microclimatic differences exist between edge and interior

forest. Thus, other explanations for why parasite infection is higher along forest edges must be

considered and one possibility is that the proximity to human activity is causing an increase in

parasite infection in colobus monkeys. This issue is important to resolve as it provides a better

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understanding of how anthropogenic habitat disturbance could increase levels of disease

transmission to and from wildlife. It is very important to point out that without genetic analysis

of the parasites, it will not be possible to know for certain if human to non-human primate

parasite transmission is occurring. For example, research by de Gruijter et al. (2005) indicated

that although both humans and non-human primates were infected with the parasite

Oesophagostomum, subsequent molecular analysis indicated that the parasites were

genotypically different and thus transmission of this parasite between the two was unlikely.

One factor that has not received sufficient attention when examining relationships

between parasitic infections and use of forest edges is whether the animals on the edges are

physiologically stressed. It is well documented that stress can suppress the immune system of

both humans and animals (Nunn & Altizer 2006; Padgett & Glaser 2003; Coe & Erickson 1997;

Black 1994; Kling et al. 1992). For example, in humans, individuals experiencing chronic stress

have been found to be more susceptible to respiratory illness than those who have experienced

short-term stress (Cohen and Miller 2001). In non-human primates, the stress of social

separation in capuchin monkeys (Cebus apella) caused a decrease in immune function (Kling et

al. 1992).

The adrenal glands produce glucocorticoids, such as cortisol and hydrocortisone, in

response to stress (Sapolsky 2004) and cortisol can be measured through fecal analysis (Mostl &

Palme 2002; Whitten et al. 1998). For example, Whitten et al. (1998) demonstrated that the

amount of cortisol excreted in feces increased approximately 2 days after a stressful experience

in captive non-human primates. Given that stress can decrease the immune response, non-human

primates experiencing increased stress may be at a higher risk of acquiring, or suffering from,

more severe parasitic infections than those that experience less stress. Such a connection

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between stress and parasite infection has been documented in wild male chimpanzees (Pan

troglodytes) by Muehlenbein (2006). Gastrointestinal parasite richness increased with higher

levels of fecal cortisol and testosterone (Muehlenbein 2006). Similarly, if colobus monkeys are

experiencing increased stress as a result of living on humanized forest edges, they may have

more severe parasite infections than those inhabiting non-humanized forest edges and interior

forest habitats.

One source of stress for monkeys inhabiting humanized forest edges may be the

interaction with local residents around the park. Animals in Kibale are known to raid crops and

many local residents have reported crop loss due to animal raiding; farms that experience crop

damage are on average about 170 m from the edge of the park (Naughton-Treves 1997).

Approximately 80% of local residents deter animals from raiding their crops (e.g., by guarding,

fences, trapping, poison, shouting and/or making noise; Naughton-Treves 1997; Hodder personal

observation). These interactions are not present along the non-humanized forest edges and it is

possible that these deterrents result in increased stress for animals inhabiting humanized edges.

This may lead to suppression of immune systems and elevated parasite infection levels.

In this study, I compared gastrointestinal parasite infections and levels of fecal cortisol of

red and black-and-white colobus monkeys inhabiting four habitat types: humanized forest edges

(two types), non-humanized forest edges, and interior forest to determine whether there are

increased levels of parasitism associated with edges as a result of human presence or a result of

some other aspect of forest edge environment. My goal was to determine whether non-human

primates living in close proximity to human activity had more severe parasite infections than

those living farther from human activity independent of whether they inhabited forest edges. I

predicted that the colobus monkeys inhabiting the humanized forest edges would have the most

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severe parasite infections and that they would be experiencing higher levels of stress than the

colobus monkeys inhabiting non-humanized edges and interior forest. This data would provide

indirect evidence to suggest whether further molecular studies identifying the relatedness of the

parasites in non-human and human populations, and thus transmission probabilities, are

warranted.

Methods

Study Site

Kibale National Park (795 km2) is located in Western Uganda (0 13' - 0 41' N and 30 19' -

30 32' E). Kibale is a moist, evergreen forest that receives 1712 mm of rainfall annually (1990-

2007; Chapman unpublished data). Two wet seasons occur over the year: March through

April/May and August/September through November (Oates 1977; Struhsaker 1975). The

annual mean daily maximum temperature is 25.5°C and daily minimum temperature is 12.7°C

(Struhsaker 1975). The land adjacent to the park has high levels of human activity. An average

farm is approximately 1.4 ha and population density is approximately 272 individuals/km2

(Naughton-Treves 1997). In addition, both large scale and small-scale tea plantations border

Kibale; plantations span hundreds of hectares to less than one hectare (Mulley and Unruh 2004).

This research complied with McGill University’s Animal Use Protocol and permission to

conduct this research was granted by the Uganda Wildlife Authority.

Forest Edge and Interior Forest Descriptions

Humanized forest edges were defined as areas where the forest was adjacent to areas of

intense human use. These edges either bordered a tea plantation (hereafter tea edges) or

bordered farmland or village area (hereafter agricultural edges). Non-humanized edges were

defined as forest edges that did not have evidence of human activity. Non-humanized edges

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were located in a grassland area in the interior of the park approximately 4 km from the park

boundary (hereafter grassland edges). These areas were formerly forest but were cut down for

pasture prior to the 1900s. They were abandoned when rinderpest devastated the livestock in the

area shortly after 1900 (Wing & Buss 1970; Kingston 1967; Osmaston 1959) and were

maintained as grassland by anthropogenetically set fires. Finally, the interior forest habitat was

an area of relatively undisturbed forest adjacent to Makerere University Biological Field Station

in Kanyawara.

Fecal Sample Collection - Kibale

We (four field assistants and I) located groups of colobus monkeys by walking along

forest edges or along the trail system until a group of colobus monkeys were encountered. We

remained with the group for the day or until 15 fecal samples were collected. After collecting 15

fecal samples, we stopped collecting and either found a new group or we began preparing

samples in the lab. Fecal samples were collected between April and July 2007 and were stored

in 15 ml plastic centrifuge tubes. The date, time of collection, species, sex (if known), and

location (edge type) were recorded. At the end of each day, 1 gram of feces was placed in a 15

ml centrifuge tube along with 2 ml of 10% formalin. Samples were transported to McGill

University for parasitological analyses. A total of 238 fecal samples were analyzed at McGill

University (29 - 30 fecal samples per species per edge type).

