Assessment of gastrointestinal parasites with zoonotic potential in two communities of chimpanzees (Pan troglodytes verus) from Guinea-Bissau and its implications for conservation.

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DIPARTIMENTO DI BIOLOGIA

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ESI DI

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AUREA

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AGISTRALE IN

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ONSERVAZIONE ED

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VOLUZIONE

ASSESSMENT OF GASTROINTESTINAL PARASITES WITH ZOONOTIC POTENTIAL IN TWO

COMMUNITIES OF CHIMPANZEES (PAN TROGLODYTES VERUS) FROM GUINEA-BISSAU AND ITS

IMPLICATIONS FOR CONSERVATION.

Candidata Relatori Sylvie Grange Fabio Macchioni

Damiano Marchi Rui M. Sá

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There is a pleasure in the pathless woods, There is a rapture on the lonely shore, There is society, where none intrudes, By the deep sea, and music in its roar: I love not man the less, but Nature more.

C'è una gioia nei boschi inesplorati, C'è un'estasi sulla spiaggia solitaria, C'è vita, dove nessuno arriva, Vicino al mare profondo, e c’è musica nel suo boato: Io non amo l’uomo di meno, ma la Natura di più.

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ABSTRACT

All chimpanzee species are classified as endangered by IUCN Red List. Their survival is menaced by ongoing anthropogenic pressures, such as hunting, pet trade, climate change and habitat fragmentation. Moreover, infectious diseases can impact their health status, sometimes becoming life-threatening. Infections can be caused by many different microorganisms, including gastrointestinal parasites. The aim of this study is to evaluate the gastrointestinal symbionts of two communities of chimpanzees from Guinea-Bissau inhabiting different areas: one inside a protected area (Cufada Lagoons Natural Park) and one outside (Tite sector). We will test two hypotheses in this study: 1. statistically significant differences in terms of prevalence and richness of gastrointestinal symbionts would be found between the two communities, in particular for zoonotic parasites; 2. chimpanzees inhabiting outside the protected area should be more infected, because of their closer contact with human communities. Fresh faecal samples (N = 155) were collected opportunistically from unhabituated chimpanzees during the dry season (March-April 2017) and analyzed performing standard parasitological analyses of flotation and sedimentation. Descriptive statistics and multivariate inference was used to interpret the results. Results in terms of prevalence and richness of gastrointestinal microorganisms suggest that the community living inside the natural park could suffer of higher anthropogenic pressures. We suggest that the role of park be revised to provide more successful strategies in chimpanzee conservation efforts.

KEYWORDS

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RIASSUNTO

Gli scimpanzé sono classificati come in pericolo di estinzione nella lista rossa dell‟IUCN. La loro sopravvivenza è minacciata dalle pressioni antropogeniche, come la caccia, il pet trade, il cambiamento climatico e la frammentazione dell‟habitat. Inoltre, le malattie infettive possono influenzare negativamente lo stato di salute degli animali e arrivare a minacciarne la sopravvivenza. Tali malattie possono essere causate da diversi microorganismi, inclusi i parassiti gastrointestinali. L‟obiettivo di questo studio è quello di valutare la composizione dei simbionti gastrointestinali di due comunità di scimpanzé che vivono in diverse aree in Guinea-Bissau, una all‟interno di un‟area protetta (Parco Naturale Lagoas de Cufada) e una all‟esterno (regione di Tite). L‟ipotesi è che differenze statisticamente significative siano presenti tra le due comunità in termini di prevalenza e ricchezza di parassiti, in particolare di quelli con potenziale zoonotico; gli animali abitanti all‟esterno dell‟area protetta dovrebbero essere più parassitati, a causa del maggior contatto con le popolazioni locali. Sono stati raccolti opportunisticamente durante la stagione secca (marzo-aprile 2017) 155 campioni fecali, provenienti da scimpanzé non abituati alla presenza dell‟uomo. I campioni sono stati analizzati utilizzando analisi parassitologiche di flottazione e sedimentazione. Analisi di statistica descrittiva e inferenza multivariata sono state usate per descrivere i risultati. I risultati ottenuti in termini di prevalenza e ricchezza, suggeriscono che la comunità stanziata all‟interno del parco naturale possa essere più soggetta alle pressioni antropogeniche. Il ruolo del parco deve quindi essere rivisto, per poter adempiere ai suoi obiettivi di conservazione degli scimpanzé.

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PAROLE CHIAVE

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LIST OF TABLES

TABLE 1:MORPHOMETRIC MEASURES. ... 85

TABLE 2:OVERALL PREVALENCE. ... 86

TABLE 3:PREVALENCE OUTSIDE AND INSIDE THE PROTECTED AREA. ... 88

TABLE 4:COMPARISON OF PREVALENCE IN SUB-AREAS OUTSIDE THE PROTECTED AREA. ... 89

TABLE 5:COMPARISON OF PREVALENCE IN SUB-AREAS INSIDE THE PROTECTED AREA. ... 90

TABLE 6:OVERALL PREVALENCE OF INFRACOMMUNITIES. ... 91

TABLE 7:PREVALENCE OF INFRACOMMUNITIES OUTSIDE AND INSIDE THE PROTECTED AREA. ... 91

TABLE 8:COMPARISON OF INFRACOMMUNITIES AMONG SUB-AREAS OUTSIDE THE PROTECTED AREA. ... 92

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LIST OF FIGURES

FIGURE 1:CHIMPANZEE MODERN TAXONOMY.FROM:DONSNOTE,2006. ... 21

FIGURE 2:CHIMPANZEE DISTRIBUTION IN AFRICA.FROM:CLEE ET AL.,2015. ... 22

FIGURE 3:CAPTIVE CHIMPANZEE,BUBA,GUINEA-BISSAU.FROM:SYLVIE GRANGE,2017. ... 23

FIGURE 4:CHIMPANZEES‟ NESTS ON TREES,TITE REGION,GUINEA-BISSAU.FROM:SALVATORE IZZO,2017. ... 26

FIGURE 5:THE "ONE HEALTH TRIAD", FIGURING THE INTERCONNECTION BETWEEN HUMANS, ANIMALS AND ENVIRONMENT.FROM:THOMPSON,2013. ... 34

FIGURE 6:CYST AND TROPHOZOITE OF ENTAMOEBA COLI WITH IODINE STAINING.FROM:ATLAS PROTOZOA,2010. ... 40

FIGURE 7:CYST AND ROPHOZOITE OF ENTAMOEBA HISTOLYTICA/DISPAR/MOSHKOVSKII UNSTAINED.FROM:ATLAS PROTOZOA,2010. ... 41

FIGURE 8:CYST AND TROPHOZOITE OF ENTAMOEBA HARTMANNI UNSTAINED.FROM:ATLAS PROTOZOA,2010. ... 41

FIGURE 9:CYST AND TROPHOZOITE OF ENDOLIMAX NANA UNSTAINED.FROM:MEDICAL-LABS,2014. ... 42

FIGURE 10:CYST AND TROPHOZOITE OF IODAMOEBA BÜTSCHLII WITH IODINE STAINING.FROM:ATLAS PROTOZOA,2010. ... 43

FIGURE 11:CYST AND TROPHOZOITE OF GIARDIA DUODENALIS WITH IODINE STAINING.FROM:CDC,2016. ... 45

FIGURE 12:CYST AND TROPHOZOITE OF CHILOMASTIX MESNILI WITH IODINE STAINING.FROM:ATLAS PROTOZOA,2010. 46 FIGURE 13:CYST AND TROPHOZOITE OF BALANTIDIUM COLI UNSTAINED.FROM:CDC,2016. ... 48

