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Host-specific Cryptosporidium, Giardia and Enterocytozoon bieneusi in shelter dogs from central Europe

Published online by Cambridge University Press:  02 February 2024

Magdalena Szydłowicz*
Affiliation:
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland
Żaneta Zajączkowska
Affiliation:
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland
Antonina Lewicka
Affiliation:
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland
Błażej Łukianowski
Affiliation:
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland Department of Pathomorphology, 4th Military Clinical Hospital, Wroclaw, Poland
Mateusz Kamiński
Affiliation:
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland
Nikola Holubová
Affiliation:
Biology Centre, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic
Bohumil Sak
Affiliation:
Biology Centre, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic
Martin Kváč
Affiliation:
Biology Centre, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic Faculty of Agriculture and Technology, University of South Bohemia in České Budějovice, České Budějovice, Czech Republic
Marta Kicia
Affiliation:
Department of Biology and Medical Parasitology, Wroclaw Medical University, Wroclaw, Poland
*
Corresponding author: Magdalena Szydłowicz; Email: [email protected]

Abstract

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Type
Research Article
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.
Copyright
Copyright © The Author(s), 2024. Published by Cambridge University Press

Introduction

Dogs play an important role in human life as companion animals; however, they may be carriers of various pathogens, constituting a potential reservoir of zoonotic infections for their owners. Many of these infectious agents reside in the intestinal tract; therefore, their dispersive forms are excreted with animal stool and may easily be spread to other hosts by fecal–oral transmission through direct contact or indirectly via water or food contamination. This includes unicellular enteric organisms such as Giardia intestinalis (syn. duodenalis or lamblia), Cryptosporidium spp. or microsporidia from genera Encephalitozoon and Enterocytozoon, which can be responsible for gastrointestinal symptoms like abdominal pain, diarrhoea or flatulence (Xiao, Reference Xiao2010; Liao et al., Reference Liao, Lin, Sun, Qi, Lv, Wu, Li, Hu, Yu, Cai, Xiao, Sun and Li2020). Since these organisms belong to the group of opportunistic pathogens, infection is of particular importance for individuals with impaired immunity (e.g. HIV-infected patients, cancer-treated patients or transplant recipients), in whom it may lead to the development of hazardous, even life-threatening symptoms.

