**2.2.1 Yeasts in the GIT of pigs**

Comparatively to all animals investigated in the study of Van Uden et al. (1958), the most frequent occurrence (88.8%) of yeasts was detected in the caecum of pigs (horses 52.4%, cattle 46.8%, sheep 6.8%, and goats 6.4%). The yeasts *Candida slooffiae*, *Candida krusei*, *Saccharomyces telluris*, *Candida albicans*, *Candida* (*Torulopsis*) *glabrata*, were commonly found in the porcine gut. However, *C. slooffiae* was isolated most frequently (48.4%). A few other yeasts such as *Saccharomyces* spp., *Pichia membranifaciens, Pichia farinose* and *Candida mycoderma* could also be identified. Roughly the same situation has been confirmed in the following studies (Van Uden & Carmo-Sousa, 1962; Mehnert & Koch, 1963), where the scientists isolated almost the same variety of yeasts from the different parts of porcine GIT. After investigation of digesta samples collected from stomach, three parts of small intestine as well as caecum and rectum of healthy 57 pigs, Van Uden & Carmo Sousa (1962) reported high animal-individual qualitative and quantitative variability if the yeast occupation. In total 15 yeast species were identified; while *C. slooffiae* was present in 27 pigs, many other species mentioned above occurred only sporadically. Moreover, *C. slooffiae* was highly abundant, from 102 to 103 CFU/g of chyme in the stomach and up to 106 CFU/g intestine contents in the rectum. A still higher occurrence of yeasts in the gut of pigs was detected by Mehnert & Koch (1963), up to 107 CFU/g in rectum. They isolated 292 yeasts from 200

Nowadays, there are over one million of accepted insect species; however, their number has yearly increased and is still largely undiscovered (Chapman, 2009). Thus, it can be supposed that the number of yeasts would tremendously rise, even if only the intestinal tracts of the

The more intensive investigations of the yeast population present in the GIT of vertebrates, based on various cultivation procedures, began in the fifties of the XXth Century. Van Uden et al. (1958) and Van Uden & Carmo Sousa (1957b) examined yeasts in the caecal samples of large number of animals: 252 cattle, 252 horses, 503 sheep, 250 goats and 250 pigs. Yeasts were also studied by Parle (1957) in the digestive tract of cows, rabbits, sheep, guinea pigs, opossums, monkeys, cats, dogs, hedgehogs, mice, pigs and rats. Lund (1974) explored yeasts and moulds in the bovine rumen. Lately, yeasts were also described in the intestinal tract of reptiles (Kostka et al., 1997), birds (Cafarchia et al., 2006; 2008; Brilhante et al., 2010; Costa et al., 2010), mice (Scupham et al., 2006), dogs (Brito et al., 2009) and fish (Gatesoupe, 2007). In these studies, the scientists have detected various ascomycetous and basidiomycetous yeasts chiefly representing the genera *Candida*, *Trichosporon*, *Pichia*, *Rhodotorula, Debaryomyces,* 

In general, the diversity of the yeast population depended on the host; but many species occurred at diverse, also not intestinal ecosystems; and several exhibited direct relationship to the individual animal. It should be noticed, however, that yeasts could not be always isolated from the investigated GIT and often they were present in small numbers. Nevertheless, taking into consideration the scarce information existing on yeasts in the gastrointestinal ecosystems of vertebrates, it is well known that relatively high variety as

Here, the yeasts diversity in the GIT of farm animals representing diverse nutritional types: omnivores (pig), monogastric herbivores (horse) and ruminants will stay in focus and will