FIGURE 14:TROPHOZOITE OF TROGLODYTELLA ABRASSARTI WITH IODINE STAINING.FROM:SYLVIE GRANGE,2017. ... 49

FIGURE 15:TROPHOZOITE OF TROGLOCORYS CAVA WITH IODINE STAINING.FROM:SYLVIE GRANGE,2017. ... 49

FIGURE 16:VACUOLAR AND GRANULAR FORM OF BLASTOCYSTIS SPP. WITH IODINE STAINING.FROM:CDC,2016. AMEBOID FORM OF BLASTOCYSTIS SPP.UNSTAINED.FROM:CASERO ET AL.,2015. ... 51

FIGURE 17:EGG OF BERTIELLA SPP. WITH IODINE STAINING.FROM:SYLVIE GRANGE,2017.PROGLOTTIDS OF BERTIELLA ADULT WORM.FROM:CDC,2016. ... 55

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FIGURE 18:EGG OF DICROCOELIUM SPP. UNSTAINED.FROM:CDC,2016.ADULT WORM STAINED TO SHOW THE INTERNAL

STRUCTURES FROM:UNIVERSITY OF PENNSYLVANIA,2008. ... 57

FIGURE 19:GENERAL PATTERN OF NEMATODE LIFE CYCLE.FROM:JOHNSTONE ET AL.,1998. ... 59

FIGURE 20:EGG OF HOOKWORM UNSTAINED.FROM:CDC,2016.ADULT FEMALE AND MALE.FROM:BETHONY ET AL., 2006. ... 61

FIGURE 21:EGG UNSTAINED AND ADULT OF OESOPHAGOSTOMUM SPP.FROM:CDC,2016. ... 63

FIGURE 22:EGG OF TRICHOSTRONGYLUS SPP. WITH IODINE STAINING.FROM:SYLVIE GRANGE,2017.ADULT WORM. FROM:VETBOOK,2010... 65

FIGURE 23:FERTILIZED EGG AND UNFERTILIZED EGG AND OF ASCARIS SPP. UNSTAINED.ADULT WORM.FROM:CDC,2016. ... 66

FIGURE 24:EGG OF TRICHURIS SPP. UNSTAINED.FROM:CDC,2013.ADULT WORM.FROM:MEDICAL-LABS,2014. ... 68

FIGURE 25:EGG OF ENTEROBIUS SPP. UNSTAINED.ADULT WORM.FROM:CDC,2016. ... 70

FIGURE 26:EGG OF STRONGYLOIDES FUELLEBORNI WITH IODINE STAINING.FROM:SYLVIE GRANGE,2017.LARVA AND ADULT OF STRONGYLOIDES STERCORALIS UNSTAINED.FROM:CDC,2016. ... 72

FIGURE 27:POLITICAL MAP OF GUINEA-BISSAU.FROM:VECTORMAP,2017. ... 75

FIGURE 28:STUDY AREAS OUTSIDE AND INSIDE THE PROTECTED AREA. ... 76

FIGURE 29: A FRESH FAECAL SAMPLE AND AN OLD FAECAL SAMPLE.FROM:SYLVIE GRANGE,2017. ... 79

FIGURE 30:SAMPLE SET.FROM:SYLVIE GRANGE,2017 ... 79

FIGURE 31:BÙ IN THE HEAD-QUARTER OF CLNP.MANO ZÉ ON A TREE.FROM:SYLVIE GRANGE,2017 ... 80

FIGURE 32:COLOR PLATE OF CHIMPANZEE SYMBIONTS RECOVERED.FROM:SYLVIE GRANGE,2017. ... 84

FIGURE 33:RICHNESS TREND. A-OVERALL (RANGE:0-8; MEAN±SD=4.1±1.7). B-OUTSIDE THE PROTECTED AREA (RANGE:0-7; MEAN±SD=4.1±1.5). C-INSIDE THE PROTECTED AREA (RANGE:0-8; MEAN±SD=4.1±1.9). D-HERGA (RANGE:2-7; MEAN±SD=4.4±1.5). E-NOVA SINTRA (RANGE:0-7; MEAN±SD=3.9±1.5). F-NHALA (RANGE:0-6; MEAN±SD=2.9±1.6). G-BUBA TCHINGUE (RANGE:2-8; MEAN±SD=5.2±1.3). H-BUBA (RANGE:3-8; MEAN±SD= 4.8±1.9) ... 94

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LIST OF ABBREVIATIONS

AMA: American Medical Association

ANOVA: Analysis of Variance

AVMA: American Veterinary Medical Association

CDC: Centers for Disease Control and Prevention

CITES: Convention on International Trade in Endangered Species of Wild Fauna and Flora

CLNP: Cufada Lagoons Natural Park

EID: Emerging Infectious Disease

IBAP: Institute for Biodiversity and Protected Area

IUCN: the International Union for the Conservation of Nature

MIFC: Merthiolate-Iodine-Formaldehyde Concentration

ND: Neglected Diseases

QP: Quantitative Parasitology

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ACKNOWLEDGEMENTS

I would like to thank all the people that helped to the success of this project. I am grateful for their help in the collection of samples to my field assistant in Nova Sintra, Mussa Djassi, and to Umaro Candé and Iéro Embaló. I am grateful to all the Guinean people I met during my stay in Guinea-Bissau, they all contributed in their way to this amazing experience. I would like to thank especially Universidade Lusófona da Guiné for the welcoming and the supply of the material and the laboratory. Thank you to Salvatore and Joana, for sharing this incredible adventure with me, supporting my fragile and emotional soul. The biggest help from abroad came of course from Rui, my supervisor and friend. I do not have enough words for you, but as I said many times, you know that without your support none of this would have been possible. Thanks to the ASP society for a part of the funds needed for this project.

A Pisa, vorrei ringraziare i miei relatori, che mi hanno seguita e supportata lungo tutto il percorso, in particolare il professor Macchioni, pronto a rispondermi a qualsiasi ora e con qualunque mezzo disponibile, e l‟Università di Pisa (dipartimento di Scienze Veterinarie) che mi ha permesso di terminare il lavoro di tesi. Grazie ai miei amici, spesso più lontani che vicini, ma che sanno come essere presenti. Grazie quindi soprattutto alle Paxxissime, per essere il mio punto di riferimento e supporto morale, a Simone, a cui voglio sempre molto bene anche se sono più le volte che gli tirerei il collo, a Salvatore, per la breve ma intensa avventura condivisa a Pisa, a Francesca, per le passioni nerd e un po‟ maniache che condividiamo, a Eluardo, il mio grande amico trovato un po‟ per caso dall‟altra parte del mondo, a Martina, che c‟è da sempre, nonostante le nostre strade abbiano preso direzioni diverse. Grazie ai miei coinquilini, per avermi fatto capire che non voglio mai più vivere con altri studenti, in particolar modo a Gian Michele e Claudia. Grazie alla mia splendida famiglia che mi incoraggia, anche se non sempre d‟accordo con le mie scelte, e crede in me più di quanto io potrò mai fare. E infine grazie a te, Lorenzo, che mi hai cambiato la vita. Ringrazio l‟universo per avermi donato quell‟incontro, davanti a un grande tonno appena pescato, su quella piccola isola sperduta, che ricorda tanto il paradiso.

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CHAPTER 1 INTRODUCTION

1.1 Chimpanzee social-ecology: an overview

1.1.1 Taxonomy

Chimpanzees are our closest relatives in evolutionary terms as they share with humans more than 98% of their genetic information (Gonzalez et al., 2012). Chimpanzees belong to the order of Primates, that is subdivided in fourteen families and eight super-families. According to the modern classification, based on genetic studies performed in the last decades, chimpanzees belong to the Hominoidea superfamily, that includes the less and the great apes, which in turn is composed of the taxonomic families: Hylobatidae (i.e. gibbons and siamangs) and Hominidae (i.e. great apes and humans). The taxonomic family of Hominidae includes the extant subfamilies: Ponginae (i.e. orangutans) and Homininae (i.e. chimpanzees, bonobos and humans) (Feagle, 2013) (Fig. 1).

