There are two stages in the life cycle of Giardia species: trophozoite and cyst (infective stage) ^3,7,8. Infection occurs by the fecal-oral route by ingestion of feces, fomites, or food contaminated with cysts ^7,9. The disease shows a diverse clinical course in humans, ranging from an asymptomatic state to an acute state. Diarrhea, vomiting, nausea, abdominal cramps, anorexia, and weight loss can be seen ¹⁰.

The disease may be asymptomatic in cats, or diarrhea may occur due to maldigestion, malabsorption, and increased motility ^2,8,10,11. Most infections in cats occur at an early age ^2,3. The chronic form of the disease is more common in immunocompromised cats and can continue for years ^2,11. Microscopic, serological and molecular methods are used in the diagnosis of the disease ^1,7,9,11. Since molecular techniques are more sensitive, they are more widely used in diagnosis ^2,7. In studies conducted around the world using the PCR method on cats, positivity rates of 3.9%, 6.1%, 8.18%, 9.2%, 13.1%, and 80% were reported in Poland, Italy, Japan, Spain, China, and Australia, respectively ^2,8,12-15. In studies to figure out the prevalence of giardia on cats in Turkey, positivity has been reported between 4% and 50% ^9,16,17.

The reports on the presence of G. duodenalis in cats in Turkey are quite limited. This study aimed to investigate the prevalence of Giardia duodenalis in stray cats in Mardin province of Turkey by microscopic and molecular methods.

The Study Area and Animal Material: This study was carried out in Mardin province, located in the Southeastern Anatolia Region of Turkey (370 17' 52" N, 400 45' 36" E). The animal material of the study consisted of a total of 48 cats, 21 male, and 27 female, in the Mardin Metropolitan Municipality Stray Animal Rehabilitation Center. The cats have not been treated for giardiasis. Fresh fecal samples from cats were placed in individual specimen containers. Sex, age, and fecal type information were recorded. All samples were transferred to the parasitology laboratory of the veterinary faculty within the iceboxes.

Microscopic Examination: One drop of physiological saline solution and one drop of lugol solution were placed in different areas on the slide. One or two gr stool samples were taken by the plastic stick and mixed first with physiological saline and then with lugol solution. Covered with a coverslip, specimens were examined under a microscope (Leica, Hamburg, Germany) at x40 magnification.

Genomic DNA Extraction: Genomic DNA (gDNA) extraction from the samples was performed by using the GeneMATRIX Stool DNA Purification Kit (Gdansk, Poland) according to the manufacturer protocol and the obtained gDNAs were stored at -20°C until further analysis.

Nested-PCR Reaction: In the first PCR, a 753 bp β-giardin gene fragment was amplified by using the published primers (G7 F5'-AAGCCCGACGACCTCACCCGCAGTGC-3' and G759R 5'-GAGGCCCGCCCTGGATCTTCGAGACGAC-3') ¹⁸. The PCR mix was inculded 6 pmol of forward and reverse primers, 4 μl of 5x FIREPol® Master Mix (7.5 mM MgCl2, Solis BioDyne, Estonia) 1.6 μl of gDNA and 13.2 μl Nuclease Free Water. The reaction consisted of pre-denaturation for 5 minutes at 95°C, followed by each cycle of denaturation (1 min at 95°C), annealing (1 min at 60°C), and extension (1 min 30 seconds at 72°C), 35 cycles and a final extension of 7 minutes at 72°C. Subsequently, using the following primers (BG1F 5'-GAACGAGATCGAGGTCCG-3' and BG2R 5'-CTCGACGAGTTCGTGTT-3') a nested-PCR reaction was performed. (19). For this aim, 6 pmol of forward and reverse primer in 20 μL of mastermix, 4 μl of 5x FIREPol® Master Mix (7.5 mM MgCl2, Solis BioDyne, Estonia) 1 μl of PCR product and 13.8 μl Nuclease Free Water were used. The reaction was carried out followed by pre-denaturation at 95°C for 5 minutes, with each cycle consisting of denaturation (1 min at 95°C), annealing (1 min at 55°C) and extension (1 min at 72°C), 35 cycles and at 72°C with a final extension of 7 minutes. PCR products were stained with RedSafe™ Nucleic Acid Staining Solution and images were obtained on 1.5% agarose gel.