Giardia intestinalis is the most common intestinal pathogenic protozoan in humans and animals (Kváč et al., Reference Kváč, Hofmannová, Ortega, Holubová, Horčičková, Kicia, Hlásková, Květoňová, Sak and McEvoy2017), consisting of 8 distinct assemblages (A–H) differing in host specificities. Assemblages A and B display a broad host range and are most commonly reported in humans, while the remaining 6 seem to be host-specific for non-human species, with assemblages C and D predominantly found in dogs (Bouzid et al., Reference Bouzid, Halai, Jeffreys and Hunter2015; Ryan and Zahedi, Reference Ryan and Zahedi2019). Among nearly 51 valid Cryptosporidium species (Tůmová et al., Reference Tůmová, Ježková, Prediger, Holubová, Sak, Konečný, Květoňová, Hlásková, Rost, McEvoy, Xiao, Santín and Kváč2023), Cryptosporidium hominis and Cryptosporidium parvum represent the major causes of human cryptosporidiosis, whereas Cryptosporidium meleagridis, Cryptosporidium mortiferum, Cryptosporidium felis and Cryptosporidium canis are rare causative agents of zoonotic infections, with the latter being the most prevalent in dogs (Xu et al., Reference Xu, Jin, Wu, Li, Wang, Li, Feng and Xiao2016; Li et al., Reference Li, Ryan, Guo, Feng and Xiao2021; Alderisio et al., Reference Alderisio, Mergen, Moessner and Madison-Antenucci2023). Out of over 1200 microsporidian species described so far, Enterocytozoon bieneusi and Encephalitozoon genus, including Encephalitozoon intestinalis, Encephalitozoon cuniculi and Encephalitozoon hellem, represent the species causing human microsporidiosis (Didier et al., Reference Didier, Didier, Snowden and Shadduck2000), especially in persons with impaired immunity (Kicia et al., Reference Kicia, Wesolowska, Jakuszko, Kopacz, Sak, Květonova, Krajewska and Kváč2014, Reference Kicia, Wesolowska, Kopacz, Jakuszko, Sak, Květonová, Krajewska and Kváč2016). These species may also be detected in a broad range of other hosts (livestock, wildlife and domesticated animals) (Dengjel et al., Reference Dengjel, Zahler, Hermanns, Heinritzi, Spillmann, Thomschke, Löscher, Gothe and Rinder2001; Mathis et al., Reference Mathis, Weber and Deplazes2005). The phylogenetic analysis of E. bieneusi allows for distinction of various genotypes differing in a host specificity. Genotypes D, EbpC and type IV are characterized by the widest host range and are also most frequently reported in humans (Li et al., Reference Li, Feng and Santin2019). In turn, genotype PtEbIX seems to be restricted to canine host population (Li et al., Reference Li, Feng and Santin2019), although dogs can serve as a reservoir of many other zoonotic genotypes as well, including those most often reported in humans (Li et al., Reference Li, Feng and Xiao2020). Regarding the genus Encephalitozoon, the ability of these 3 species to inhabit a wide variety of organisms has been shown, with E. cuniculi having the widest host range, mainly among mammals and birds. Four E. cuniculi strains have been identified (I–IV); ‘canine’ strain III has been shown to cause high mortality in dogs, while the recently discovered ‘human’ strain IV has so far been documented in humans, cats and dogs. Although there appears to be some host preference in each strain, this specificity is not exact; humans have been found to be infected with all known strains (though rarely with strain III). In turn, E. hellem is the most common species among birds, while E. intestinalis is the most prevalent Encephalitozoon species in humans (Hinney et al., Reference Hinney, Sak, Joachim and Kváč2016).

The dispersive forms of the discussed pathogens are very resistant, and many species of wild and domesticated animals, as well as humans, may serve as their hosts which facilitates their spread and maintenance in the environment. Previous research on the occurrence of these species in central Europe, including Poland and the Czech Republic, shows different data depending on the population studied and the detection methods used. According to a review by Plutzer et al., the reported incidence of Cryptosporidium spp. and G. intestinalis per 100 000 inhabitants is 0.006 and 5.43 for Poland and 0.01 and 0.51 for the Czech Republic, respectively (Plutzer et al., Reference Plutzer, Lassen, Jokelainen, Djurković-Djaković, Kucsera, Dorbek-Kolin, Šoba, Sréter, Imre, Omeragić, Nikolić, Bobić, Živičnjak, Lučinger, Stefanović, Kučinar, Sroka, Deksne, Keidāne, Kváč, Hůzová and Karanis2018). In Poland, estimates of the prevalence of these 2 species in humans are available based on the results of research limited to specific population groups and regions (Plutzer et al., Reference Plutzer, Lassen, Jokelainen, Djurković-Djaković, Kucsera, Dorbek-Kolin, Šoba, Sréter, Imre, Omeragić, Nikolić, Bobić, Živičnjak, Lučinger, Stefanović, Kučinar, Sroka, Deksne, Keidāne, Kváč, Hůzová and Karanis2018). However, studies of Czech residents regarding the seroprevalence of Cryptosporidium spp. show the frequency of antibodies at the level of approximately 67–72% (Kozisek et al., Reference Kozisek, Craun, Cerovska, Pumann, Frost and Muller2008). In turn, the prevalence of Cryptosporidium spp. in dogs in Poland ranges from approximately 3.5 to 12.5% (Bajer and Bednarska, Reference Bajer and Bednarska2007; Piekara-Stępińska et al., Reference Piekara-Stępińska, Piekarska and Gorczykowski2021a), and G. intestinalis from 6 to 36%, with mainly canid-specific genotypes detected, which suggests that they do not represent an important source of Giardia infection for humans (Bajer and Bednarska, Reference Bajer and Bednarska2007; Piekarska et al., Reference Piekarska, Bajzert, Gorczykowski, Kantyka and Podkowik2016; Piekara-Stępińska et al., Reference Piekara-Stępińska, Piekarska, Gorczykowski and Bania2021b). A similar prevalence refers to dogs from the Czech Republic, especially those from animal shelters (Zemanová et al., Reference Zemanová, Husník and Svobodová2005; Dubná et al., Reference Dubná, Langrová, Nápravník, Jankovská, Vadlejch, Pekár and Fechtner2007). Microsporidia, however, occur in dogs at a low level, only a few per cent, which may nevertheless pose a risk for immunodeficient individuals, as zoonotic genotypes are often detected (Piekarska et al., Reference Piekarska, Kicia, Wesołowska, Kopacz, Gorczykowski, Szczepankiewicz, Kváč and Sak2017). Importantly, frequent exposure to microsporidia has been confirmed among immunocompetent people in the Czech Republic (Sak et al., Reference Sak, Brady, Pelikánová, Květoňová, Rost, Kostka, Tolarová, Hůzová and Kváč2011), while in studies conducted in Poland, up to 26% of tested immunocompromised individuals were found to be infected with at least 1 microsporidian species (Kicia et al., Reference Kicia, Wesolowska, Kopacz, Jakuszko, Sak, Květonová, Krajewska and Kváč2016, Reference Kicia, Szydłowicz, Cebulski, Jakuszko, Piesiak, Kowal, Sak, Krajewska, Hendrich, Kváč and Kopacz2019).