Figure 1: Chimpanzee modern taxonomy. From: Donsnote, 2006.

1.1.2 Distribution

Chimpanzees, Pan troglodytes ssp., are geographically distributed from a West-East tropical belt in Africa, from Senegal in the West to Tanzania in the East. There are four recognized subspecies of common chimpanzees, that present small differences in terms of morphology (PIN, 2006): Pan troglodytes verus, or Western chimpanzees, inhabiting in West Africa, where they are

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present in Senegal, Mali, Guinea-Bissau, Guinea, Sierra Leone, Liberia, Ivory Coast and Ghana (Inskipp, 2005). They were present, but now considered extinct, in Togo and Benin (Brownell, 2003); Pan troglodytes ellioti inhabiting in Nigeria and Cameroon (Morgan et al., 2011); Pan troglodytes troglodytes inhabiting Central Africa; Pan troglodytes schweinfurthii inhabiting in East Africa (Morgan et al., 2011) (Fig. 2).

Figure 2: Chimpanzee distribution in Africa. From: Clee et al., 2015.

1.1.3 Habitat and ecology

The home range of chimpanzees can change considerably in relation to the ecological and environmental characteristics of the habitat, such as: the presence of predators, the presence of other chimpanzee communities (Goodall, 1986) and food availability (Netwon-Fisher, 2003). According to the type of habitat, it was observed that chimpanzee communities living in more continuous habitats (e.g. forests) showed smaller home ranges, generally lower than 10 km2 (e.g. Kano, 1972; Newton-Fisher, 2003; Yamagiwa and Basabose, 2009) while communities living in a more open and fragmented environments (e.g. savannahs) showed bigger home ranges of hundreds of km2, with a maximum estimated of 400 km2 (e.g. Baldwin et al., 1982; Ogawa et al., 2006).

Chimpanzees can live in different types of habitats, from tropical forests, where they play an important role for the vegetation restoration, as they can act as seed-dispersers (Mittermeier, 1988), to more extreme areas, such as hot, dry and open arboreal savannahs in Niokolo-Koba National

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Park, Senegal (McGrew et al., 1981; Hunt and McGrew, 2002). Important differences, in terms of morphology, behavior and sociality, are presented by chimpanzees living in these two different types of habitats. For this reason, some authors such as Moore (1996) make distinctions between the “forest chimpanzee” and the “savannah chimpanzee”.

Chimpanzees are able to travel for 5 km/day (ADV, 2005) into the forest branches and canopies to protect themselves from predators (Tutin et al., 1981) or searching for food (Chance and Jolly, 1970). Despite this, they are also capable to move on the ground, where they can pass a good period of time socializing, jumping from one tree to another, and moving sometimes on a bipedal way (Chance and Jolly, 1970). When they walk with all the four limbs they use a locomotion type called “knuckle-walking”, which is also present in bonobos and gorillas (Tuttle, 1986).

1.1.4 Group composition

Chimpanzees (Fig. 3) live in multi-male-multi-female groups composed by 35 members in average (IUCN, 2016) and they are characterized by a fission-fusion social dynamics (Goodall, 1986). This means that, in some moment, usually during the day, the components of the community are subdivided into small subgroups that are called “parties”. The parties are not stable and can change frequently in number (Ghiglieri, 1984), although stable parties were sometimes reported, for example in Bossou, Guinea (Sugiyama and Koman, 1992).

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The average number of individuals present in a party is five, but this number can vary depending on population composition and it was highlighted that smaller communities are characterized by less flexibility (Boesch, 1996). Moreover, the size but also the composition of parties is influenced by other factors, for example, Wrangham et al. (1996) demonstrated how grouping patterns can be influenced by food distribution while Goodall (1986) proposed that the most influent factor is sex. Usually, the parties are composed of adults of both sexes and their offspring, although more rarely parties composed by only adults of the same sex or only adults can be observed (Boesch, 1996). Among chimpanzees, affiliative behavior between males is reported, because the necessity of defense of the territory and maintenance of domination rank (Palagi, 2006). According to this behavior, the community presents female exogamy and male retention in their natal group to avoid incest (Ghiglieri, 1987). According to de Waal (1996), chimpanzees can be considered an egalitarian species but, in fact, males are usually dominants on females and allow them to access to some resources only in the period of reproduction (Hemelrijk, 2002). Even if they present a clear social hierarchy (de Waal, 1986) subordinate individuals can join forces and create alliances to take off the power of dominant members of the community such as the alpha males (de Waal, 1982). However, even if related males are supportive to each other and can defend together their territory, they can kill other males that are not related or belong to a different community (Ghiglieri, 1987). Agonism is, in fact, a common behavior, especially between and within the communities, as chimpanzees are very territorial and hostilities can occur because of the defense of their territory and their resources, such as food and females (Farley, 2016).

1.1.5 Diet

According to a study performed by Stumpf (2011), the chimpanzee diet is highly variable, as it depends on the distribution of the natural resources in the area. Their diet is mainly composed of fruits, but they can also include terrestrial herbaceous vegetation, such as leaves, and animal protein, that are necessary to supply essential nutrients (Yamagiwa and Basabose, 2009). Differences in the consumption of mammals and insects among populations of chimpanzees in different parts of Africa was investigated by McGrew (1983), that observed that in general chimpanzees rarely eat meat and when they do, they usually prey small mammals, while birds are very rarely hunted. He concluded that the most part of differences in chimpanzee diet composition can be explained by the characteristics of the habitat and other environmental factors. The

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consumption of mammals was observed by other studies, e.g. Nishida and Uehara (1983), and in some cases, chimpanzees were observed to kill and eat other species of primates, such as colobines (e.g. Goodall, 1986; Busse 1977; Morris and Goodall 1977). Despite these evidences, chimpanzees eat more insects than meat of mammals. Insects are richer in terms of nutrients and calories and, for this reason, insect-eating can be observed as a chimpanzee daily activity in some communities (Nishida and Hiraiwa, 1982). They consume mainly social insects such as ants (Order Hymenoptera) and termites (Order Isoptera), because they provide more energy in a single consumption, even if they can consume non-social species as well (McGrew, 1983). In particular, it was proved that in more difficult habitats, like in Senegal, chimpanzees spend more time eating termites than in other populations (Bogart and Pruetz, 2010).

1.1.6 Behavior

Important cultural differences can be observed in the behavior of communities inhabiting different areas of Africa (Farley, 2016). Whiten et al. (1999) examined and analyzed data coming from the six most long chimpanzee study sites (Bossou, Guinea; Taï Forest, Ivory Coast; Gombe, Tanzania; Mahale Forest, Uganda; Kibale Forest, Uganda; Budongo Forest, Uganda), finding 39 different behavior patterns, including tool using, grooming and courtship behaviors. Some of these behaviors, such as start playing or investing probes were common to all sites, while others such as ant-fish or bee-probe were found only in some communities (Whiten et al., 1999). Pan troglodytes ssp. are characterized by the biggest spectrum of behavior among wild animals and for some authors this resembles a typical characteristic of humans (Murdock, 1967). Chimpanzees and other great apes present a building nest behavior (Goodall, 1962) (Fig. 4). In particular, Pan troglodytes verus from Guinea-Bissau seem to show a preference for some species of trees to nest in relation to their abundance: Sousa et al. (2011), found in Cantanhez National Park, Guinea Bissau, a 92% of preference to construct their nests on oil-palms trees (Elaeis guineensis), a high nest prevalence in this area. Another example came from Senegal, where Ndiaye et al. (2013), highlighted that Western chimpanzees in this area prefer to use Pterocarpus erinaceus to build their nests. The preference of trees for nesting can have different causes: (i) they can choose some trees because of their height and wood hardness (Ndiaye et al., 2013) or (ii) because they are important food resources (Basabose and Yamagiwa, 2002). The nest building behavior influence the distribution of

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chimpanzees, as some habitats, like canopy forest or savannah-woodlands have suitable trees for building nests, while herbaceous savannahs, swamp forests and mangroves have not.