Parasitological Analyses:

To increase the accuracy of the estimate of parasite infections, two parasitological

methods were used to extract parasite eggs from the fecal samples: sodium nitrate flotation

(Greiner and McIntosh, in press) and fecal sedimentation (Garcia 1999). Twenty-five samples

from each edge type for both species were examined using saturated sodium nitrate flotation (n =

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200). Five samples from each edge type for the red colobus were examined using the more time

consuming fecal sedimentation method (n = 20). For the black and white colobus, five samples

from the interior forest and tea were examined using fecal sedimentation and four samples from

the agriculture and grassland were examined using this method (n = 18).

1. Sodium Nitrate Flotation: The fecal/formalin mixture in each tube was homogenized

and 0.5 g of the fecal/formalin mixture was placed in a small paper cup. A small amount of

sodium nitrate was added and the contents were stirred using a wooden stir stick. The mixture

was then poured through 2 layers of cheesecloth into a second paper cup. The strained mixture

was then poured into a 15 ml centrifuge tube and saturated sodium nitrate was added until a

positive meniscus was formed at the top of the centrifuge tube. A 22 x 22 mm cover-slip was

placed on the top of the meniscus and the tube was left for 10 min to allow the parasite eggs to

float to the top. After 10 min, the cover-slip was placed on a microscope slide. Slides were

examined using a 10 x objective lens. Photographs were taken (at 40 x magnification) of all

parasites except Trichuris sp., which was very easy to recognize.

2. Fecal Sedimentation: The fecal/formalin mixture in each tube was homogenized and

0.5 g of the fecal/formalin mixture was placed in a small paper cup. A small amount of water (1-

2 ml) was added to the mixture and then stirred using wooden stir stick. Next the mixture was

filtered through 2 layers of cheesecloth and transferred to a 15 ml centrifuge tube. Water was

added to each centrifuge tube so that all tubes had approximately 10 ml of the mixture in them.

Tubes were centrifuged at 2500 rpm for 2.5 minutes. The supernatant was poured off, leaving a

small amount of the fecal sample in the bottom of each tube. Seven milliliters of water were

added followed by 3 ml of ethyl acetate. The tube was shaken to re-suspend the fecal sample

and then centrifuged at 3000 rpm for 2.5 minutes. The supernatant was poured off, leaving a

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fecal sample in the bottom of the tube. Excess droplets of fatty material on the inside of the tube

were removed with a cotton-tipped stir stick. One milliliter of 10% formalin was added to each

tube to preserve the sample. Samples were stored at room temperature until microscopic

examination. Slides for microscope examination were prepared using a pipette to extract 4-5

drops of the formalin/feces mixture and placing these on microscope slides. This continued until

all of the formalin/feces mixture had been examined. Slides were scanned under 10 x

magnification and photographs of parasites were taken at 40 x magnification. Approximately 10

– 20 slides were examined per fecal sample using the sedimentation method.

Fecal processing for Cortisol

A total of 182 fecal samples were analyzed for cortisol. Samples were collected from unknown

individuals and were collected throughout the day. Samples were divided as follows: red

colobus tea (n = 16), red colobus grassland (n = 17), red colobus agriculture (n = 24), red colobus

interior (n = 25), black and white colobus tea (n = 25), black and white colobus grassland (n =

25), black and white colobus agriculture (n = 26), and black and white colobus interior forest (n

= 24). At the time of collection, fecal samples were placed in 15 ml plastic centrifuge tubes and

then placed in coolers to ensure they remained cold until the end of the day. Preliminary

laboratory analysis followed Wasserman and Chapman (unpublished lab guide). At the end of

each field day, 1 g of feces was weighed out and stored in 15 ml centrifuge tubes in a -20°C

freezer. To prepare for cortisol analyses, fecal samples were thawed and 0.5 g was weighed out.

Following thawing, a 5.0 pH citrate buffer and 95% ethanol solution were added to the 0.5 g of

feces and then mixed continuously for 21 – 27 hr using a mechanical shaker. This allowed time

for the cortisol to be extracted from the fecal material. At the end of this period, the mixture of

feces, citrate buffer, and ethanol was placed in a centrifuge for 30 min. After centrifuging, the

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supernatant (containing the cortisol hormone) was filtered through an Altech maxi-clean

cartridge. These cartridges were sent to the University of Wisconsin-Madison and were further

processed at the National Primate Research Center. Enzymeimmunoassays (EIA) were

preformed at the Research Center following Ziegler et al. (1995) and the metabolites of cortisol

were reported in nanograms per gram.

Analyses

Four indices were used to describe infection: prevalence, multiple infections, richness, and load

(following Chapman et al. 2006a). Prevalence is a count of how many monkeys are infected

with one parasite type. Multiple infections is the number of monkeys infected with two or more

parasites. Richness is the number of different parasite species observed, and load is the number

of eggs per gram of feces for each parasite. Statistical analyses were carried out using SPSS

14.0. The four questions I addressed were:

1. Do colobus monkeys inhabiting forest edges experience more severe parasite infections than

those inhabiting the interior forest?

2. Do colobus monkeys inhabiting humanized habitats experience more severe parasite

infections than those inhabiting non-humanized habitats?

3. Do colobus monkeys inhabiting edge habitats experience greater stress than those inhabiting

interior forest?

4. Do colobus monkeys inhabiting humanized habitats experience greater stress than those

inhabiting non-humanized habitats?

Data were fitted to a log-linear model to determine whether habitat type(s) and parasite

infection were related. This analysis reduces the number of comparisons that must be made if

differences are non-significant and this is analogous to using an ANOVA versus multiple t-tests.

