USE OF PARASITOLOGICAL AND MOLECULAR METHODS IN GIARDIA SP. DETECTION IN ANIMALS HELD IN CAPTIVITY

Protozoa of the genus Giardia cause the disease giardiosis in a large number of animal species, but also in humans. This disease can be either subclinical or followed by diarrhea, malabsorption, weight loss and stunted growth. The aim of this study was to examine the presence of Giardia sp. in different species of animals in Belgrade Zoo (asymptomatic infection) and to compare the sensitivity of parasitological and molecular-genetic methods. Examination of the presence of Giardia sp. protozoa was performed in 52 fecal samples, using fecal ﬂ otation and molecular-genetic PCR method. No positive samples were detected when deploying the parasitological method, while PRC analysis revealed the presence of Giardia sp. in one animal - ring-tailed lemur ( Lemur catta ), which indicates the advantage of the molecular-genetic method over the parasitological one. The absence of Giardia sp. in other samples can be explained by adequate housing conditions, successful implementation of hygienic and sanitary measures, as well as good veterinary practice. Use of PCR method for testing the presence of Giardia sp. showed a higher sensitivity when compared to the fecal ﬂ otation method.


INTRODUCTION
Parasites of the genus Giardia commonly cause diarrhea in humans, as well as in domestic and wild animals.Probably the most common intestinal protozoa infecting humans, Giardia sp. also appears to be common in some zoo-housed primates [1].The clinical presentation in domestic animals infected by Giardia sp. is most often characterized by diarrhea, stunted growth, weight loss, decreased reproductive capacity and even death, which can consequently lead to signifi cant economic losses [2,3].In humans, these parasites can cause persistent diarrhea that can last for 3 weeks or longer [4].Giardia sp. is a parasitic fl agellate which morphologically has a bilaterally symmetrical body with eight fl agella.They have an adhesive disk on the ventral surface of the body which enables them to attach to the epithelial cells of the intestinal mucosa.Trophozoites, vegetative forms of Giardia sp.multiply in the small intestine where they cause diarrhea and malabsorption, the main clinical signs of giardiosis.Cysts are responsible for the transmission of the infection.They are formed and released from the gastrointestinal system of the host, after which a new susceptible individual can be infected.Healthy animals can be infected through contaminated water, food, or direct fecal-oral route [5].There are several pathways for the introduction of Giardia sp. in zoos, which include: animal food, newly acFquired infected animals, staff and visitors [6].Among the eight species within the genus Giardia, the most important in the fi eld of veterinary medicine is Giardia duodenalis.There are eight assemblages (A -H) of this species that do not differ morphologically but show more or less host specifi city [7].Zoonotic assemblages A and B do not show host specifi city and can be found in humans, as well as in a wide range of animals.Remaining assemblages (C-H) show certain host specifi city: assemblages C and D are mainly found in canids, assemblages E in equines and ruminants, assemblages F in felines, assemblages G in rodents, and assemblages H in marine mammals [7].Various studies examined the occurrence of Giardia sp. in animals kept in captivity [1,4,8,9], using different diagnostic methods, while centrifugal fecal fl otation (using saturated aqueous zinc sulphate solution) is one of the most used methods to detect Giardia cysts in fecal samples.However, the disadvantage of this method can be found in the success rate of cysts detection, being 90% after consecutive examination of at least three fecal samples.This method lacks liability in differentiating species of the genus Giardia [10].On the contrary, molecular genetic methods such as those based on polymerase chain reaction (PCR) are undoubtedly more reliable and can detect not only the presence or absence of parasites' DNA in the investigated sample, but can also determine the species within the genus Giardia [11].
Currently, PCR-based methods are generally used in the detection of Giardia parasites in humans and animals.The use of these methods has affected the sensitivity of determination of Giardia sp.[12].
Over the years, there has been an increase in the number of visitors in the zoos, which prefer interaction (direct contact) with animals.As a result, the risk of zoonosis transmission has also grown.From a public health point of view, these protozoa have a high zoonotic potential, being among the most common intestinal parasites in humans worldwide.Thus, the importance of wild animals kept in captivity must be taken into consideration since the increasing number of infectious diseases of animals poses a great health risk for humans.For this reason, research on animal diseases is one of the most important factors in maintaining human health [13].
Gi ardia sp. has been detected in zoos across Europe [1,8,14,15].There are reports of their high prevalence in wild ruminants kept in captivity which can serve as reservoirs for zoonosis transmission [16].Giardia sp. was detected in the Zagreb Zoo in Artiodactyla, Carnivora, Hyracoidea, Perisodactyla, Primates and Rodentia, [8], while in two zoos in Spain (Madrid and Valencia) their presence was confi rmed in 16 different species of primates [14].
Having in mind the zoonotic potential of Giardia sp. and the fact that animals often do not show clinical signs, as well as the limitations of parasitological methods in Giardia sp.detection, the aim of this study was to investigate the occurrence of Giardia sp. in various mammal species in Belgrade Zoo, using centrifugal fecal fl otation and PCR methods and to compare the sensitivity rate of those methods.