Figure 4: Chimpanzees‟ nests on trees, Tite region, Guinea-Bissau. From: Salvatore Izzo, 2017.

1.2 Major threats to chimpanzee conservation

Although chimpanzees can be very adaptable to different types of habitats, their survival is threatened. Chimpanzee conservation status is classified as “Endangered Species” by IUCN Red List of Endangered Species, since 1996 and the subspecies of Pan troglodytes verus was recently updated to “Critically Endangered Species” (IUCN 2016). Today, the estimated number of wild chimpanzees is between 173,000 and 300,000 individuals (Butynski, 2003), representing a loss of 66% of individuals in 30 years (Butynski, 2001). All the chimpanzee populations are showing a general decrease and Pan troglodytes ellioti is the least numerous subspecies (IUCN, 2016). Western chimpanzees are estimated to be between 18,000 and 65,000 individuals (Khül et al., 2017) with a population estimated between 600 and 1,000 individuals living in Guinea-Bissau (Gippoliti et al., 2003). Applying measures for their conservation is difficult, because less than a half of them inhabits in protected areas (Kormos and Boesch, 2003). Formally, chimpanzees are legally protected over the countries where they live due to national and international protection laws (GRASP, 2015). For example, they are listed in Appendix 1 of CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) and in Class A under the African Convention on the Conservation of Nature and Natural Resources. Due to those conventions, every activity involving primates needs to be authorized by the local government. In

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particular, their hunting and killing are forbidden, but also the capture and trade of live chimpanzees or of their body parts (IUCN, 2016). Nevertheless, the enforcement of the laws is not effective. They have the aim to protect chimpanzees from major threats, that are usually caused, directly or indirectly, by human activities, such as: habitat destruction and fragmentation (Ndiaye et al., 2013), bushmeat hunting (Amador et al., 2015), climate change (Chapman et al., 2005) and infectious diseases (Alehegn et al., 2014).

1.2.1 Habitat destruction and fragmentation

The ongoing unsustainable exploitation of resources by humans is modifying the natural environment, causing important loss of habitat and fragmentation of suitable areas in patches (Hartter and Southwoth, 2009), where the physical and biogeographical conditions can be different from the native environments (Saunders et al., 1991). Habitat destruction and fragmentation increase the rate of species extinction (Ferraz et al., 2003). Particularly, the fragmentation of the habitat can have negative effects on wildlife behaviors such as on foraging and ranging patterns and on anti-predation strategies (Caro and Sherman, 2011). Habitat fragmentation also increases the proximity with humans, causing a higher risk of interspecific contact and increasing the probability of disease transmission (Schwitzer et al., 2010). The creation of fragments also creates barriers to animals that are not able to surpass, causing the formation of sub-populations and limiting the gene flow and the dispersal between them, and thus, decreasing their genetic diversity (Oklander et al., 2010). In the end, logging activities results in the creation of separate patches of suitable habitat with a higher percentage of edges, increasing the "edge effect" (Chapman et al., 2006): the life on the edge is more difficult than in the core of forest, because they are characterized by different biotic and abiotic conditions, such as the density of the forest and the microclimate (Murcia, 1995).

1.2.2 Hunting

Hunting, involves tracking, capturing, handling, transporting, preparing and consuming meat (Wolfe et al., 1998). It can be the most important cause of the decline of populations of wildlife species, as proposed in Guinea Bissau (Sá et al., 2012; Carvalho, 2014). It has always been an important way of pathogen transmission (Wolfe et al., 1998), because it increases the risk of

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contagion, in particular for human beings (i.e. zoonotic spilling over), leading humans and non-human primates to enter in more close contact with zoonotic pathogens. Bushmeat consumption also increases the possibility for humans to become in contact with body fluids of living or recently dead individuals (Chapman et al., 2005). Furthermore, illegal human hunting can cause traumatic injuries on animals, that influence feeding behavior or other activities (Munn, 2006; Hermans, 2011). Hunting is a very stressful experience for animals, and some authors demonstrated that reduces their immune system to fight against infections (Yersin et al., 2016). It is important to underline the different impact that village and commercial hunting have on chimpanzee populations and to understand which hunting type is occurring in the region of the study, in order to better apply conservation measures (Kuehl et al., 2009). Chimpanzees can also be hunted to be sold as pets (e.g Kabasawa, 2009) or their body parts can be used for traditional medicine (Sá et al., 2012).

1.2.3 Climate change

Climate change is an additional threat to primates and also to chimpanzees, mainly because it affect disease transmission and vector dynamics (Chapman et al., 2005). Departing from some climate models, it is possible to expect changes in climatic variables, especially for rainfall and temperatures (IPCC, 2007). The higher frequency of extreme meteorological events, such as hurricanes and floods, can directly injure chimpanzees leading to a decrease in their communities (Korstjens and Hillyer, 2016). Climate changes can also have important negative consequences on the flora providing food for chimpanzees and other primates, humans included (Butt et al., 2015). The increase or decrease in rainfall could promote transmission of waterborne diseases and the more high temperatures and humidity could have direct impacts on the range area of vectorborne diseases (Haines and Patz, 2004). Many diseases can in fact be observed only in particular season or under particular climatic conditions (Chapman et al., 2005).

1.3 The plummeting impact of infectious diseases

The impact of infectious diseases on non-human primate populations is an important conservation issue as infectious diseases can, in a short period of time, have a great influence on the genetic diversity of a population and on its population size (Altizer et al., 2003). The number of

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studies investigating this topic grown considerably during the last decade (e.g. Nunn and Altizer, 2006; Jin et al., 2015). The high risk of infectious diseases is mainly caused by anthropogenic alterations, such as introduction of non-native species, pollution and climate change (Martin et al., 2010). The transmission of diseases is possible due to the contact that occurs between different species and the number of species involved depends on the type of pathogen (Estrada-Peña et al., 2014). Moreover, the probability of infection among individuals of the same population is not always equal and can be quantified through social network analysis, that try to understand how individuals interact, in order to put in evidence how diseases spread in a population (Rushmore et al., 2013; Godfrey, 2013). When a pathogen start to be infective for species different from the usual host species (i.e. host specificity), the term “cross-species transmission” is used (Kowalewski et al., 2011). In case the disease is naturally exchanged between humans and animals, the term “zoonoses” is used (Marí, 2015). Particular attention must be posed in order to avoid the emerging of new diseases: Emerging infectious diseases (EID) which can be defined as diseases that have spread around the world, increasing their incidence in the last 20 years or possibly increasing in the near future (CDC, 2017). Moreover, EID are considered those that: have not spread through a particular species before; have spread in the past, but having consequences only in a low number of individuals; have spread previously but only in the recent past (NIH, 2007). They can be caused by the closer proximity of livestock and wild animals, by the displacement of host or pathogens mobilized by humans or they can simply arise without the involvement of humans or livestock (Daszak et al., 2000). At the same time, it‟s essential to not allow the spread of the diseases known as Neglected tropical diseases (NTD), a group of diseases affecting people living in poor conditions that have high possibility to enter in contact with vectors of infection (WHO, 2017). The knowledge about some neglected diseases is lacking, because they receive little attention (Liese et al., 2010) and because the distribution of funds for their research is unequal, when compared with malaria, tuberculosis or HIV, which in turn are receiving more than 80% of the global funding (Moran et al., 2009).