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Residuals from the log-linear model allowed us to determine more specifically which habitat(s)

had the greatest influence in the chi-square/Fisher’s exact tests (Agresti 1996). A residual of

between 2 and 3 indicated that the null hypothesis (lack of association) was not supported

(Agresti 1996). Multiple infections and prevalence were addressed using chi-square/Fisher’s

exact tests. Mann-Whitney U tests and Kruskall-Wallis tests were used to compare parasite load

and fecal cortisol levels among habitats. Non-parametric statistics were used because of the non-

normal nature of the data. Where directional hypotheses were present (comparing an individual

habitat to another habitat), one-tailed tests are reported. Where non-directional hypotheses were

present (comparing all 4 habitats to each other), two-tailed tests are reported; this is clearly

reported for each test.

In this study I made two main assumptions. First, I assumed that the colobus monkeys

whose ranges included humanized edges would experience higher levels of stress than the

colobus whose ranges did not include humanized edges. This increase in stress for the colobus

alongside the humanized edges would likely be a result of more frequent interactions with local

residents in the humanized areas, possibly leading to chronic stress in these monkeys. Whitten et

al. (1998) noted the potential usefulness of fecal cortisol measures for documenting chronic

stress, as well as its value in field settings. Thus I assumed that chronic stress would be evident

in the colobine fecal samples collected from humanized edges. The second assumption I made

was that the parasite infections would correspond to the habitat that the colobines were found in

at the time of fecal sample collection. However, it is possible that ranging behaviour of the

colobus monkeys may have spanned more than one habitat type. This is further discussed below

in the section on Ranging and Behaviour.

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Results

Nematode eggs identified as Trichuris sp. (Superfamily Trichinelloidea), strongyle

(Superfamily Strongyloidea), and Strongyloides sp. (Order Rhabditida) were observed in the

fecal samples (Bowman 1999). Larvae were observed in some fecal samples, but were not

included in the analysis because it was often very difficult to determine whether they were free-

living (i.e., picked up when the fecal material hit vegetation or the ground) or whether they had

hatched in the gastrointestinal tract of the monkey. Both species of colobus were infected with

all three parasite types.

In the following section I present the results for prevalence, load, multiple infections and

parasite species richness in turn. I present the data on the black and white colobus followed by

the data for the red colobus. I end by contrasting cortisol levels among habitats.

Parasite Prevalence in Black and White Colobus

Prevalence of Trichuris sp. in black and white colobus monkeys differed among habitat types (X2

= 11.926, two-tailed p = 0.008). Residuals from the log-linear model indicated that more

monkeys were infected with Trichuris sp. in the agricultural habitat than expected if parasite

infection and habitat type were not associated (residual = 2.275 for agriculture). In contrast,

residuals for the tea habitat indicated that fewer monkeys were infected with Trichuris sp. than

expected (residual = -2.249 for tea). Each habitat was then compared individually to each of the

other habitats and the agricultural habitat was found to have significantly more monkeys infected

with Trichuris sp. than both the grassland habitat (Fisher’s exact test, one-sided p = 0.012) and

the tea habitat (Fisher’s exact test, one-sided p = 0.006). Thus, counter to what was predicted, a

difference was found between the two humanized habitats with respect to Trichuris prevalence.

Also, counter to what was expected, the interior forest habitat had more monkeys infected with

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Trichuris sp. than both grassland (Fisher’s exact test, one-sided p = 0.046) and tea habitats

(Fisher’s exact test, one-sided p = 0.026). No significant differences were found for prevalence

of Trichuris sp. between agriculture and interior forest habitats (Fisher’s exact test, one-sided p =

0.508) or between grassland and tea habitats (X2 = 0.060, one-tailed p = 0.404).

Unlike infection with Trichuris sp., infection with strongyles was not related to habitat

type (X2 = 6.012, two-tailed p = 0.111). However, differences between the habitats were found

in the prevalence of Strongyloides sp. (X2 = 7.853, two-tailed p = 0.049). Residuals from the

log-linear model indicated that more monkeys were infected with Strongyloides sp. in the

grassland habitat than would be expected if they lacked association (residual = 2.769). When the

habitats were individually compared, more monkeys were found to be infected with

Strongyloides sp. in the grassland than the agriculture (X2 = 4.350, one-tailed p = 0.019), the tea

habitats (Fisher’s exact test, one-sided p = 0.035), and the interior forest habitat (X2 = 3.007, one-

tailed p = 0.042). No significant differences were found between agriculture and interior forest

habitats (Fisher’s exact test, one-sided p = 0.516), between agriculture and tea habitats (Fisher’s

exact test, one-sided p = 0.681) or between interior forest and tea habitats (Fisher’s exact test,

one-sided p = 0.500).

Parasite Prevalence in Red Colobus

Prevalence of Trichuris sp. in red colobus also differed according to habitat type (X2 = 9.293,

two-tailed p = 0.026). Residuals from the log-linear model indicated that fewer monkeys were

infected with Trichuris sp. in the grassland habitat than would be expected if they lacked

association (residual = -2.653). When each habitat was individually compared the samples from

the agriculture habitat did not differ from those of the tea habitat (X2 < 0.001, p = 0.5) in terms of

Trichuris sp. prevalence. However, both the agricultural samples and the tea samples had higher

15

Trichuris sp. prevalence measures than the grassland samples (X2 = 6.944, p = 0.004; X2 = 6.944,

p = 0.004 respectively). Similarly, both the agriculture and tea samples had marginally higher

Trichuris sp. prevalences than those from the interior forest (X2 = 1.669, p = 0.098; X2 = 1.669, p

= 0.098 respectively). Samples from the interior forest had a marginally higher Trichuris sp.

prevalence than those from the grassland (X2 = 1.926, p = 0.083).

Like the black and white colobus, prevalence of infection with strongyles did not differ

among habitat types for red colobus (X2 = 2.667, two-tailed p = 0.446). The prevalence of

infection with Strongyloides sp. also did not vary among habitat type for red colobus (X2 = 6.102,

two-tailed p = 0.107).

Parasite Load in Black and White Colobus

Significant differences in load for Trichuris sp. were observed between habitats for black and

white colobus (Kruskal-Wallis test, p <0.001). When each habitat type was compared, to the

other habitat types, black and white colobus in the agricultural habitat were found to have greater

Trichuris sp. loads than those from both the grassland habitat (Mann-Whitney U Test, one-tailed

p < 0.001) and the tea habitat (Mann-Whitney U test, one-tailed p < 0.001). Black and white

colobus in the interior forest also had greater Trichuris sp. loads than those in both the grassland

(Mann-Whitney U, one-tailed p < 0.001) and the tea habitats (Mann-Whitney U test, one-tailed p

< 0.001). However, no differences were found between the agricultural and interior forest

habitats (Mann-Whitney U Test, one-tailed p = 0.306) or between the grassland and tea habitats

(Mann-Whitney U, one-tailed p = 0.179).

