Indian Journal of Animal Research

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Full Research Article

Molecular Characterization and Zoonotic Significance of Cryptosporidium spp. and Giardia duodenalis in Asymptomatic Adult Stray Cats and Dogs in Turkey

Duygu Neval Sayın Ipek1,*
1Department of Parasitology, Faculty of Veterinary Medicine, Dicle University, Diyarbakýr-21000, Turkey.

ABSTRACT

Background: Cryptosporidium species and Giardia duodenalis are important protozoan parasites due to their wide host range and potential as zoonotic diseases. These parasites, responsible for Cryptosporidiosis and Giardiasis, are significant health concerns and have garnered attention from both the public and veterinary fields. Pets, such as dogs and cats, are susceptible to these infections and play a crucial role in transmitting these protozoans to humans. This study was conducted to determine the prevalence, species, genotypes and zoonotic significance of Cryptosporidium and Giardia in asymptomatic adult stray cats and dogs. 

Methods: Fecal samples from 75 adult stray animal, 42 dogs and 33 cats, were collected in Diyarbakýr city, southeastern Anatolia, Turkey. Direct immunofluorescence test was used to confirm the presence of G. duoenalis cysts and cryptosporidium spp. oocysts in collected fecal samples. Microscopic analysis was used to count the number of cysts/oocysts per gram as specified by the manufacturer. Molecular confirmation was done with Polymerase chain reaction (PCR) using Giardia SSU-rDNA and Cryptosporidium spp. SSU rRNA gene. The secondary PCR products of all positive samples were sequenced in one directions on an automated sequencer. Nucleotide sequence analysis was performed by BLAST alignment using the National Center for Biotechnology Information database. 

Result: The prevalence of Cryptosporidium was 7.14% in 42 cats and 9.09% in 33 dogs. The prevalence of G. duodenalis was 23.80% in cats and 54.54% in dogs. The average number of Cryptosporidium spp. oocysts per gram of cat and dog feces was 1866.3 and 1813.3, respectively. The average number of G. duodenalis cysts per gram of cat and dog feces was 1011.6 and 4786, respectively. All Cryptosporidium isolates were determined as C. canis in dogs and C. felis in cats. C. canis and C. felis isolates identified in the study, MT329018.1 and MN696800.1, AF159113.1 and KM977642.1 showed similarity with Genbank number, respectively. Assemblages A, E, C, D were found in dogs and assemblages A and E in cats in the result of the sequence of 130 bp gene fragments obtained from G. duodenalis isolates. This study sequence analysis of G. duodenalis cat isolates identified assemblages A (50%/10) and E (50%/10). In this study, assemblage E was the most common genotype isolated in dogs (38.8%), followed by Assemblage C (27.7%), Assemblage D (16.6%) and Assemblage A (15.5%). Assemblage A, which has zoonotic importance, was detected in cats and dogs, while assemblage E in cats and dogs was reported for the first time in Turkey. 

INTRODUCTION

Gastrointestinal system diseases, particularly infections that cause diarrhea, are critical for cat and dog health. These infections include cryptosporidiosis and giardiasis caused by Cryptosporidium spp. and Giardia duodenalis, respectively, which are zoonotic and usually asymptomatic in cats and dogs. The diseases of cats and dogs, which are in close contact with humans and farm animals, are as important as zoonotic aspects of the health of these animals (Feng and Xiao, 2011; Xiao, 2010). Cysts and oocysts excreted with the host feces handle the spread of infection in humans and animals and the infection occurs through the consumption of contaminated water and food with cysts and oocysts (Heyworth, 2016; Ryan et al., 2018).
       
There are around 40 different species of Cryptosporidium and over 21 of those have been linked to human infections. The majority of infections in dogs and cats are caused by C. canis and C. felis, respectively, according to molecular investigations (Ballweber et al., 2010; Lucio-Forster et al., 2010; Rossle and Latif, 2013; Ryan et al., 2018) but C. hominis, C. parvum, C. muris and C. ubiquitum have sporadically been found in these species (Alves et al., 2018; Feng et al., 2018; Xiao, 2010).
 
G. duodenalis has eight different genotypes with varying host ranges. Assemblages C to H have a limited host range, while assemblages A and B are zoonotic and can be found in humans and many animal species. The majority of assemblages C and D are found in canines, while the assemblages F are mostly found in cats. Assemblage E primarily affects cattle, sheep, goats and pigs, unlike assemblages C, D, E and F, which have been recorded in humans. (Ballweber et al., 2010; Fantinatti et al., 2016; Lucio-Forster et al., 2010; Tungtrongchitr et al., 2010; Vivancos et al., 2018; Xu et al., 2016; Zahedi et al., 2017).
       
This study was conducted to determine the prevalence, species and genotypes and zoonotic significance of Cryptosporidium and Giardia and in asymptomatic adult stray cats and dogs in Diyarbakýr, Turkey.