It has been previously shown that specific genotypes and assemblages of these enteric pathogens may be detected in both humans and animals (Xiao et al., Reference Xiao, Cama, Cabrera, Ortega, Pearson and Gilman2007; Soliman et al., Reference Soliman, Fuentes and Rubio2011; Karim et al., Reference Karim, Dong, Yu, Jian, Zhang, Wang, Zhang, Rume, Ning and Xiao2014a, Reference Karim, Wang, Dong, Zhang, Li, Zhang, Rume, Qi, Jian, Sun, Yang, Zou, Ning and Xiao2014b; Hinney et al., Reference Hinney, Sak, Joachim and Kváč2016), suggesting the possible zoonotic route of transmission. One of their sources may be shelter dogs, which often live in poor sanitary conditions and crowded spaces that favour the spread of such microorganisms (Raza et al., Reference Raza, Rand, Qamar, Jabbar and Kopp2018). Currently, there are 226 registered animal shelters in Poland (General Veterinary Inspectorate, 2023) and 248 in the Czech Republic (State Veterinary Administration, 2024). Therefore, the aim of the present study was to investigate the occurrence of Cryptosporidium spp., G. intestinalis, Encephalitozoon spp. and E. bieneusi in dogs living in animal shelters in central Europe (Poland and the Czech Republic) and to assess the host specificity and zoonotic potential of these organisms at the genotype level.

Materials and methods

Samples

Individual fresh fecal samples were collected from dogs in animal shelters in Poland and the Czech Republic. Samples were collected directly from the floor by study research staff immediately after defecation, with care taken to avoid sampling fecal material that came into contact with the ground (concrete surface, without contact with the soil). They were collected in the morning, before daily cleaning routinely performed in each shelter. Each sample was individually placed in a sterile tube with animal ID, refrigerated at 4°C without preservatives and transported to laboratory. None of the collected stool had an apparent diarrhoeal symptom at the time of sampling. Where possible, information about the animal, such as sex and age, was also recorded during material collection (see Supplementary Table 1). Control of intestinal protozoa in dogs and cats in both Polish and Czech shelters is carried out according to current ESCCAP guidelines (ESCCAP, 2018) – in all facilities, pyrantel and fenbendazole were routinely used once every 3 months as part of antiparasitic prophylaxis.