While the prevalence of strongyle infections did not show significant differences among

habitats, strongyle load marginally differed among habitats (Kruskal-Wallis test, p = 0.094).

Strongyle load in the tea habitat was higher than that of the interior forest habitat (Mann-

16

Whitney U test, one-tailed p = 0.015), the agricultural habitat (Mann-Whitney U test, one-tailed

p = 0.038) and was marginally higher than the strongyle load of the grassland habitat (Mann-

Whitney U test, one-tailed p = 0.087). No significant differences were found in comparisons of

strongyle load between the other habitats (Mann-Whitney U tests, one-tailed p-values =

agriculture and grassland p = 0.269; agriculture and interior forest, p = 0.328; grassland and

interior forest, p = 0.145).

Significant differences in load for Strongyloides sp. were also observed among habitats

(Kruskal-Wallis test, p = 0.045). However, close examination of the data revealed one fecal

sample from the grassland habitat with a high egg output (96 eggs per gram). All other fecal

samples in all other habitats had fewer than 16 eggs per gram of fecal/formalin mixture. Thus

the sample with 96 eggs was considered as an outlier. I present the analysis with the outlier

excluded and included, but in interpreting differences the reader should recall that this is an

analyses of ranks, thus the sample with 96 eggs per gram is just one rank higher than the next

highest values. When this outlier was removed from the analysis, no significant differences

between habitats were found (Kruskal-Wallis, p = 0.106). When the outlier was included, the

samples from the grassland habitat had a higher egg output than the agricultural habitat (Mann-

Whitney U test, one-tailed p = 0.019), the tea habitat (Mann-Whitney U test, one-tailed p =

0.017), and the interior forest habitat (Mann-Whitney U test, one-tailed p = 0.038). No

differences were found between samples from the agriculture and interior forest habitats (Mann-

Whitney U test, one-tailed p = 0.342), between samples from agriculture and tea habitats (Mann-

Whitney U test, one-tailed p = 0.486), or between samples from the interior forest habitat and tea

habitats (Mann-Whitney U test, one-tailed p = 0.328).

17

Parasite Load in Red Colobus

Marginal differences in Trichuris sp. egg output were found among red colobus in different

habitats (Kruskal-Wallis test, p = 0.077). When samples from each habitat were compared, the

samples from both the agricultural habitat and the tea habitat had significantly higher Trichuris

sp. loads than those from the grassland habitat (Mann-Whitney U tests, one-tailed p-values =

0.010 and 0.010 respectively). The interior forest habitat had marginally higher Trichuris sp.

loads than those from the grassland habitat (Mann-Whitney U test, one-tailed p = 0.081). No

significant differences were observed between samples from the agriculture and tea habitats

(Mann-Whitney U test, one-tailed p = 0.5), between agricultural and interior habitats (Mann-

Whitney U test, one-tailed p = 0.180), or between interior forest and tea habitats (Mann-Whitney

U test, one-tailed p = 0.199).

Strongyle load did not differ for red colobus among habitat types (Kruskal-Wallis, p =

0.434). Likewise, the Strongyloides sp. load did not differ among habitats for red colobus

(Kruskal-Wallis, p = 0.109).

Multiple Infections in Black and White and Red Colobus

The frequency of multiple infections did not differ among the habitats for black and white

colobus (X2 = 3.145, p = 0.370) or for red colobus (X2 = 3.710, p = 0.295).

Parasite Richness in Black and White and Red Colobus

As noted above, both colobus species were infected with all three parasites that were identified;

thus richness did not differ between colobus species. For black and white colobus, no

differences in richness were observed among the four habitats. For the red colobus, only two

parasites were found in all three habitat types: Trichuris sp. and strongyles. Strongyloides sp.

was only found in fecal samples from the agricultural habitat.

18

Cortisol Levels in Black and White and Red Colobus

No differences were found among cortisol levels for black and white colobus in the different

habitats (Kruskall-Wallis, p = 0.261). However, significant differences were found among

cortisol levels for red colobus in different habitat types (Kruskall-Wallis, p < 0.001). The

cortisol levels in the tea samples were less than those in the agricultural (Mann Whitney U test,

one-tailed p < 0.001), grassland (Mann-Whitney U test, one tailed p < 0.001), and interior forest

habitats (Mann-Whitney U test, one tailed p < 0.001). No significant differences were found for

red colobus in comparisons of cortisol levels between agriculture and grassland habitats (Mann-

Whitney U test, one-tailed p = 0.331), between agriculture and interior forest habitats (Mann-

Whitney U test, one-tailed p = 0.187), or between grassland and interior forest habitats (Mann-

Whitney U test, one-tailed p = 0.117).

Discussion

Although not all indices of colobus parasite infection were found to be greater in the

forest edges in the research by Chapman et al. (2006a), my approach in this research was to test a

general hypothesis about whether parasite infection is more severe in humanized or forest edge

habitat. To address this question, I examined whether any of the parasites observed in the fecal

samples were higher in colobus found alongside forest edges or in those found alongside human

activity. The edge samples collected by Chapman et al. (2006a) were collected in areas most

similar to the agriculture edges in this study. Chapman et al. did not collect samples from areas

that bordered tea plantations. Results of their study indicated that they did not find a significant

difference in Trichuris sp. prevalence and load between black and white colobus from the forest

edge and interior forest. Similarly, I did not find differences in Trichuris sp. prevalence and load

between black and white colobus samples from the agriculture edge and those from the interior

19

forest. However, I did find differences in Trichuris sp. prevalence and load among habitats for

black and white colobus when the other types of forest edges (tea and grassland) were included

in the comparison. The differences among habitats were not consistent with a pattern that would

suggest forest edges increase parasite infection. If proximity to forest edges were to cause a

significant increase in prevalence and load of Trichuris sp., then these measures of Trichuris sp.

infection in samples from each of the three edge habitats should be greater than the prevalence

and load in samples from the interior forest. This was not the case for the black and white

colobus monkeys. The interior forest had a higher prevalence and load of Trichuris sp. than the

grassland and tea which was counter to what was expected.