MATERIAL AND METHODS
Fecal samples of 52 animals belonging to 19 species were collected in Belgrade Zoo (Table 1).All animals were clinically healthy.Samples were collected in sterile plastic bags during morning hours and were transported to the Laboratory for Animal Genetics, Department of Biology and Laboratory for parasitology, Department of Parasitology, Faculty of Veterinary Medicine, University of Belgrade, where they were stored at 4°C until further processing.Positive control was obtained from the laboratory of the Department for Parasitology in the same institution.

Centrifugal fecal fl otation with zinc sulphate solution
Fecal samples were analyzed by using centrifugal fecal fl otation method with saturated aqueous zinc sulfate solution (ZnSO4, specifi c gravity 1.18 at 20°C), following the procedure described by Bogunović et al. [17].Each fecal sample was examined in duplicate by two experienced specialists under light microscope (Olympus CX 23, Japan) using 100× and 400× magnifi cations.

DNA extraction
The extraction was performed using a Quick-DNA TM Faecal/Soil Microbe Miniprep kit (Zymo Research, USA).From each collected sample, 200 mg of feces was added to ZR BashingBead TM Lysis Tube and mixed with 800 µl of Lysis solution.The Lysis Tube was placed in TissueLyser II (Qiagen, USA) and processed at maximum speed for 5 minutes.The Lysis Tube was then centrifuged at 10000 x g for 1 minute.Acquired supernatant in the volume of 400 µl was transferred to a Zymo-Spin TM IV Spin Filter placed in a collection tube and centrifuged at 7000 × g for 1 minute.Further on, 1200 µl of Faecal DNA Binding Buffer was added to the fi ltrate in the collection tube.The mixture acquired in the previous step in the volume of 800 µl was transferred to a Zymo-Spin TM IIC Column, which was placed in a collection tube and centrifuged at 10000 × g for 1 minute.The fl ow-through was discarded and the step was repeated.Zymo-Spin TM IIC Column was placed in a new collection tube, 200 µl of DNA Pre-Wash Buffer was added in the column and centrifuged at 10000 × g for 1 minute, while the fl ow-through was discarded.Further on, 500 µl of Faecal DNA Wash Buffer was added to the Zymo-Spin TM IIC Column and the fl ow through was discarded.The column was transferred to a sterile 1.5 ml tube and 100 µl of DNA Elution Buffer was added directly to the column matrix and centrifuged for 30 seconds at 10000 × g in order to elute the DNA.In order to purify the DNA sample, a Zymo-Spin TM IV-HRC Spin Filter was placed in a sterile 1.5 ml tube and the eluted DNA from the previous step was added inside and centrifuged at 80000 × g for 1 minute.The quality and quantity of extracted DNA were determined using BioSpec-nano Micro-volume UV-Vis Spectrophotometer (Shimadzu, Japan).

PCR amplifi cation
In order to amplify a 292-base pair (bp) long ssrRNA fragment, the following primers were used: RH11: 5`-CATCCGGTCGATC-CTGCC-3` and RH4: 5`-AGTCGAAC-CCTGATTCTCCGCCCAGG-3` [18].The PCR mixture was prepared using a commercial set of KAPA2G Robust HotStart ReadyMix PCR mixture (Kapa Biosystems, USA) according to the manufacturer's instructions.PCR was performed in a fi nal volume of 25 µl containing: 10 µl KAPA2G Robust HotStart ReadyMix, 1.25 µl of each primer, 10 ng of a DNA sample and 2.5 µl of ddH2O.PCR amplifi cation was performed in a T100 Thermal Cycler device (Bio-Rad, USA), while the PCR parameters were as follows: activation at 95° C for 5 minutes, followed by 40 cycles of denaturation at 95 °C for 30 seconds, primer annealing at 65 °C for 30 seconds, extension at 72 °C for 60 seconds and fi nal extension at 72 °C for 7 minutes.

Visualization of the PCR products
The amplifi cation products (5 μl) were mixed with 1 μl 6X DNA Loading Dye (Thermo Scientifi c, USA), subjected to electrophoresis in 2% agarose gel, stained with ethidium bromide, visualized under UV light, examined for different band patterns and documented.

RESULTS
When the centrifugal fecal fl otation method was deployed, no Giardia cysts were found in any of the analyzed samples (Table 2).Unlike this classical parasitological procedure, molecular analysis using PCR has shown different results.Namely, PCR analysis revealed one positive sample.An electrophoretic band of 292 bp shown in Figure 1 indicates a PCR product amplifi ed the fecal sample no. 5, originating from ring-tailed lemur (L.catta).Further on, centrifugal fecal fl otation revealed other parasitic forms as a side fi nding in seven out of 52 inspected fecal samples.These were identifi ed as: trichostrongyle and oxyurid eggs in a southern three-b anded armadillo (Tolypeutes matacus); trichostrongyle eggs and coccidian oocysts in four red kangaroos (Macropus rufus); coccidian oocysts in one parma wallaby (Macropus parma); Sarcocystis spp.oocysts in one arctic wolf (Canis lupus arctos) (Table 2, Figure 2).