Molecular analysis

Stool samples were stored up to 2 months in 4°C without preservatives until DNA extraction. Initial homogenization of 200 mg of each stool sample was performed by bead disruption for 60 s at 5.5 m s−1 with 0.5 mm glass beads using a Precellys 24 Instrument (Bertin Technologies, Montigny le Bretonneux, France), followed by genomic DNA (gDNA) extraction using a GeneMATRIX Stool DNA Purification Kit (EurX, Gdańsk, Poland). Molecular detection was based on the nested polymerase chain reaction (PCR) protocols for the amplification of the chosen genes of E. bieneusi (ITS), Encephalitozoon spp. (ITS), Cryptosporidium spp. (18S rRNA) and G. intestinalis (TPI) (Didier et al., Reference Didier, Vossbrinck, Baker, Rogers, Bertucci and Shadduck1995; Katzwinkel-Wladarsch et al., Reference Katzwinkel-Wladarsch, Lieb, Helse, Löscher and Rinder1996; Xiao et al., Reference Xiao, Morgan, Limor, Escalante, Arrowood, Shulaw, Thompson, Fayer and Lal1999; Buckholt et al., Reference Buckholt, Lee and Tzipori2002; Sulaiman et al., Reference Sulaiman, Fayer, Bern, Gilman, Trout, Schantz, Das, Lal and Xiao2003). Additional PCRs amplifying selected loci were performed for subtyping in order to assess intra-species genetic diversity in the case of samples positive for Cryptosporidium spp. (partial 60-kDa glycoprotein gene – gp60) and G. intestinalis (β-giardin – BG and glutamate dehydrogenase – GDH) (see Supplementary Table 2) (Cacciò et al., Reference Cacciò, De Giacomo and Pozio2002, Reference Cacciò, Beck, Lalle, Marinculic and Pozio2008; Lalle et al., Reference Lalle, Jimenez-Cardosa, Cacciò and Pozio2005; Jiang et al., Reference Jiang, Roellig, Guo, Li, Feng and Xiao2021). Each PCR contained 0.25–2.0 μL of DNA, 200 μm each of deoxynucleoside triphosphate (dNTP), 1× PCR buffer (DreamTaq™ Green Buffer, ThermoFisher Scientific, Waltham, MA, USA), 3.0 mm MgCl2, 0.125 U of Taq polymerase (ThermoFisher Scientific), 10 μg of bovine serum albumin (BSA) and 200 nm of each primer in a total of 20–25 μL reaction. The reactions were performed in a C1000 Bio-Rad thermocycler, with an initial hot start (94°C for 5 min) and a final extension (72°C for 10 min), according to the conditions described in Supplementary Table 2. An aliquot of primary PCR was used as a template for the secondary PCR. Its conditions were identical to the primary PCR, except that BSA was not added to the secondary reaction. Negative (molecular grade water) and positive controls (DNA extracted from E. bieneusi genotype CZ3, E. cuniculi genotype III spores, Cryptosporidium serpentis oocysts or G. intestinalis assemblage F cysts) were included in each PCR amplification. Secondary PCR products were electrophoresed on a 1% agarose gel containing 0.2 mg mL−1 Midori Green DNA stain in TAE buffer at 75 V for approximately 1 h. Bands of the predicted sizes were visualized using a UV light source, cut from the gel, extracted using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) and sequenced bi-directionally by a company offering this service commercially (Genomed S.A., Warsaw, Poland). The nucleotide sequences obtained were processed using Chromas Pro 2.4.1 software (Technelysium, Pty, Ltd., South Brisbane, Australia). Subsequently, BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to verify the identity of the sequences. The edited and aligned sequences were further processed using BioEdit v.7.0.5 (Hall, Reference Hall1999). To align the obtained sequences with reference sequences from GenBank, the online server MAFFT version 7 was used (http://mafft.cbrc.jp/alignment/software). The best model for DNA/protein phylogeny for each alignment was selected based on the Bayesian information criterion in MEGA 7 (Kumar et al., Reference Kumar, Stecher and Tamura2016). Tamura's 3-parameter model + G + I was used for the alignments. The maximum likelihood (ML) approach was carried out in MEGA7 software. Bootstrap support was calculated based on 1000 replications to evaluate the robustness of tree branching. Finally, the resulting trees were visualized using Corel Draw X7 software (https://www.corel.draw.com). Representative nucleotide sequences of all loci used as markers for subtyping of isolates obtained in the current study were deposited in GenBank with the accession numbers OR791083, OR791084, OR791770, OR791659, OR791771–OR791785, OR807726 and OR807727.