Infectious primate diseases are mainly caused by viruses and bacteria, which can have a human origin (e.g. Palacios, 2011) and that can spread in non-human primate populations due to their closer proximity to human villages (Gillespie, 2006). Among viruses, Ebola Virus Disease and Acute Respiratory Infections had high consequences on primates‟ health (Nunn and Gillespie, 2016). Ebola viruses belong to the family of Filoviridae that includes five viruses that cause haemorrhagic fevers. They are present only in the African continent, although one strain, non

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pathogenic for humans, was isolated in Philippines (Tu et al., 2009). The natural reservoir of the virus seems to be bat fruits (Leroy et al., 2005). It can cause the death of thousands of apes and a big decline of populations, in particular of chimpanzees and gorillas (Bermejo et al., 2006; Huijbregts et al., 2003). Ebola outbreaks among chimpanzees were mainly reported in Gabon and Congo (e.g. Walsh et al., 2003; Leroy et al., 2004). This pathogen has a very high virulence, or rather a high ability to infect the host (Ewald, 1993), and a great mortality rate causing a high number of deaths among non-human primate populations (Gilardi et al., 2015). The fight against Ebola is particularly difficult because the transmission and contamination are fast, and, despite all scientific efforts no medical treatments or vaccines are available for the moment (Leroy et al., 2004). Respiratory diseases are another threat for primate‟s health and they are principally caused by the proximity to humans, such as tourists and researchers (Tegner, 2013). They can cause symptoms in non-human primates similar to those observed in humans, such as coughing and sneezing but also appetite loss (Gilardi et al., 2015). The first evidence of a case of transmission of respiratory disease was reported by Kondgen et al. (2008), on a chimpanzee that died in Taï National Park, in Ivory Coast, due to the infection of two human paramyxoviruses, probably deriving from the researchers working in that area. Afterwards, other studies described possible outbreaks from human origins (i.e. anthropozoonosis) in chimpanzees and gorillas (e.g. Kaur et al., 2008; Palacios, 2011). Respiratory diseases can be easily monitored during field surveys involving the surveillance of illness (e.g Lonsdorf et al., 2016) and some authors advocate that great apes populations should be controlled with treatments or vaccines by veterinarians, in order to protect them (Gilardi et al., 2015).

Bacterial diseases can be transmitted in the same way from humans to non-human primates, when they live in close proximity due to forest fragmentation, as proved by Goldberg et al. (2008), in a study on red-tailed guenons that harbored a strain of Escherichia coli similar to the human type, and confirmed in other researches (e.g. Rwego et al., 2008). In particular Goldberg et al. (2007) showed that the bacteria found in chimpanzees were more similar to those harbored by tourists and researchers of the area than those harbored by local people. The more difficult infectious diseases to prevent are those coming from tourists. In fact, a survey conducted by Muehlenbein (2017) highlighted how tourists, even if they are aware of the possibility to exchange disease with primates, would like to have contact with them. Many precautions could be taken in order to prevent the spread of diseases and every person interacting directly or indirectly with non-human primates must follow them strictly (Gilardi et al., 2015). First rules for reducing the risk of infection

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for gorillas were produced in 1985 and revised in 1999 by a team of specialists, before being established in other countries of Africa for all non-human primate species, directed to tourists and researchers. For example: tourists cannot approach chimpanzees with a distance less than 10 meters and researchers less than 7.5 meters and no one can visit chimpanzees if they show signs of sickness; moreover, they all need to wear surgical masks while visiting animals, in order to reduce the risk of infection and cross-transmission (Lukasik-Braum and Spelman, 2008). Other pathogens that can cause important diseases in primate species are intestinal parasites (Thompson et al., 2010).

1.4 Parasitism and its effects on chimpanzee populations: a literature review

Primate populations harbor a great number of organisms in their gastrointestinal apparatus, showing different relationships with the host, known as “symbionts” (Combes, 2001; Douglas, 2010). Symbionts can be subdivided into (i) mutualists (when the relationship with the host is positive for both of them), (ii) commensals (when the relationship is neutral) (Hooper and Gordon, 2001) and (iii) parasites. Parasites are usually defined as organisms inhabiting the body of the host, taking nutrition from it, having particular characteristics to adapt to this symbiotic interaction and causing some damage (Poulin, 2011). Some of the emerging and neglected infectious diseases harming primates are caused by gastrointestinal parasites (e.g. Giardia duodenalis and Cryptosporidium spp.; Savioli, 2006). The interaction between hosts and parasites has a particular dynamics and follows its own mechanisms that parasitologists are still trying to understand (Combes, 2001). At the beginning, the relationship between hosts and parasites was considered the same as the one between prey and predators, without arguing that the situation was more complex (Anderson and May, 1978). The preponderant idea was that parasites were not so important, because of their low biomass; but this idea was refuted with the discoveries of their essential role in ecosystem dynamics (Hudson et al., 2006). Parasites can have different effects (i) they can influence the genetic composition of the host population (Altizer et al., 2003), (ii) they can modify the host lifecycle (Dobson and Hudson, 1992), (iii) they can interact with pollutant substances (Vidal-Martinez et al., 2010) and, (iv) they can cause important diseases, directly, or indirectly acting as vector for other pathogens (Mehlhorn, 2015) leading to the demographic decline of the host populations (Gillespie et al., 2008). The infections caused by parasites are common in all wildlife species and several studies were already performed on non-human primates (Terio et al., 2016) but compared to other pathogens, such as bacteria and virus (also considered microparasites),

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gastrointestinal parasites are less studied (Guillot et al., 2011). In general, some parasites can live in a kind of equilibrium with the host, without causing immediate consequences for its health (Leendertz et al., 2006; Gillespie et al., 2008). Conversely, other gastrointestinal parasites can cause important consequences on primate health, like pain, diarrhea, blood loss, ulceration of the mucosa, weight loss, abortion and in some case even death (Krief et al., 2008; Drakulovski et al., 2014; Cibot et al., 2015; Lonsdorf et al., 2016). Normally, if damage is caused by parasites, the disease observed in the animal will be more probably chronic than acute (Gilardi et al., 2015).

The exponential growth of the human population and the exploiting of suitable areas and natural resources, are causing the loss and fragmentation of habitat for primates, which in turn can increase their risk for parasite infection (e.g Schwitzer et al., 2010; Gillespie and Chapman, 2008; Valdespino et al., 2010). Parasitism in chimpanzees can be influenced by population size (Altizer et al., 2007; Snaith et al., 2008; Helenbrook et al., 2015); host density (Nunn et al., 2003); intraspecific contact (Rimbach et al., 2015); habits, such as the use of soil or the use of nests (Zommers et al., 2013; Bakuza and Nkwengulila, 2009); daily home ranges (Mbora et al., 2009); proportion of forest edges (Chapman et al., 2006); proximity to other species (Ebbert et al., 2015; Howells et al., 2011); and proximity to human villages (Foitová et al., 2009, Gillespie and Chapman, 2006; Narat et al., 2015). Due to their close genetic and taxonomic relationship, probably humans and non-human primates share some important characteristics that make easier the parasite transmission, as it was demonstrated that the most part of the human infectious diseases has a zoonotic origin (approximately 75%) (Osburn et al., 2009). Making general rules could be difficult, as the same parasites can have different effects depending on how the infected individual reacts and this can explain why parasites do not always have the same effect on the population size of the host (Ebert et al., 2000). Parasitological studies are important to understand the dynamics of the infection (i.e. epidemiology) and to shed light on host specificity (Cooper et al., 2012).