MATERIALS AND METHODS

Study areas and animal sources
 
Stool samples were collected from sterile gloves and swaps from the rectum of 42 cats and 33 dogs randomly selected to determine Cryptosporidium spp. and Giardia duodenalis in adult stray cats and dogs at in Diyarbakýr city, southeastern Anatolia, Turkey.
 
Microscopic analysis
 
The Crypto/Giardia-Cel FITC Staining Kit (Cellabs Inc. Brookvale, Australia) was used to confirm the presence of G. duodenalis cysts and Cryptosporium spp. oocysts. 1g of fecal material was treated as directed by the manufacturer. A fluorescent microscope (Carl Zeiss Microscopy GmbH. Jena, Germany) was used to analyze each sample at 200x, 400x and 1000x magnifications. At a magnification of 200x, the oocytes and cysts discovered in each fecal sample were tallied and the number of oocysts per gram feces (opg) was calculated as follows:
 
 
 
DNA extraction
 
Total Genomic DNA was extracted from of each fresh 200 mg fecal sample with the direction of suggestion of kit by using ZR Fecal DNA MiniPrep kit (Zymo Research, Irvine, C.A.). Purified DNA samples (100 mL) were stored at -20oC.
 
Molecular detection of cryptosporidium spp.
 
To determine the species, all DNA extracts were submitted to a nested PCR procedure to amplify a fragment of the Cryptosporidium spp. SSU rRNA gene (830 bp). A PCR product of 1325 bp was amplified for the first PCR using the forward primer 5'-TTCTAGAGCTAATACATGCG-3' and the reverse primer 5'-CCCTAATC CTTCGAAACAGGA-3'. The PCR reactions were performed in a total volume of 50 mL containing 10X PCR buffer, 3 mM MgCl2, 0.2 mM each dNTP, 20 pmol of each primer and 2 U of Taq DNA polymerase under the following conditions: initial denaturation at 94oC for 3 min, 35 cycles at 94oC for 45 s, 55oC for 45 s and 72oC for 1 min and a final extension step at 72oC. For the second round of PCR, reactions were carried out using primers forward primer 5' GGAAGGGTTGTATTTATTAGATAAAG-3' and reverse primer 5'-AAGGAGTAAGGAACAACCTCCA-3' (Xiao et al., 1999). The nested PCR mixture and conditions were identical to the primary PCR except that a concentration of 1.5 mM MgCl2 was used. The PCR products were subjected to electrophoresis in a 1.4% agarose gel and visualized by staining with ethidium bromide.
 
Molecular detection of giardia duodenalis
 
A nested PCR was utilized to amplify a 130 bp region of the SSU-rDNA gene using four primers (RH11, RH4, GiarF and GiarR) as previously described by Hopkins et al., (1997) and Read et al., (2002) (Hopkins et al., 1997; Read, 2002). First and second PCR amplifications were performed in 25 ml volumes with the final mix containing, 2 ml Q solution, 10 pmol of each primer, 1.25 unit DNA polymerase, 0.2 mM of each dNTP, 2.5 mM MgCl2, 10x PCR buffer and H2O. First PCR reaction was heated to 96oC for 2 min followed by 35 cycles of 96oC for 4 s, 62oC for 30s and 72oC for 45s and one cycle of 72oC for 4 min and second PCR reaction was heated to 96oC for 5 min followed by 35 cycles of 96oC for 45 s, 55oC for 30 s and 72oC for 45 s and one cycle of 72oC for 7 min. The PCR products were subjected to electrophoresis in a 2% agarose gel and visualized by staining with ethidium bromide.
 
Sequence analysis
 
The secondary PCR products were sequenced in one directions on an automated sequencer (ABI PRISM 310 model, Perkin-Elmer, USA).

RESULTS AND DISCUSSION

The prevalence of Cryptosporidium was 7.14% (3/42) in 42 cats, whereas G. duodenalis was 23.80% (10/42). In 33 dogs, Cryptosporidium was found to be present in 9.09% (3/33) and G. duodenalis in 54.54% (18/33). The average number of Cryptosporidium oocysts per gram of cat and dog feces was 1866.3 and 1813.3, respectively. The average number of G. duodenalis cysts per gram of cat and dog feces was 1011.6 and 4786, respectively (Table 1).
       

Table 1: Occurrence species and genotypes distributions of Cryptosporidium and G. duodenalis in stray dogs and cats.


 
As a result of the sequence of PCR products obtained from all positive samples, it was determined that Cryptosporidium was determined as C. canis in dogs and C. felis in cats. C. canis and C. felis isolates identified in the study, MT329018.1 and MN696800.1, AF159113.1 and KM977642.1 showed similarity with Genbank number, respectively. Assemblage A, E, C, D were found in dogs and assemblage A, E in cats in the result of the sequence of 130 bp gene fragments obtained from G. duodenalis isolates (Table 1).
       