Statistical Analysis: The studied cats were divided based on sex (female, male), age (≤1, >1), fecal type (diarrheic and non-diarrheic), and technique (Microscopy, PCR) into two groups. Age was estimated by the dental formulary. The data obtained in the study were analyzed using the SPSS V16.0 (IBM, Chicago, IL, USA) program. The chi-square test was used to compare differences in infection rates among the investigated groups. The differences were considered significant at P≤ 0.05 ⁹.

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Table 1: Infection rates of G. duodenalis in cats according to sex, age, fecal type, and used technique

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Figure 1: G. duodenalis cyst in stool samples, x40 magnification

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Figure 2: PCR amplification products of some selected G. duodenalis samples. Lanes M: Marker, N: Negative control, P: positive control, Lanes 2,4,5,17,19 represent G. duodenalis positive samples (511 bp)

In the world, different results have been reported in studies to determine the prevalence of G. duodenalis in cats. Respectively, 5% and 80% by microscopic and PCR methods in Australia ^2, 13.1% by PCR method in China ^15, 8.18% in Japan ^14, in Italy 4.4% and 6.1% by IFAT and PCR method respectively ^13, 9.2% by PCR method in Spain ^12, 3.9% by PCR method in Poland ⁸ and a prevalence of 2% and 3.33%, respectively were reported in Iran by microscopic and immunochromatography assay methods ⁷.

In Turkey, studies to determine the prevalence of Giardia spp. in cats are very limited. Some studies report its presence at the rate of 4% as G. cati ¹⁶ in Ankara, and 8% as G. intestinalis in a study including the Samsun and Kayseri provinces ¹⁷. In another study in the Central Anatolia Region where microscopic examination, rapid immunochromatographic test, and PCR methods were used, the prevalence of G. duodenalis was reported to be 37.3%, 50% and 29.4%, respectively ⁹.

Microscopic, serological and molecular methods are used in the diagnosis of the disease ^1,7,9,11, among which is reported that the PCR method is more sensitive ^2,7. In this study, microscopic examination and PCR methods were used for diagnosis. As a result of the study, 8.33% positivity was detected by microscopic examination and 16.67% by PCR method. The results of this study are similar to the results of the studies carried out by Önder et al. ¹⁷ and Xu et al. ¹⁵. However, it is nonetheless lower than the results of the study by McGlade et al. ^2, while it was higher than the ones carried out by Sursal et al. ^9, Suzuki et al. ^14, Paoletti et al. ¹³ and Gil et al. ¹². Differences between studies may be caused by different geographical conditions, the diagnostic procedure used, the age of the animals and the population.

While some researchers ^7,12,20 found more positivity in males, some ^6,9 reported more positivity in females. In this study, compatible with the findings of Sursal et al. ⁹ and Procesi et al. ^6, higher positivity was detected in females.

In the study by Li et al. ²⁰ a statistically significant difference between the sexes was reported, while other researchers ^6,7,9,12 stated no significant difference. In this study, no statistically significant difference was found between the sexes, as well. (P>0.05).

Procesi et al. ⁶ reported a higher prevalence in those older than one year and stated that this difference was statistically significant. On the other hand, some other researchers ^7,8,12,14,15 show that the prevalence is higher in those under the age of one, and there is no statistically significant difference between age groups. In this study, a higher prevalence was found in cats under one year of age, as well (P>0.05). The low prevalence in cats over one year old may be due to the development of humoral immunity with advancing age.

In a study by Li et al. ^20, more positivity was found in the non-diarrheic group than in the diarrheic group (P>0.05). On the other hand, in a study by Mosallanejad et al. ^7, more positivity was found in the diarrheic group and a statistically significant difference was reported. In this study, more positivity was detected in the diarrheic group (17.65%) than in the non-diarrheic group (16.13%), but no statistically significant difference was detected.

As a result, the prevalence of G. duodenalis in stray cats in Mardin province was revealed in this study. The results show that cats can be a potential reservoir for human infection and demonstrate a potential risk to public health. Further studies are needed to investigate the general epidemiological status of giardiasis in domestic and stray cat populations.

Acknowledgement
The author expresses her deepest thanks to the Assoc.Prof. Adnan AYAN for supporting this study.

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