Statistical analysis

Statistical analysis was performed using χ 2 or Fisher's tests to compare the frequency of occurrence of the tested pathogens between Polish and Czech shelters (Statistica software, TIBCO Software Inc., USA). A P < 0.05 was considered significant.

Results

A total of 187 apparently healthy dogs from 10 shelters (Fig. 1), 5 in Poland (101 dogs) and 5 in the Czech Republic (86 dogs), were studied (Table 1). Specific DNA of targeted parasites was detected in 50 of 187 animals (26.7%), with higher occurrence observed in Polish than in Czech animals (32.6 vs 24.4%, χ 2 = 1.0254; P = 0.3112; Table 1). Most of the detected infections were monoinfections; only 2 dogs (ID 2966_CZ.1 and 3031_CZ.5) had a coinfection, E. bieneusi and G. intestinalis. Giardia intestinalis (29 dogs, 15.5%) followed by E. bieneusi (19 dogs, 10.2%) were the most frequently detected parasites, whereas each Encephalitozoon spp. and Cryptosporidium spp. were found in 2 dogs (Table 1). Overall, there was a significant trend towards more frequent occurrence of G. intestinalis in Polish (21.8%) vs Czech animals (8.1%, χ 2 = 6.5979; P = 0.0102), while E. bieneusi was seen more often in dogs from Czech shelters (16.3%) than in Polish ones (4.9%, χ 2 = 6.5305; P = 0.0106). Since detailed demographic data were obtained only from a small number of individuals, they were not subjected to statistical analysis.

Figure 1. Schematic arrangement of shelters in Poland (P.1–P.5) and the Czech Republic (CZ.1–CZ.5) from which samples were collected.

Table 1. Occurrence of Cryptosporidium spp., Giardia intestinalis, Encephalitozoon cuniculi and Enterocytozoon bieneusi in individual dogs kept in animal shelters in Poland and the Czech Republic

The results of genotyping of all pathogens performed in this study are presented in Table 2. Phylogeny analysis of partial sequences of 18S rRNA of Cryptosporidium showed the presence of C. canis identical to C. canis dog genotype (GenBank Acc. No. AB120909) in isolate 2806_P.1 and in a Cryptosporidium sp. isolate 2735_P.2, phylogenetically clustered near the gastric Cryptosporidium spp. (Fig. 2). Cryptosporidium sp. isolate 2735_P.2 differed from Cryptosporidium proliferans in 3 single-nucleotide polymorphisms (SNPs) with 99.6% sequence identity. Based on C. canis gp60 locus subtyping, isolate 2806_P.1 was assigned to the XXe family (Fig. 3). Genotype II, determined at ITS sequences, was detected in both dogs positive for E. cuniculi (Table 2; phylogeny is not shown). Phylogenetic analysis of Giardia showed different results in 12 isolates, depending on the marker used (Table 2). Subtyping based on TPI gene revealed the presence of only assemblage C in all samples examined (Fig. 4), while for BG (Fig. 5) and GDH (Fig. 6) loci the presence of both C and D assemblages has been shown. Subtyping of BG and GDH failed in 2 and 3 isolates, respectively. With the exception of genotype D, which was detected in dog 2699_P.5, all other E. bieneusi sequences were identical to the PtEbIX genotype (Fig. 7).