Two interesting results were found for the prevalence of Trichuris sp. in red colobus.

First, Trichuris sp. prevalence differed among habitats but, like the black and white colobus, the

differences were not consistent with a pattern that would suggest that forest edges increase the

prevalence of Trichuris sp. infection. I found Trichuris sp. prevalence in red colobus samples to

be marginally higher in the agricultural and tea edges when compared to the interior forest

samples. However, Trichuris sp. prevalence in the grassland edge was marginally lower than that

of the interior forest. Thus, while agriculture, tea, and grassland were all considered a forest

edge, a corresponding increase in Trichuris sp. prevalence in red colobus was not observed in all

three habitats. Second, my results for agriculture were not consistent with Chapman et al.

(2006a) which found no significant difference in Trichuris sp. prevalence between edge and

interior forest samples. Some possible reasons for the differences between my results and those

by Chapman et al. are discussed below.

I did not find significant differences between Trichuris sp. load in samples from the

agriculture and those from the interior forest and this was consistent Chapman et al. (2006a). I

20

found the samples from the interior forest to have a marginally higher Trichuris sp. load than

those from the grassland edge and they did not differ from the tea edge. Results for Trichuris sp.

load for the red colobus did not correspond to the expectation that the three edge habitats would

have a greater Trichuris sp. load than the interior forest. However, specific edge type does seem

to be important in determining how colobus parasite infection is altered. In some cases, living

next to the forest edge was related to decreased measures of the Trichuris sp. infection. In other

cases, edge habitat was related to a marginal increase in measures of Trichuris sp. infection.

Like my expectation for Trichuris sp., if strongyle prevalence in black and white colobus

monkeys were to increase as a result of proximity to forest edges, then I would expect the

samples from the three edge habitats to have greater strongyle prevalence and load measures than

the samples from the forest interior. However, I did not find significant differences between any

of the habitat types for strongyle prevalence in black-and-white colobus. The results for

strongyle load indicated that the samples from the tea edge tended to have higher or marginally

higher strongyle loads than the other habitats and that the two other edge habitats, agriculture and

grassland, did not differ from the forest interior. My results for strongyle infection in black-and-

white colobus are interesting for two reasons. First, they suggest that edge habitat is not

associated with an increase in either the prevalence or load of strongyles in these monkeys.

Second, these results suggest that the number of black and white colobus infected with

strongyles may be similar among habitats, but some habitats may be associated with higher egg

loads in infected black and white colobus.

For the red colobus, neither strongyle prevalence nor load differed among habitat types,

suggesting that edge habitat is not associated with an increase in their strongyle infections. My

results for strongyle prevalence were unexpected because Chapman et al. (2006a) found a

21

significant difference in prevalence and load of strongyles for red colobus on the forest edge and

those in the interior forest. This suggests that factors other than edge type may influence parasite

infection; these are discussed below.

Prevalence and load (outlier included) of Strongyloides sp. in black and white colobus

differed among the habitats. The samples from the grassland edge tended to have a greater

prevalence and load of Strongyloides sp. than all of the other habitats (both edge and interior).

Furthermore, the prevalence and load of Strongyloides sp. in the two other edge habitats

(agriculture and tea) did not differ from the interior forest. This suggests that while some aspect

of the grassland habitat may increase both the number of black and white colobus infected with

Strongyloides sp. and the Strongyloides sp. egg load, factors other than proximity to the forest

edge should be considered. Strongyloides sp. prevalence and load in red colobus monkeys did

not differ among the habitats and this was not consistent with the expectation that the three edge

habitats would have more severe parasite infections than the interior forest habitat.

My results suggest that broad classifications such as “edge” or “interior” are too general

and that parasite infection may be affected by more specific conditions than just habitat

classification. I return to this point after discussing my second question, whether proximity to

human presence results in more severe parasite infections in colobus monkeys. I expected both

humanized habitats (agriculture and tea) to have more severe parasite infections than the non-

humanized habitats (interior forest and grassland). In general, I found that parasite infection was

not more severe in colobus found in humanized habitat. The expectation that black and white

colobus in the two humanized habitats would have more severe parasite infections than black and

white colobus in the two non-humanized habitats was not met for any of the parasites studied.

The prevalence and load of Trichuris sp. was found to be greater in a non-humanized habitat

22

(interior forest) than it was in one of the humanized habitats (tea). In another instance, the

prevalence and load of Trichuris sp. infection in a non-humanized habitat (interior forest) did not

differ from those of a humanized habitat (agriculture). Similarly, my results for strongyles also

did not correspond to the pattern that would be expected if humanized habitats caused an

increase in strongyle prevalence or load. Unlike Trichuris sp., none of the habitats had a greater

prevalence of strongyles than any other habitat. Strongyle load, on the other hand, appeared to

be greatest in one of the humanized habitats (tea), yet the other humanized habitat (agriculture)

did not differ from the non-humanized habitats. Finally, the prevalence and load of

Strongyloides sp. in black and white colobus samples from the interior forest did not differ from

either humanized habitat. Measures of infection with Trichuris sp., strongyles, and

Strongyloides sp. did not support my hypothesis that black and white colobus found in the two

humanized habitats would have more severe parasite infections than those found in the two non-

humanized habitats.

If proximity to humanized habitat was associated with increased prevalence of Trichuris

sp. infection in red colobus, I would expect samples from the two humanized habitats to have

more monkeys infected with Trichuris sp. than the two non-humanized habitats. The pattern of

Trichuris sp. prevalence in red colobus supported my expectation. Trichuris sp. prevalence in

the two humanized habitats did not differ from each other and both had a higher prevalence of

Trichuris sp. than the grassland habitat. The prevalence of Trichuris sp. in the agriculture and

the tea were also marginally higher than the interior forest. Thus the pattern of Trichuris sp.

prevalence among the different habitats appeared to be consistent with the hypothesis that

colobus found alongside humanized habitat have more severe infections than those in the non-

humanized habitat.