Comparatively to all animals investigated in the study of Van Uden et al. (1958), the most frequent occurrence (88.8%) of yeasts was detected in the caecum of pigs (horses 52.4%, cattle 46.8%, sheep 6.8%, and goats 6.4%). The yeasts *Candida slooffiae*, *Candida krusei*, *Saccharomyces telluris*, *Candida albicans*, *Candida* (*Torulopsis*) *glabrata*, were commonly found in the porcine gut. However, *C. slooffiae* was isolated most frequently (48.4%). A few other yeasts such as *Saccharomyces* spp., *Pichia membranifaciens, Pichia farinose* and *Candida mycoderma* could also be identified. Roughly the same situation has been confirmed in the following studies (Van Uden & Carmo-Sousa, 1962; Mehnert & Koch, 1963), where the scientists isolated almost the same variety of yeasts from the different parts of porcine GIT. After investigation of digesta samples collected from stomach, three parts of small intestine as well as caecum and rectum of healthy 57 pigs, Van Uden & Carmo Sousa (1962) reported high animal-individual qualitative and quantitative variability if the yeast occupation. In total 15 yeast species were identified; while *C. slooffiae* was present in 27 pigs, many other species mentioned above occurred only sporadically. Moreover, *C. slooffiae* was highly abundant, from 102 to 103 CFU/g of chyme in the stomach and up to 106 CFU/g intestine contents in the rectum. A still higher occurrence of yeasts in the gut of pigs was detected by Mehnert & Koch (1963), up to 107 CFU/g in rectum. They isolated 292 yeasts from 200

**2.2 Yeasts' diversity in the GIT of vertebrates with focus on farm animals** 

currently known insects were explored.

*Kluyveromyces* and *Saccharomyces*.

**2.2.1 Yeasts in the GIT of pigs** 

be compared.

well as quantity of yeasts can be found in the GIT of pigs.

digesta samples collected from stomach and rectum from 98 (of 100 examined) pigs. Apart from the *C. slooffiae* which was detectable in 75% of pigs, yeast species such as *C. krusei*, *S. telluris*, *C. albicans*, *C. glabrata, C. tropicalis, C. parapsilosis* and *C. pintolopesii* (60%, 26%, 9%, 4%, 3%, 3% and 2% respectively) were isolated, too. Also in this study the appearance of yeasts was variable within a part of the GIT and among examined animals. Thus, stomach was generally colonized by yeasts at lesser intense than rectum. In most animals, *C. slooffiae*  and *C. krusei* were detected both in stomach and rectum, while just in a few cases the yeasts could be found only in a single part of the GIT. *C. slooffiae* and its closely related species: *S. telluris* and *C. pintolopesii* have been newly molecularly investigated and based on multigene sequence analyses they were assigned to the teleomorphic genus *Kazachstania* (Kurtzman et al., 2005).

Recently, Urubschurov et al. (2008) described yeasts' diversity in the gut of piglets around weaning which were reared at two facilities: at experimental farm (EF) with improved husbandry conditions than at commercial farm (CF). Most piglets, 33 at CF and 35 at EF, were weaned at 28 days (d) of age and fed with the same diet until 39 d in both farms. A number of piglets, namely 18 at CF and 9 at EF, were left by the sows without additional feeding. All piglets were sacrificed at 39 d of age and digesta samples from GIT were collected. D1&D2 domains of 26S rRNA gene from 173 yeast isolates obtained from 95 piglets were sequenced. The alignment to known sequences revealed close relationship to 17 species, of which the most dominated are presented in figure 2. Urubschurov et al. (2008) observed distinction of yeasts variety between both facilities that were proven by calculation of different similarity and diversity indices. In piglets from CF *Galactomyces geotrichum*, *Kazachstania slooffiae* and *Candida catenulata* were the most abundant ones and the other were present only at low abundances. Unlike at CF, at EF two species, namely *K. slooffiae* and *C. glabrata* were found to be the most dominating ones and the others were rarely isolated. Some of the other species could be found in piglets either only at the EF (*P. fermentans*, *C. tropicalis, C. oleophila*, *C. parapsilosis*, *P. guilliermondii*, *Rh. mucillaginosa*, *T. montevideense*) or at the CF (*C. silvae* and *P. farinose*). This study provided evidence for association of *K. slooffiae* with the porcine GIT. *K* (*C.*) *slooffiae* was found for the first time in 6 of 252 examined horses (Van Uden & Carmo-Sousa, 1957a), however, due to frequent occurrence and high concentration in the porcine digestive tract it can be considered to be specific for pigs.

Fig. 2. Dominated yeasts isolated from the gut of 39 d old piglets, kept at experimental (EF) and commercial farm (CF), in the study of Urubschurov at al. (2008).