Parasitological studies on chimpanzees started on 1970's, but only around the year 2000' begun to gain more interest. The most part of them was performed on chimpanzees inhabiting forested environment and just some authors focused on savannah chimpanzees (McGrew et al., 1989; Howells et al., 2011; Kalousová et al., 2014). Parasitological studies started in Gombe National Park, Tanzania, and at first, researchers simply try to describe the parasitofauna of wild chimpanzees (e.g. File et al., 1976). Other authors compared and described the differences in terms of prevalence and richness of parasitofauna of wild chimpanzees in different studies or with different species inhabiting the same areas (McGrew et al., 1989; Ashford et al., 2000; Murray et

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al., 2000; Lilly et al., 2002; Bakuza and Nkwengulila, 2009; Kooriyama et al., 2012; Ebbert et al., 2015). Some studies tried to investigate the general health of chimpanzees, not only by parasitological analysis, but also with urine analysis and behavioral observations (Krief et al., 2005; Lonsdorf et al., 2016). During the years, researches tried to find a correlation between the level of parasitism and the ingestion of medical plants (Kawabata and Nishida, 1991; Huffman et al., 1997); the season of the year (Gillespie et al., 2010); the immunological status of chimpanzees (Drakulovski et al., 2014; Lonsdorf et al., 2016); the age and sex of individuals (Gillespie et al., 2010); the anthropogenic pressure which they are subjected (Muehlenbein, 2006; Krief et al., 2010; Zommers et al., 2013; Sá et al., 2013; Cibot et al., 2015; Debenham et al., 2015; Yersin et al., 2016). When possible, molecular analysis was performed to help the identification of parasite taxa (Hasegawa et al., 2010; 2016). On a parallel set of studies performed on wild chimpanzees, researches on captive primates are still conducted (e.g. Shemshadi et al., 2015). The parasitofauna of captive chimpanzees will present important differences from those living in the wild, mainly because of the treatments performed and given to the animals, such as antihelmintics (Petrželková et al., 2010).

1.5 The “One Health” approach

The relationship that occurs between parasites, humans and animal species is so strict and interconnected that, in order to better understand this topic, the concept of “One Health” was conceived (Thompson, 2013). The idea of “One Health” is not new, as in fact the term “One Medicine” was used for the first time in 1984 by Calvin Schwabe, the father of veterinary epidemiology, in his book Veterinary Medicine and Human Health (Schwabe, 1984). The “One Health” project take rise in 2007, at the American Veterinary Medical Association (AVMA) Annual Convention, proposed by AVMA, American Medical Association (AMA) and Centers for Disease Control and Prevention (CDC) (Dhama et al., 2013). The One Health project had the objective to ensure the optimal connection between humans, animals and environment, instead of on focus on a unique component (Thompson, 2013) (Fig. 5).

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Figure 5: The "One Health Triad", figuring the interconnection between humans, animals and environment. From: Thompson, 2013.

The “One Health” approach tries to fight against the anthropocentric concept, deconstructing the idea that it is possible to look and talk about human health without considering environment and animal health (Lee and Brumme, 2013). Due to the different subjects involved, a multidisciplinary approach is needed, involving different specialists, such as veterinarians, ecologists, biologists, epidemiologists, engineers, doctors, managers of wildlife, public health laborers, appliance officers, policy responsible and others professionals (Mazet et al., 2009; Gilardi et al., 2015). Particular attention was given to the education of the future professional health staff (Barrett et al., 2011). The high number of zoonoses existing (Taylor et al., 2001), obliges the international health organizations to find restraint measures at the local level, to fight the disease where it is already present, but also at international level, to avoid its spread in other parts of the world (Marì, 2015). Several fields needs to be accurately monitored, because of their role on influencing the spread of diseases: climate change (Pounds et al., 2006), destruction of the habitat (Mbora et al., 2009), translocation of wildlife for conservation programs (Daszak et al., 2000), ecotourism (Muehlenbein and Ancrenaz, 2009), overpopulation and overlap of human/wildlife areas (Johnston et al., 2010) and food supply (Osburn et al., 2009). By monitoring chimpanzee gastrointestinal parasites it is possible not only to understand their health status but also to understand which parasites with zoonotic potential they have and if any cross-transmission between humans and chimpanzees are occurring.

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1.6 Aim, objectives, hypotheses and predictions

Aim:

The aim of this dissertation is to investigate and to characterize the symbiont fauna, with particular attention to zoonotic parasites, of two chimpanzee populations from Guinea-Bissau located in the southern part of the country; one living inside a protected area and the other living outside.

General objectives:

(i) To assess the difference in terms of gastrointestinal symbiont prevalence and richness of two chimpanzee populations from Guinea-Bissau;

(ii) To identify parasites with zoonotic potential in these populations;

(iii) To infer the health status of these two chimpanzee populations and contribute to their conservation plan;

(iv) To establish baseline data for future monitoring studies.

Specific objectives:

(i) To conduct fieldwork in two areas with different anthropogenic pressures, Nova Sintra (Tite sector) and Cufada Lagoons Natural Park, from Quinara region, Guinea-Bissau;

(ii) To perform standard parasitological analysis by using a combination of flotation and sedimentation techniques in order to identify and shed light about the symbiont fauna of these chimpanzees;

(iii) To compare statistically the results obtained in terms of symbiont prevalence and richness from both areas;

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(iv) To consider the obtained results and put them into perspective with previous findings.

Hypotheses:

H1. Less zoonotic parasites will be found in the chimpanzee population living inside Cufada Lagoons Natural Park, comparing to the chimpanzee population inhabiting outside the park (i.e. Nova Sintra). Assuming that the population from the natural park will suffer less anthropogenic pressures, the chimpanzees living in it will harbor less zoonotic parasites, as expected according to with previous studies (e.g. Cibot et al., 2015; Sá et al., 2013; Hasegawa et al., 2016).

H2. Similarly, a healthier status of the chimpanzee population living inside the natural park is expected compared to the one inhabiting outside (e.g. Rangel-Negrín et al., 2014; Krief et al., 2005; Howells et al., 2011).

H3. It is also expected a lower parasite richness and a lower prevalence, in chimpanzees living inside the natural park, with a high level of host specificity compared to those inhabiting outside. Assuming that habitat fragmentation is facilitating the transmission of more generalist parasite as demonstrated by previous studies (e.g. Kooryiama et al., 2012; Schwitzer et al., 2010; Gillespie and Chapman, 2008).

Predictions:

(i) If H1 is true, then it is not expected to find a high prevalence of parasites with zoonotic potential, such as Giardia duodenalis, Oesophagostomum spp., Strongyloides spp. and Strongylida nematodes in the population of chimpanzee living inside Cufada Lagoon Natural Park.

(ii) If H2 is true, then it is expected to find more mutualistic and commensal symbionts and less parasite species in the chimpanzee community living inside the protected area.

(iii) If H3 is true, then it is expected to find a higher symbiont prevalence and richness in the chimpanzee population of Nova Sintra.

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CHAPTER 2 PARASITES IN CHIMPANZEES

The parasite category includes a large number of species that can cause diseases of varying severity in primate species. In particular, gastrointestinal symbionts that inhabit the gut of primates include protozoan species, which can live inside the host asparasites or commensals, and helminth species, where the majority has zoonotic potential. In this section, the most common parasite species found in the gastrointestinal tract of wild chimpanzees will be described.