23

Trichuris sp. load in the red colobus samples differed among habitats but not in a manner

that was consistent with the expectation that both humanized habitats would have more severe

parasite infections than both non-humanized habitats. Trichuris sp. load for one non-humanized

habitat (the grassland) was lower than both humanized habitats and was marginally lower than

the interior forest. However, the Trichuris sp. load for the other non-humanized habitat (interior

forest) did not differ from the Trichuris sp. load for either of the humanized habitats. Neither

strongyles nor Strongyloides sp. differed among habitats for red colobus; this suggests that

neither of these parasite types are worsened by proximity to humans.

To summarize, significant differences were found among habitats, but the differences

lacked clear associations with specific habitat classifications such as ‘edge’ or ‘humanized’.

Rather, habitat types appeared to affect parasite infections individually. Three conclusions were

suggested by the results. First, the black and white colobus samples collected in the tea habitat

tended to have a higher load of strongyle eggs than the other habitats. Second, Strongyloides sp.

prevalence and load (with the outlier included) in the black and white colobus samples tended to

be greater in the grassland habitat (but this was not the case when the outlier was excluded).

Third, Strongyloides sp. was only found in red colobus samples from the agriculture habitat. The

results of my study suggest that there may be other aspects of primate habitat and/or aspects of

their behaviour that affect the prevalence and load of gastrointestinal parasites. It is possible that

differences in parasite infection among groups of colobus are due to factors other than the habitat

(see below).

Fecal cortisol did not differ between habitats for the black and white colobus, suggesting

that proximity to edges and/or proximity to humans did not result in increased stress for these

monkeys. Differences in cortisol levels were found between habitats for the red colobus

24

samples, but the differences were not related to specific habitat classifications such as “edge”

and “humanized”. Fecal samples from the agriculture (humanized, edge), grassland (non-

humanized, edge) and interior forest (non-humanized, non-edge) all demonstrated significantly

higher cortisol levels than the fecal samples from the tea (humanized edge) which was not

expected. No other differences were found which was not consistent with my expectation that

the humanized/edge habitats may be more stressful than non-humanized or non-edge habitats.

A number of factors may affect parasite infections (Nunn & Altizer 2006; Stuart & Strier

1995) and here five factors are discussed in detail. Nutrition, ranging, microclimate, the effect of

season and yearly variation in parasite infection may all contribute to the pattern of parasite

infection observed. I examine the potential impact of each of these four factors in turn and then

discuss conclusions and suggest ways forward for primate-parasite research.

Nutrition

Aspects of the host’s diet may affect gastrointestinal parasite infection (Rothman et al. in

press; Petkevicius 2007; Nunn & Altizer 2006; Ezenwa 2004; Coop and Kyriazakis 2001;

Crompton 1987). Ezenwa (2004) examined the gastrointestinal parasite infection of nine species

of wild bovid in Kenya. The bovid species with the lower quality diets demonstrated the highest

parasite egg output during drought, a time of nutritional stress. In primates, a connection

between diet and gastrointestinal parasite infection was suggested by Rothman et al. (in press) in

a study on mountain gorillas (Gorilla beringei) that examined the role of dietary protein and

tannins on gastrointestinal parasite infection. They found a reduction in the larval load of

Probstmayria sp. after some of the gorillas ate foods higher in tannins. Similarly, other primate

research has also suggested that parasite infection may be affected by changes in food/diet. For

example, Chapman et al. (2006b) found that loss of food trees in forest fragments around Kibale

25

was associated with a decline in the overall number of red colobus, as well as higher levels of

parasite infection in these monkeys. In a study on baboons, Appleton and Henzi (1993) found

baboons living at a higher altitude had greater parasite infections than those at a lower altitude.

Differences in foraging behaviours, food accessibility, and time spent traveling were considered

as contributing factors to the dissimilarities in the parasite infections. Finally, Weyher et al.

(2006) compared the parasite infections in a troop of crop-raiding baboons to a troop of baboons

that did not raid crops. Non-crop raiding baboons had higher parasite outputs of Physaloptera

sp. and Trichuris sp., whereas the troop that raided crops had a higher output of Balintidium coli.

The researchers suggested the dissimilarity in parasite infection between the two troops may be

related to nutritional differences resulting from access to the crops. All of these studies indicated

that I should consider whether there were nutritional differences among the groups inhabiting the

four habitat types in this study.

Variations in the diets of black-and-white colobus exist among groups. Harris and

Chapman (2007) studied the diet of multiple groups of black-and-white colobus and found the

proportion of each group’s diet comprised of certain tree species was related to which tree

species were found within the group’s range. Furthermore, variation in nutrition of food trees

ingested by red colobus and black-and-white colobus was documented by Chapman et al. (2003).

They found between-species nutritional differences as well as spatial and temporal variation in

the nutritional quality of the same species of food trees for both red colobus and black and white

colobus. Based on this research, it is quite likely that there were differences in diets between the

groups that I sampled, which may have affected their patterns of parasite infection. Likewise,

Chapman et al. (2006a) also speculated as to whether nutritional differences may have been

26

present between the forest edges and interior forest.

Ranging Patterns/Behaviour

A number of researchers have suggested that ranging patterns may also affect primate

parasite infections (Nunn & Dokey 2006; Nunn & Altizer 2006; Stoner 1996; Freeland 1976). In

a review of 119 primate species, Nunn and Dokey (2006) found increased helminth richness in

primates that have a longer day range within their home ranges. Nunn and Dokey (2006)

suggested that the higher parasite richness was due to increased contamination of areas that were

used more often. The researchers did not find home range overlap or territorial behaviour to be

correlated with parasite richness. Thus, species with greater daily travel within a small range

may have increased parasite infections compared to species that travel less each day within a

larger range.

Generally, the home ranges of red colobus are about 50% greater than the home ranges of

black and white colobus monkeys (Oates 1994). Oates (1994) provides home and day ranges

estimates for two colobus groups from Kibale (one black-and-white colobus group and one red

colobus group). The group of black and white colobus were found to have a mean daily travel

distance of approximately 535 m and a home range of approximately 28 ha (Oates 1977; Oates,

1994). The group of red colobus monkeys were found to have a mean daily travel distance of

about 649 m, but a home range of about 65 ha (Oates 1994). Based on Nunn and Dokey’s

(2006) research, differences in ranging between the two species may produce differences in their

parasite infections. While I expected parasite infections in red colobus and in black and white

colobus to respond similarly to edge or humanized habitat (e.g., both to increase as a result of

proximity to edge/humanized areas), it may be that other aspects of each colobine’s behaviour

27

(e.g., ranging patterns) will produce differences in their parasite infections, even though they are

found in the same habitat.