Biodiversity of Yeasts in the Gastrointestinal

spp. and *Pichia* spp. have been found only occasionally.

Ecosystem with Emphasis on Its Importance for the Host 291

from a much higher number of cattle (252) as well as sheep (503) and goats (250). Among the investigated animals, cattle showed the highest (46.8%) occurrence of yeasts, whereas just a few yeasts could be found in sheep and goats, 6.8% and 6.4% of the animals, respectively. The most frequently isolated yeasts were *C. tropicalis* and *C. krusei* in cattle, and *C. albicans* in sheep. These species were also isolated from the goats, but just two times each; and *C. glabrata* four times. A few other yeasts identified as members of *Saccharomyces* spp.*, Candida* 

Quite similar results regarding yeast colonization have been obtained in the cultivation dependent studies (Clarke & Menna 1961; Lund 1974; 1980; Van Uden et al. 1958) from different geographical regions. Shin et al. (2004) explored different rumen samples (fluid, solid and epithelium) from one cow, examined for yeasts population using molecular approaches. Shin et al. (2004) have succeeded to obtain 97 clones containing 26S rRNA gene fragments from the three types of samples and to assign them to the different phylogenetic groups. Compared to 4 phylotypes from the rumen epithelium showing the closest relatedness to *Geotrichum silvicola*, *Acremonium alternatum*, *Pseudozyma rugulosa* (up to 99%) and *Galactomyces* sp. (97%), and 2 phylotypes (*Geotrichum silvicola*, 99% and *Galactomyces* sp., 97%) from the rumen solid, the highest yeast' diversity was observed in the samples of rumen fluid revealing presence of 15 various phylotypes. Only 5 (*Setosphaeria monoceras, Raciborskiomyces longisetosum, Magnaporthe grisea, Ustilago affinis* and *Pseudozyma rugulosa*) of the 15 phylotypes showed 99% identity with the sequences deposited at the NCBI GenBank. The identification rate of the others belonging also to the classes *Pezizomycotina, Urediniomycetes, Saccharomycotina* and *Hymenomycetes* ranged from 91 to 98%. These phylotypes could represent new species, because in yeasts more than 1% of the nucleotide divergence in D1&D2 domains of the 26S rRNA gene may represent a separate species (Kurtzman & Fell, 2006). In spite of the lack of inter-individual comparison, this study

showed a potential existence of the other yeasts that have not been discovered yet.

From the cited references it is obvious, the biodiversity studies depend very much on the applied method. However, this is beyond the scope of this chapter to provide very detailed description of all possible methods that could be used for studies on yeasts' diversity. Nor calculation of the different biodiversity indices is in the focus of the paragraph. This paragraph is meant to provide short discussion on the existing possibilities, their limitations and advantages, and provide the reader with some input for consideration which methods

Any application of either method mentioned below requires correct sampling of the material. Studying the biodiversity of the yeasts harbouring the GIT the dominating yeasts are in focus of most studies, as well as their abundance and changes of the abundance in time and in relation to the diet. For these purposes faecal or digesta samples have been collected from large animals (Urubschurov et al., 2008; 2011) or whole intestines from e.g. insects have been dissected (Suh et al., 2004b; 2005a; Nguyen et al., 2007). Whereas rain worms, termites or other small animals can provide the whole GIT for the studies, only part of contents of wall of the GIT can be studied in large animals. Therefore the choice of sampling is the first bottle neck in the studies on yeasts biodiversity in the GIT. Following proceedings such as homogenization, concentration or dilution of the samples must be

**3. Methods for investigating biodiversity of the yeasts from GIT** 

he or she would choose for his/her studies.

hereby additionally considered.

Furthermore, compared to other yeasts occurring in the porcine GIT, which can survive also in other ecological niches outside the animals, *K* (*C.*) *slooffiae* seems to be well adapted to the porcine gastrointestinal habitat, as this species is one of those that need high temperature to grow, comparable to the temperature of animal body, being characterized as thermophilic or psychrophobic (Travassos & Cury, 1971).