2.1 Protozoa

Kingdom: Protista

Sub-kingdom: Protozoa

Historically, Protozoa belonged to the Kingdom Protista. Protista taxon includes small organisms, that have a complexity level no higher than a single cell, which can vary greatly, especially from a phylogenetic point of view. These include autotrophic photosynthetic organisms, known as Protophytes, and heterotrophic organisms, known as Protozoa (Urquhart et al., 1998). Protozoa are eukaryotic unicellular microorganisms, most of which measure less than 50 µm, with cells that possess all the organelles. They do not have a true cell wall, but in some cases they are covered by a structure composed of proteins or polysaccharides. Moreover, the contractive vacuole, the citoscheletrum, the micronucleus (one or more) and the structures allowing motility are always present, while the macronucleus is present only in ciliate species (Polsinelli et al., 1993). Protozoa can be found in all kinds of habitat. Most of them are free-living, nonetheless, protozoa commonly infect animals, causing infections that may be asymptomatic or lead to clinical signs, and in some cases they may be life-endangering. Reproduction generally takes place asexually (binary fission), but it is possible to come across more complex methods of reproduction, such as multiple asexual division and sexual reproduction. Protozoa usually have two life cycle stages: the trophozoite and the cyst. The role of the trophozoite is feeding, reproduction and movement, and usually is the

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pathogenic stage. Conversely, the cyst is not motile and is characterized by a thick protective wall, allowing it to survive in the external environment, and usually is the infective stage (Yaeger, 1996). Trophozoites can be found in faeces in the event of diarrhoea, but only cysts are usually observed in healthy subjects with formed stools (CDC; 2016). It is important to remember that Protist and Protoza taxa are no longer found in more recent classification system and nowadays these two terms are only used as descriptive terms. According to the revised classification of eukaryotes presented by Adl et al. (2012), the organisms in question were grouped together and defined as “protozoa” in the past, but today are subdivided into different phylogenetic groups. The sub-groups are: Amoebozoa (which includes the genus Entamoeba, Endolimax and Iodamoeba); Excavata (which includes the genus Giardia and Chilomastix); SAR (which includes the genus Balantidium, Troglodytella, Troglocorys and Blastocystis). Amoebozoa includes organisms that generally form cysts that exhibit varying morphologies. They can be uninucleate, binucleate or multinucleate and reproduce sexually or asexually. All of them are characterized by the “ameboid” movement, with the formation and retraction of pseudopodia. The Excavata group includes organisms that are characterized by a distinctive feeding method. They possess a feeding groove that permits ingestion of small pieces of food, which approach the groove by means of a feeding current generated by a cilium, situated on the posterior part of the body. SAR is a clade that includes Alveolata, Rhizaria and Stramenopiles organisms. Alveolata are characterized by cortical alveoli, Rhizaria possess pseudopodia that are sometimes supported by microtubules, while Stramenopiles are motile biciliate cells also called heterokonta because of the presence of two different flagella. Despite the complexity of the new classification proposed, in most cases parasitologists still subdivide intestinal parasite protozoa on the basis of their movement technique simply into different descriptive categories: amoeba (move by means of pseudopodia), flagellates (move by means of flagella), ciliates (move by means of cilia) and sporozoa (whose adult stages are not motile) (Garcia, 1999). It is important to remember that these categories does not have taxonomical value. Moreover, parasitologists usually refers to the older classifications based on morphology and accepted by the Society of Protozoologists, such as this proposed by Honigberg et al. (1964) and by Levine et al. (1980). According to this latter classification, the Subkingdom of Protozoa is subdivided into different phyla: Sarcomastigophora (which includes the organisms usually called amoebas and flagellates), Labyrinthomorpha, Apicomplexa, Microspora, Ascetospora, Myxozoa and Ciliophora (which includes the microorganisms usually called ciliates). I will examine amoebae, flagellates and ciliates belonging to the protozoan species that is likely to infect chimpanzees, classifying them according to Levine et al. (1980).

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2.1.1 Amoeba Kingdom: Protista Sub-kingdom: Protozoa Phylum: Sarcomastigophora Sub-phylum: Sarcodina Class: Lobosea Order: Amoebida Family: Endamoebidae

Genus: Entamoeba; Endolimax; Iodamoeba

According to their classical definition, amoebae are organisms able to alter their body shape due to the creation and retraction of pseudopodia (Singleton, 2006). Species that are usually observed in the faeces of wild chimpanzees are: Entamoeba coli, Entamoeba histolyitica/dispar/moshkovskii, Entamoeba hartmanni, Endolimax nana and Iodamoeba bütschlii.

Entamoeba coli: the cyst measures from 10 to 30 µm and is usually oval but may have a distorted shape owing to the stain method. There are from 1 to 8 nuclei, but occasionally supernucleate cysts (16 to 32 nuclei) can be found. Immature cysts present a lower number of nuclei and are larger. The karyosome is large and may be eccentric or located in the central part of the nuclei. The chromatin is not evenly granular. The trophozoite measures from 15 to 50 µm, has one single nucleus and a number of vacuoles that contain debris and food. In the nuclei, the karyosome is eccentric and the chromatin is peripheral and irregurarly distribuited. In fresh samples, it is possible to observe the appearance of pseudopodia (Ash and Orihel, 2007) (Fig. 6). E. coli is illustrated in most parasitological studies of wild chimpanzees, with prevalence ranging from 1.6% (n=123. Ashford et al., 2000) to 53% (n=79. Gillespie et al., 2010).

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Figure 6: Cyst and trophozoite of Entamoeba coli with iodine staining. From: Atlas Protozoa, 2010.

Entamoeba histolytica/dispar/moshkovskii: the cyst measures from 10 to 20 µm, it is round and less variable in shape than E. coli. It contains from 1 to 4 nuclei, and supernucleated cysts are rare. Immature cysts have a lower number of nuclei and are larger. In contrast to E. coli, the karyosome is small, compact and in most cases visible in the centre of the nuclei. Moreover, the granules of the peripheral chromatin are more uniform. The trophozoite is difficult to distinguish from the E. coli trophozoite, given that their dimensions are similar (20-40 µm), it possesses one single nucleus and a number of food vacuoles. The nuclei has a small karyosome centrally located. Pseudopodia can be observed in fresh samples. In the case of E. histolytica, it is possible to find the presence of ingested red blood cells, which helps differentiate it from the other species of the complex (Ash and Orihel, 2007) (Fig. 7). This complex is not so common in wild chimpanzees, but when recorded, it shows a high prevalence (e.g. 70%; n=74. Gillespie et al., 2010).

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Figure 7: Cyst and rophozoite of Entamoeba histolytica/dispar/moshkovskii unstained. From: Atlas Protozoa, 2010.

Entamoeba hartmanni: the cyst is morphologically similar to those of E. coli and E. histolytica, but its small dimensions (from 5 to 10 µm) help identify the symbiont. It is round and can have from 1 to 4 nuclei. The karyosome is small and compact, and located in the centre of the nuclei. The peripheral chromatic is organized in equally distributed fine granules. In the same way, the trophozoite is similar to that of other Entamoeba, but different in size - usually ranging from 5 to 12 µm (Ash and Orihel, 2007) (Fig. 8). This amoeba is rarely reported during researches, maybe because, being small, it is difficult to identify. When it is present, its prevalence is around 15% (n=37. Muehlenbein, 2005).

Figure 8: Cyst and trophozoite of Entamoeba hartmanni unstained. From: Atlas Protozoa, 2010.

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Endolimax nana: the cyst is usually oval and has from 1 to 4 nuclei. It generally measures between 5 and 10 µm and observation with 100X magnification is recommended in order to distinguish it from E. hartmanni. These two species can chiefly be distinguished by the position of the nuclei. Its karyosome is usually larger than that registered in Entamoeba spp. and there is no peripheral chromatin. The trophozoite ranges from 5 to 12 µm and is granular. Fresh samples show that more than one pseudopod is emitted at the same time (Ash and Orihel, 2007) (Fig. 9). It is rarely recorded in the faeces of wild chimpanzees and when present, its prevalence is low (e.g. 5%, n=37. Muhelenbein, 2005).

Figure 9: Cyst and trophozoite of Endolimax nana unstained. From: Medical-Labs, 2014.