Cryptosporidium species and G. duodenalis, which are the most important protozoan parasites transmitted by water in developed countries, are crucial for both human and animal health due to their wide host range and their expression as a zoonotic disease.
       
Studies on the number of Cryptosporidium spp. and G. duodenalis oocysts/cysts excreted by cats and dogs are limited (Enemark et al., 2020; Kostopoulou et al., 2017; Yang et al., 2015). This study has shown that the average number of Cryptosporidium oocysts and G. duodenalis cysts per gram of cat feces was 1866.3 and 1011.6, respectively. The average number of Cryptosporidium oocysts is higher than that reported in Western Australia and Greece but lower than that reported in Danish cats G. duodenalis cyst numbers are lower than those reported in the Greece and much lower than those reported in the Australian and in the Danish. The average number of Cryptosporidium oocysts and G. duodenalis cysts per gram of dog feces were 1813.3 and 4.817.7 respectively. Our results are higher than that reported in Greece (Enemark et al., 2020; Kostopoulou et al., 2017; Yang et al., 2015). These differences are likely related mainly to differences in methodologies and the health status of the animals between studies. However, oocysts and cysts shed by asymptomatic dogs and cats show that they are an important risk factor for the spread of the disease.
       
There are studies on the prevalence of Cryptosporidium and G. duodenalis in dogs and cats in many countries. The prevalence of G. duodenalis have been reported in dogs 6.3% and cats 2.0% in Australia (Palmer et al., 2008), in dogs 25.2% and cats 20.5% in Greece (Kostopoulou et al., 2017), in dogs 33% and cats 9.2% in Spain (Gil et al., 2017), in dogs 64.0% and cats 87.0% in Canada (McDowall et al., 2011), in dogs 19.6% in Brazil (Fava et al., 2016; Wang et al., 2021), in dogs 4.5-26.2% and cats 1.2-13.1% in China (Wang et al., 2021), in dogs 16.2% in Saudi Arabia (Malki, 2021), in dogs 16.4-18.8% and cats 8-29.4% in Turkey (Çelik, 2022; Önder et al., 2021; Sursal et al., 2020). In our study, G. duodenalis was found in asymptomatic cats and dogs at a rate of 23.8% and 54.54%, respectively and the rate detected in dogs is higher than in cats, similar to many studies conducted worldwide (Gil et al., 2017; Kostopoulou et al., 2017; Palmer et al., 2008; Wang et al., 2021). While the rate we found in cats is higher than the rates reported in Australia, Spain, Brazil, China, Saudi Arabia and some cities in Turkey (Mardin, Samsun and Kayseri), it is similar to the rate reported in Greece (Çelik, 2022; Fava et al., 2016; Gil et al., 2017; Kostopoulou et al., 2017; Malki, 2021; Önder et al., 2021; Palmer et al., 2008; Wang et al., 2021). On the other hand, it was lower than the rate reported in asymptomatic cats in the Central Anatolian Region of Turkey and Canada (McDowall et al., 2011; Sursal et al., 2020). The rate detected in dogs in our study is the highest reported in Turkey. It is higher than the studies conducted in Australia, Spain, Brazil and China in the world and lower than the rate reported by McDowall et al. in Canada (Fava et al., 2016; Gil et al., 2017; McDowall et al., 2011; Palmer et al., 2008; Wang et al., 2021). These differences in the rates reported in the studies are due to factors such as the diagnosis method used in the study, geographical area, feeding areas, age, the health status of the animals, population density, ownership, or not. However, the molecular prevalence of Giardia duodonalis varies considerably depending on the method used.
       