DISCUSSION
The absence of Giardia sp.DNA in most of analyzed animals can be explained by good hygiene measures, as well as good practice of animal health control applied by the staff in the Belgrade Zoo.Additionally, the absence of mutual contact between individuals of different mammal species and communication of animals with humans (excluding workers that are in direct or indirect contact during cleaning, feeding and other technical work) are also factors that explain the low prevalence of infection.
As previously described, infection occurs after the consumption of contaminated water/food or by the fecal-oral route.The fi nding of Giardia sp. in only one sample, which originated from the ring-tailed lemur, indicates that animals, although housed close to each other, have not come into contact with contaminated water or food, suggesting very successful hygienic and sanitary measures and good veterinary practice implemented by the employees of the Belgrade Zoo.Furthermore, we should have in mind that in our study samples were collected only once from each animal, which can affect the acquired results, having in mind that Giardia sp. is excreted intermittently in the feces.In the study conducted by Chagas et al. [19] in São Paulo Zoo (Brazil), the prevalence of Giardia sp. was evaluated using passive fl otation and was 1.5% while it was highest (2%) within the class of Mammals.This fi nding is in accordance with our results, in which the prevalence was 3.1%.Research conducted by Matsubayashi et al. [4] in Osaka Zoo (Japan), similarly to our results, showed a low prevalence of Giardia sp.Namely, out of 284 examined samples, Giardia sp. was detected only in one mandarin duck (Aix galericulata) and two ruddy shelducks (Tadorna ferruginea).The authors suggest that the low prevalence may be due to the good quarantine system conducted by veterinary surgeons in the Zoo.Beck et al. [8] conducted an investigation on 131 asymptomatic animals in Zagreb Zoo (Croatia) and revealed 38 positive fi ndings.The highest Giardia sp.prevalence among the investigated animals in the research of Beck et al. [8] was found within Artiodactyla, while in our research there were no positive samples within this order.Further on, in the same study high prevalence of Giardia sp. was found in animals belonging to orders of Carnivora, Primates and Rodentia, while in our research there was only 1 positive sample originating from a primate.
Fomsgaard et al. [1] conducted a comparative study of the prevalence and intensity of G. duodenalis infection in samples of ring-tailed lemurs from fi ve zoos in Denmark and those living freely in nature.Quantitative PCR results revealed that the prevalence (88.6%) and the degree of infection (median Ct value 31.1) in lemurs from zoos that have an area for contact with visitors (without a fence) is signifi cantly (P>0.01)higher than in lemurs living freely in nature (prevalence 20.0%, median Ct value 37.7).Within the same study, a comparison was made between different types of accommodation spaces within zoos.It was determined that both the prevalence (89.0%) and the degree of G. duodenalis infection (median Ct value 28.6) in zoo accommodation spaces that have an area for contact with visitors (without fence) were statistically signifi cantly (P>0.005)higher than in those with traditional accommodation (cages), in which the prevalence of 65% was determined, while the intensity of infection expressed over a median Ct value was 35.2 [1].Analysis of the results of previously mentioned studies, as well as the results of our study indicate that low occurrence of Giardia sp. is mainly due to the absence of contact of animals with visitors, along with good hygiene and veterinary practice applied by the Belgrade Zoo.In our study, no positive samples were revealed when the centrifugal fecal fl otation method was deployed for the detection of Giardia sp.cysts, while one positive sample was detected by the PCR method, which indicates a higher sensitivity of molecular genetics over fecal fl otation method.On the contrary, in a study conducted by Solarczyk and Majewska [15], who analyzed 242 samples originating from 131 species from Poznan Zoo (Poland), parasitological analysis of feces revealed the presence of Giardia sp.cysts in fi ve samples, while PCR detected only one positive sample originating from anteater (Tamandua tetradactyla).The low sensitivity of PCR in the study of Solarczyk and Majewska [15] can be explained by inadequate DNA extraction method and primers that amplify long fragments of DNA, {i.e., 753 bp [20]; 530 bp [21]; 430 bp [22]}.Unlike Solarczyk and Majewska [15] we used Quick-DNA TM Faecal/Soil Microbe Miniprep set (Zymo research, USA) for DNA extraction and primers designed by Hopkins et al. [18] that provide a relatively short amplifi cation product (292 bp).Side fi nding of trichostrongyle eggs and coccidian oocysts in red kangaroos in our study is not surprising, having in mind they are commonly found parasites in marsupials [23,24].Sarcocystis spp.oocysts found in one arctic wolf are probably caused by infection through raw feed diet which occurs in captivity.Having in mind that the feces was collected from a three-banded armadillo which has just arrived in the Belgrade Zoo and was under anthelmintic therapy, the fi nding of oxyurid eggs, a common parasite of this animals [25], does not surprise.

Table 1 .
The number of samples and the taxonomic position of sampled animals

Table 2 .
Findings of endoparasites in the investigated fecal samples