Iodamoeba bütschlii: the cyst measures from 5 to 12 µm and contains one nucleus, with a large karyosome and no peripheral chromatin. It is easy to identify this symbiont thanks to the presence of one single nucleus and a large, clearly visible glycogen vacuole. The trophozoite ranges from 9 to 14 µm, with one nucleus and many cytoplasmic vacuoles. It is difficult to distinguish from the E. nana trophozoite (Ash and Orihel, 2007) (Fig. 10). Like other small amoebae, it is rarely reported in studies, but when present, its prevalence is high (e.g. 65% , n=74. Gillespie et al., 2010).

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Figure 10: Cyst and trophozoite of Iodamoeba bütschlii with iodine staining. From: Atlas protozoa, 2010.

Species identification can be achieved thanks to the morphology of cysts and trophozoites found in faeces using standard coprological methods of flotation and sedimentation, with the exception of Entamoeba histolytica/dispar/moshkovskii complex (CDC, 2016). Species of this complex can be distinguished using immunological analyses based on the detection of antibodies or antigens (Haque et al., 1998) or using genetic analyses, including conventional and real-time PCR (Fotedar et al., 2007). Intestinal amoebae are usually considered commensals and do not represent a

health hazard (Drakulovski et al., 2014). However, in the complex E.

histolytica/dispar/moshkovskii, E. histolytica it is pathogenic (Sodeman, 1996) but it is impossible to identify the species without using genetic analyses. E. histolytica, causes a varying extent of damage, from mild diarrhoea to severe infection leading to perforation of the mucosa and peritonitis. In some cases, infection can extend from the intestine to the liver, causing hepatomegaly. From the digestive apparatus, the infection rarely reaches the lungs, heart or brain (Sodeman, 1996). All amoeba species have a common life cycle. When cysts are accidentally ingested by the host in contaminated food or water, they undergo a process of excystation in the small intestine, producing trophozoites. These stages migrate to the large intestine and multiple by binary fission, producing the pre-cystic stage. These elements become immature cysts that mature before being expelled in the faeces. The life cycle is completed in one single host (monogenetic parasite), which may be a human or non-human primate (CDC, 2016).

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2.1.2 Flagellates Giardia duodenalis Kingdom: Protista Sub-kingdom: Protozoa Phylum: Sarcomastigophora Sub-phylum: Mastigophora Class: Zoomastigophorea Order: Diplomonadida Family: Diplomonadidae Genus: Giardia Chilomastix mesnili Kingdom: Protista Sub-kingdom: Protozoa Phylum: Sarcomastigophora Sub-phylum: Mastigophora Class: Zoomastigophorea Order: Retortamonadida Family: Retortamonadidae Genus: Chilomastix

According to their definition, flagellate species are characteristically propelled by whip-like structures, called flagella (Singleton, 2006). The flagellate species found in the faeces of wild chimpanzees are Giardia duodenalis and Chilomastix mesnili.

Giardia duodenalis: the cyst is from 8 to 19 µm in length. Two nuclei are visible in the mature form and four nuclei are visible in the immature form. The wall is smooth and colorless. The trophozoite measures from 10 to 20 µm in length. It is easy to identify due to its characteristic "kite-shape", bilateral symmetry and flat surface facilitating attachment to the mucosa of the host. It has four pairs of flagella (one anterior pair, two lateral pairs, one posterior pair) and two nuclei that are clearly visible after coloration. The movement of the flagella may be visible in fresh samples, but only for a short period (Ash and Orihel, 2007; CDC, 2016) (Fig. 10). It is rarely found in parasitological studies involving wild chimpanzees and when is present, its prevalence is around 5% (e.g. 6%, n=102. Sá et al., 2013).

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Figure 11: Cyst and trophozoite of Giardia duodenalis with iodine staining. From: CDC, 2016.

Giardia duodenalis is composed of many genetic assemblages, from A to H, and each assemblage has a specific host. Only A and B appear to be zoonotic and have the largest number of hosts, including non-human primates (Heyworth, 2016). Giardia duodenalis cysts are not always excreted in the faeces when the host is infected. For this reason, during coprological analysis, it is necessary to take multiple samples, with at least three stools collected on non-consecutive days (CLSI, 2005). Being more sensitive than coprological methods, immunodiagnostic techniques should be used (Johnston et al., 2003). Finally, to distinguish between the different assemblages, genetic analysis such as PCR is necessary (CDC, 2015). G. duodenalis may be asymptomatic, but in most cases it is not, and the damage inflicted on the host can vary widely. The main symptoms of giardiasis are intestinal disorder, including diarrhoea, abdominal pain, nausea, vomiting and weight loss (Wolfe, 1992). The faeces of infected organisms do not contain blood, as this parasite harms the host, not by directly damaging the mucosa, but simply by atrophying the microvilli. In this way, the nutritional status of the host is compromised (Taylor et al., 1987).

Chilomastix mesnili: the cysts is usually from 6 to 10 µm in diameter, and is characteristically lemon-shaped. It has only one nucleus that may be extremely large, occupying one half of the cyst. The trophozoite can measure from 6 to 24 µm in length. It is characteristically pear-shaped, has one large nucleus visible in the anterior part and three flagella that extend at the anterior end. A fourth is found in the cytostome (Ash and Orihel, 2007) (Fig. 12). This symbiont is rarely revealed in parasitological studies involving wild chimpanzees and, when present, its prevalence is low (e.g. 7% , n=102. Sá et al., 2013).

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Figure 12: Cyst and trophozoite of Chilomastix mesnili with iodine staining. From: Atlas Protozoa, 2010.

C. mesnili can be identified by standard coprological analysis of flotation and sedimentation on faecal samples (CDC, 2016). It is considered non-pathogenic (Issa, 2014) and is usually considered asymptomatic (Beaver et al., 1984), but today this is under discussion.

The two species‟ life cycles are similar. In both cases, the host is infected due to ingestion of the cysts in contaminated food or water. Cysts undergo excystation in the small intestine (C. mesnili also in the large intestine). The released trophozoites multiply by binary fission and then undergo encystation before expulsion with the faeces. In G. duodenalis, the trophozoite may be free floating or attached to the mucosa of the intestine (CDC, 2006).

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2.1.3 Ciliates Balantidium coli Kingdom: Protista Sub-kingdom: Protozoa Phylum: Ciliophora Class: Kinetofragminophorea Order: Trichostomatida Family: Balantidiidae Genus: Balantidium

Troglodytella abrassarti/Troglocorys cava

Kingdom: Protista Sub-kingdom: Protozoa Phylum: Ciliophora Class: Kinetofragminophorea Order: Entodiniomorphida Family: Troglodytellidae

Genus: Troglodytella, Troglocorys

According to their classical definition, ciliates are organisms propelled by their cilia (Sanderson, 2006). Basically, three species of ciliates are expected to be found in the faeces of wild chimpanzees: Balantidium coli, Troglodytella abrassarti and Troglocorys cava. T. abrassarti and T. cava are symbionts specific to chimpanzees and they are also known as entodiniomorphid ciliates.

Balantidium coli: the cyst is covered in cilia and contains a macronucleus, a micronucleus and contractive vacuoles, which are not always easily visible. It has a double membrane and measures from 50 to 70 µm in diameter. The trophozoite is oval and its cilia are more clearly visible than in cysts. All the organelles found in cysts can be seen, but in this form it also has a cytostome. Moreover, it is bigger than cyst, measuring from 40 to 200 µm in length (Ash and Orihel, 2007; CDC, 2016) (Fig. 13). This flagellate is rarely recorded in wild chimpanzees and its prevalence is low (e.g. 8.7%, n=23. Lilly et al., 2002).

Assessment of gastrointestinal parasites with zoonotic potential in two communities of chimpanzees (Pan troglodytes verus) from Guinea-Bissau and its implications for conservation.

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ABSTRACT

RIASSUNTO

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

ACKNOWLEDGEMENTS

CHAPTER 1

INTRODUCTION

CHAPTER 2

PARASITES IN CHIMPANZEES