With their larger home ranges, the red colobus monkeys in this study could spend more

time in the forest interior than on the forest edge and it is also possible that they could range as

far from the interior forest as the forest edge. This was less likely for the black and white

colobus as they have smaller home ranges. Thus, parasite infection may not be representative of

the habitat the colobus were in at the time of fecal sample collection. A more accurate

assessment of the effects of habitat on parasite infection could be obtained by continuously

following specific groups of monkeys and relating the amount of time spent in different habitat

types (e.g. edge habitat) to indices of parasite infections. However, this approach would take a

great investment of time since accurately determining the ranging patterns of colobus takes many

months (Chapman, unpublished data).

Differences in ranging patterns may also affect parasite infections at the group level

within a single species. For example, Stoner (1996) studied the parasite infection in two groups

of howler monkeys (Alouatta palliata) in Costa Rica. One group was found to have a higher

parasite prevalence than the other group. One of the suggestions Stoner (1996) put forth to

explain this difference was that the group with the higher prevalence of parasites had a smaller

home range. Additionally, this group reused particular arboreal pathways, which was

hypothesized to increase exposure to feces. The group with the lower parasite prevalence had a

larger home range and did not reuse arboreal pathways as frequently. It thus seems important to

consider the between-group ranging patterns within a single species.

Between-group differences in ranging behaviour have been documented in red colobus

monkeys. Gillespie and Chapman (2001) found both home and day ranges to vary between two

28

red colobus groups with a large group of red colobus (48 members) having a much larger home

range and daily travel distance than the smaller group (24 members). The smaller group re-

visited feeding trees more often than the larger group and this repeated use of food trees could

lead to an increased risk of parasitic infection. Black and white colobus also vary in their

ranging patterns between groups. Harris and Chapman (2007) found black and white colobus

ranges to vary between approximately 7 and 30 ha and that the colobus make excursions to

important food resources. Consequently, it possible that differences in the specific ranging

patterns of individual colobus groups contributed to the variation in parasite infection I observed

among habitat types.

In addition to ranging behaviour, another aspect of primate behaviour to consider with

directly-transmitted parasites is whether primates are arboreal or terrestrial. Although re-using

particular arboreal pathways may increase exposure to parasites (Stoner 1996), an arboreal

lifestyle in general may reduce exposure to parasites (Vitazkova & Wade 2007; Ekanayake et al.

2006). This was suggested by Ekanayake et al. (2006) who examined gastrointestinal parasitism

in toque macaques (Macaca sinica), gray langurs (Semnopithecus priam) and purple-faced

langurs (Trachypithecus vetulus). The macaques and gray langurs spent more time on the

ground in areas nearby human defecation sites and this was suggested as a possibility for why

Cryptosporidium sp. was more prevalent in fecal samples from these two primates. The purple-

faced langurs were more arboreal and the researchers suggested that this may have contributed to

the reduced prevalence of Cryptosporidium sp. in these monkeys (Ekanayake et al. 2006).

Similarly, Vitazkova and Wade (2006) suggested that howler monkeys (Alouatta pigra) may

become infected with Giardia sp. when traveling on the ground between forest fragments.

Perhaps an arboreal lifestyle is helping to reduce exposure to parasites in colobus monkeys and

29

this may be one reason why more severe parasite infections were not observed in the colobus

found alongside humanized habitats.

Microclimate

A number of primate studies have suggested the environmental conditions of specific

areas may affect parasite infection (Chapman et al. in press-b; Nunn and Altizer 2006; Stoner

1996; Stuart and Strier 1995; Appleton and Brain 1993; Stuart et al. 1990); including some on

colobines (Chapman et al. in press-b). For example, in an interesting study on the

gastrointestinal parasites of baboons (Papio cynocephalus ursinus), Appleton and Brain (1993)

discuss how the lifecycle of the parasites and the desert environment that the baboons inhabit

may both contribute to the parasite species infecting the host. The researchers found protozoan

parasites to be the most common in this baboon species and they point out that the protozoan

parasites do not require a development period outside of the host whereas helminth parasites do.

Briefly, protozoan parasites such as Giardia sp. Cryptosporidium sp. are infective when shed in

feces (Hunter & Thompson 2005). This is in contrast to eggs of Trichuris trichiura which take

approximately 4 weeks to develop (become infective) in a suitable environment (warm, moist,

shady soil) (Ash and Orihel 2007; Bethony et al. 2006; Stephenson et al. 2000). Appleton and

Brain (1993) mention that the number of helminth species observed in these baboons may be low

due to the combination of low soil moisture and the time helminths require outside the host. The

researchers note that the soil may not retain enough moisture for the length of time required for

eggs to develop. It therefore seems that characteristics of the parasite and the habitat(s) that a

host inhabits should be considered in studies on parasite infection. Research by Chapman et al.

(in press-b) compared the parasite infection of black-and-white colobus monkeys inhabiting wet

and dry habitats in the Kanyawara study site of Kibale. The animals that inhabited the wet

30

lowland areas had increased parasite richness and Trichuris sp. load than animals in the drier

areas. Similarly, research on howler monkeys has also suggested that howlers inhabiting wetter

habitats may have more severe infections of some gastrointestinal parasites (Stoner 1996; Stuart

et al. 1990).

In this study, there were likely differences in the microclimate between the areas of the

habitat types from which we collected colobus fecal samples. This might result in a difference in

parasite infections between a colobus group that spends a significant portion of time in a

swampy, moister area of the park versus on that inhabits a drier one (Chapman et al. in press-b).

Other differences may occur between a group that spends a lot of time on a forest edge versus

one from the forest interior as forest edges may have a different microclimate than the forest

interior (Murcia 1995).