The prevalence of Cryptosporidium has been reported 3.9% in dogs (Yoshiuchi et al., 2010) and 1.4%- 12.7% in cats  in Japan (Ito et al., 2016; Yoshiuchi et al., 2010), 5.4-5.5% in dogs and 8.8% in cats in Spain (de Lucio et al., 2017; Gil et al., 2017), 1.7%-6%  in dogs and 0.6-5.6% in cats in China (Cao et al., 2022; Xu et al., 2016; Wang et al., 2021), 0.6% in dogs in Iran (Homayouni et al., 2019), 1.6% in dogs in USA (Wang et al., 2012), 0.2-1.7% in dogs in  Ýtaly (Paoletti et al., 2015; Simonato et al., 2017),  4.2% in dogs and 8.2% in cats in Brazil (Alves et al., 2018), 5.9% in dogs and 6.8% in cats in Greece (Kostopoulou et al., 2017).  The prevalence of Cryptosporidium has been reported to be 2.1%-5% in cats and 0.5-15.78% in dogs, in Turkey (Köseoğlu  t al., 2022; Kilinç et al., 2018; Korkmaz et al., 2016; Denizhan and Karakuş, 2019; Ağaoğlu et al., 2022; Çelik et al., 2023; Görkem and Ulutaş, 2022; Ünal and Gokpinar, 2020). In our study, the molecular prevalence of cryptosporidium in cats was 7.14%. While this rate is higher than those reported in Japan, China, Greece and Turkey, (Cao et al., 2022; Ito et al., 2016;.Kostopoulou et al.,  2017; Wang et al., 2012; Xu et al., 2016; Köseoğlu et al., 2022; Kilinç et al., 2018; Korkmaz et al., 2016) it is lower than those reported in Spain and Brazil (Denizhan and Karakuş, 2019; Alves et al., 2018; de Lucio et al., 2017). In the present study, the prevalence of cryptosporidium in dogs (9.09%) is higher than in Japan, China, Iran, USA, Italy, Greece, Sivas and Ankara provinces, Turkey (Yoshiuchi et al., 2010; Ağaoğlu et al., 2022; Cao et al., 2022; Xu et al., 2016; Wang et al., 2021; Ünal and Gokpinar, 2020; Paoletti et al., 2015; Simonato et al., 2017; Homayouni et al., 2019). By contrast, higher infection rates were reported in dogs in Brazil and Van and Ege provinces, Turkey (Görkem and Ulutaş, 2022; Alves et al., 2018) These different rates reported in studies conducted in various parts of the world may be due to geographic area, feeding sites, animal health status and population densities of the animals.
       
In recent years, with the increasing use of molecular methods in studies on giardia in cats and dogs, information about the zoonotic potentials of species and genotypes related to these genera has increased. Research has shown that the most prevalent genotype present in cats is F assemblages, followed by assemblages A. In recent investigations, only a few samples showed the presence of Assemblages B, C and D (Cai et al., 2021; Gil et al., 2017; Palmer et al., 2008).  Assemble E has been reported in a limited number of studies. (Read et al., 2004). The results of studies conducted in cats in Turkey showed that all the isolates were identified as G. duodenalis assemblage B (Önder et al., 2021; Sursal et al., 2020). This study sequence analysis of G. duodenalis cat isolates identified assemblages A (%50 /10) and E (%50/10). The presence of zoonotic assemblage A in our study is consistent with many studies reported in cat worldwide (Cacciò et al., 2008; Enemark et al., 2020; Hoopes et al., 2015; McDowall et al., 2011;  Papini et al., 2007; Read et al., 2004). However, assemblage A with zoonotic importance in cats in Turkey is reported for the first time in this study. Our study found genotype assemblage E that was previously not reported in cats in Turkey.
       
The most common genotypes in dogs are C and D, followed by the zoonotic genotype A, B ,and E (Cai et al., 2021; Fantinatti et al., 2016; Zahedi et al., 2017). In addition, assemblages B and E have also been reported (Adell-Aledónet_al2018; Cai et al., 2021; Dado et al., 2012; Uehlinger et al., 2013) It has been reported that Assemblages A, B, C and D have been detected in dogs in Turkey (Çelik et al., 2022; Gultekin et al., 2017) In this study, assemblage E was the most common genotype isolated in dogs (38.8%), followed by Assemblage C (27.7%), Assemblage D (16.6%) and Assemblage A (15.5%). Although the studies reporting the presence of Assemblages E in dogs are limited, there is yet to be a study reporting Assemblages E in dogs in Turkey. (Dado et al., 2012) In our research, it is thought that the most common subspecies of assemblage E, written for the first time in dogs in Turkey, is due to the contact of stray dogs with rural areas where farm animals are located. The assemblages C, D and A that were found in our study match the subspecies that have been previously identified in dogs.
       
Cryptosporidium canis and C. felis are host-adapted to dogs and cats, respectively. However, they are also among the five most common Cryptosporidium spp. that can infect humans (Xiao and Feng, 2008; Li et al., 2021). In Our study, sequence analysis of Cryptosporidium spp. isolates revealed that the positive samples were all identified as C. canis in dogs and C. felis in cats. Our results are consistent with the studies reported in our country and in the world (Gil et al., 2017; Homayouni et al., 2019; Kostopoulou et al., 2016; Köseoğl et al., 2022; Li et al., 2019; Yoshiuchi et al., 2010).

CONCLUSION

In conclusion, the relatively high prevalence of giardiasis in dogs and cats, the presence of potential zoonotic subgenotypes A and E and the zoonotic characteristics of cryptosporidium species detected in dogs and cats indicate the importance of treatment and preventive measures. However, further studies in human and animal populations living in this region will be useful to determine the zoonotic epidemiology of G. duodenalis.

FUNDING

This work was supported by the authors’ previous project (Tubitak. 18.002). Financial support was provided by the Dicle University Scientific Research Projects.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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