Seasonal Effect

Seasonal changes in weather may influence parasite infection (Nunn and Altizer 2006;

Larsen and Roepstorff 1999; Milton 1996). For example, seasonal changes in parasite infection

have been reported for sheep, goats, and cattle (Nwosu et al. 2007; Umur and Yukari 2005; Lima

1998; Pandey et al. 1993). Research has suggested that there is also an element of seasonality to

gastrointestinal nematode infection in primates including chimpanzees (Huffman et al. 1997;

Kawabata & Nishida 1991), howler monkeys (Eckert et al. 2006), gorillas (Rothman et al. 2008),

and mandrills (Setchell et al. 2007). For example, Huffman et al. (1997) documented an increase

in prevalence and load of Oesophagostomum stephanosum in fecal samples from chimpanzees

(Pan troglodytes) collected during the rainy months of the year compared to those collected

during the drier months. It is important to note the possibility that season could affect parasite

infection because my study examined parasite infection over 3 months (April to July). This

31

coincides with the end of the wet season and start of the dry season in Kibale. The study by

Chapman et al. (2006a) examined parasite infection over a number of years (1997 – 2004) and

thus encompassed both wet and dry seasons. Consequently the dissimilarity in parasite infection

patterns between two studies may be partly due to differences in season of collection.

Previous research on colobus parasite infections did not find a seasonal effect, but

parasite infections did vary throughout the year (Gillespie et al. 2005). The researchers

examined many groups of colobus (red and black and white) throughout Kibale and noted that

the lack of a seasonal effect was unexpected. Gillespie et al. (2005) propose further examination

into this finding. Here I speculate as to whether the variation between fecal samples collected

from multiple colobus groups in the research by Gillespie et al. (2005) could obscure observation

of a seasonal pattern. Gillespie et al. (2005) collected samples from colobus groups inhabiting

forest fragments and continuous forest and all of the samples were combined. Since then,

Gillespie and Chapman (2008) have documented differences in parasite infection in red colobus

between forest fragments and the forest interior (higher prevalence and richness of some species

in the forest fragments), but not in black and white colobus. Based on the findings of this

research and on the factors discussed that may influence parasite infection, I speculate as to

whether a seasonal trend may become apparent if the parasite infection of a smaller number of

colobus groups (e.g., 1 - 2) were tracked over the course of the year, rather than pooling the

samples from multiple groups in multiple habitats. This might reduce some of the variability that

could stem from differences in habitat, microclimate, group size, ranging, etc. For example, the

work by Huffman et al. (1997) which found parasite infection varied according to season focused

on a single group of chimpanzees (Pan troglodytes) and was over a year in duration.

32

Yearly variation

Finally, another point to keep in mind is that there may be year to year variation in parasite

infections (Huffman et al. 1997). For example, Huffman et al. (1997) found Strongyloides

fulleborni infections to be more prevalent in chimpanzees in 1993/94 than they were in 1991/92.

The researchers also mention that there appeared to be a slight increase in Oesophagostomum

stephanosum and Trichuris trichiura over the study. This is important to consider in comparative

studies. For example, here I compared my results to the study by Chapman et al. (2006a).

However, I collected colobus fecal samples in 2007 whereas Chapman et al. (2006a) collected

colobus fecal samples from 1997 to 2004. Annual variation in parasite infection may thus be an

important factor to consider in comparative research.

Other Factors

Both Nunn and Altizer (2006) and Stuart and Strier (1995) provide excellent reviews on

many of the factors that may affect parasite infections in primates. They mention factors such as

group size, primate density, mating and reproduction, dominance, body mass, and other factors

to consider when investigating parasitic infections in primates. One approach may be to consider

many factors in analyses and see which factor(s) are correlated with parasite infection (Nunn and

Altizer 2006). For example, in research on parasite infection in red colobus inhabiting forest

fragments around Kibale, Gillespie and Chapman (2006) included a number of factors in their

analysis: fragment size, fragment type, distance to the park, distance to the next fragment,

number of trees/ha, number of tree species/ha, tree stump density, number of red colobus/ha and

total colobus/ha. They found tree stump density (a measure of habitat degradation) to be most

strongly correlated with parasite prevalence in red colobus. Unlike the forest fragments, the

boundary between the forest edges of Kibale and the humanized (agricultural) areas are

33

considered to be relatively undisturbed (Chapman et al. 2006a). Thus, habitat degradation is

likely not as great in these areas and this may be one reason why parasite infection did not appear

to be greater in the humanized habitats. Perhaps the degree of habitat degradation should be

considered along with proximity to humans. A number of researchers have also suggested that

parasite infection may be affected by more than one factor at a time (Vitazkova and Wade 2007;

Chapman et al. 2006b; Stoner 1996; Stuart and Strier 1995) and this may worsen parasite

infection (Chapman et al. 2006b). Analyses of primate health that takes multiple factors into

consideration may thus be one way to decipher which factors are making a significant

contribution to parasite infections in primates.

Conclusions

The aim of this study was to determine whether colobus monkeys on humanized forest

edges experience more severe parasite infections than colobus monkeys on non-humanized forest

edges. I asked four questions: Do colobus monkeys inhabiting forest edges experience more

severe parasite infections than those inhabiting the interior forest? Do colobus monkeys

inhabiting humanized habitats experience more severe parasite infections than those inhabiting

non-humanized habitats? Do colobus monkeys inhabiting edge habitats experience greater stress

than those inhabiting interior forest? Do colobus monkeys inhabiting humanized habitats

experience greater stress than those inhabiting non-humanized habitats? Neither edge habitat nor

humanized edge habitat appeared to increase parasite infection or cortisol levels. The results of

this study suggest that classifications such as “humanized” may be too broad and that many

factors may affect parasite infections of primates.

One way forward suggested by Chapman et al. (in press-a) is to incorporate disease

modeling/spatial epidemiology into primate research. In their discussion on the usefulness of

34

models, they mention that modeling diseases may allow researchers to make predictions about

the possible effects of disease(s) on a population (e.g. decline, little effect, stability, etc) or on a

segment of a population (e.g. infants) and may also allow investigation into associations between

disease and multiple factors (e.g. environmental, demographic) (Chapman et al. in press-a).

35

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Appendix – Animal Care Protocol

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