Table 2. Results of genotyping of Cryptosporidium spp., G. intestinalis, E. cuniculi and E. bieneusi in all tested samples

Accession numbers in square brackets indicate the isolates deposited in GenBank as representative nucleotide sequences derived from the present study.

TPI, triosephosphate isomerase; BG, β-giardin; GDH, glutamate dehydrogenase; 18S rRNA, small ribosomal subunit rRNA; gp60, 60-kDa glycoprotein; ITS, internal transcribed spacer region of rRNA.

a Cryptosporidium sp. isolate 2735_P.2, phylogenetically clustered near the gastric Cryptosporidium spp., differed from C. proliferans in 3 SNPs with 99.6% sequence identity of the 18S rRNA region.

b N/A, assemblage not available (subtyping failed).

Figure 2. Phylogenetic relationships between Cryptosporidium spp. detected in dogs in this study (highlighted in green) and other Cryptosporidium available in GenBank using an ML analysis of partial sequences of 18S rRNA (sequence alignment length: 820 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2806) followed by region and location (P.1, Poland location 1).

Figure 3. Phylogenetic relationship between Cryptosporidium canis detected in 1 dog in this study (highlighted in green) and other C. canis available in GenBank using an ML analysis of a region of gp60 gene (sequence alignment length: 540 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (2806) followed by region and location (P.1, Poland location 1).

Figure 4. Phylogenetic relationships between Giardia intestinalis assemblages detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other G. intestinalis assemblages available in GenBank using an ML analysis of a region of TPI gene (sequence alignment length: 467 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2966) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Figure 5. Phylogenetic relationships between G. intestinalis assemblages detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other G. intestinalis assemblages available in GenBank using an ML analysis of a region of BG gene (sequence alignment length: 820 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2966) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Figure 6. Phylogenetic relationships between G. intestinalis assemblages detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other G. intestinalis assemblages available in GenBank using an ML analysis of a region of GDH gene (sequence alignment length: 439 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2966) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Figure 7. Phylogenetic relationships between Enterocytozoon bieneusi genotypes detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other E. bieneusi genotypes available in GenBank using an ML analysis of ITS region of rRNA gene (sequence alignment length: 309 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2973) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Discussion

In the present study, the screening of fecal samples collected from shelter dogs in central Europe was performed in order to analyse the prevalence of zoonotic unicellular pathogens (Cryptosporidium spp., G. intestinalis, E. bieneusi and Encephalitozoon spp.) at the molecular level. Over a quarter of the tested dogs were carriers of at least one of the studied pathogens, among which the most often observed was G. intestinalis – one of the most common intestinal parasites infecting humans and animals (Kváč et al., Reference Kváč, Hofmannová, Ortega, Holubová, Horčičková, Kicia, Hlásková, Květoňová, Sak and McEvoy2017). Nevertheless, its prevalence in other European canine populations, including shelter dogs, has shown to be even higher [27–36.5% in various regions of Spain (Gil et al., Reference Gil, Cano, de Lucio, Bailo, de Mingo, Cardona, Fernández-Basterra, Aramburu-Aguirre, López-Molina and Carmena2017; Adell-Aledón et al., Reference Adell-Aledón, Köster, de Lucio, Puente, Hernández-de-Mingo, Sánchez-Thevenet, Dea-Ayuela and Carmena2018; Remesar et al., Reference Remesar, García-Dios, Calabuig, Prieto, Díaz-Cao, López-Lorenzo, López, Fernández, Morrondo, Panadero and Díaz2022), 33.8% in Portugal (Pereira et al., Reference Pereira, Teixeira, Sousa, Parreira, Campino, Meireles and Maia2021), over 45% in Serbia (Sommer et al., Reference Sommer, Zdravković, Vasić, Grimm and Silaghi2017), while in central Italy this value ranged from about 7% (Scaramozzino et al., Reference Scaramozzino, Carvelli, Iacoponi and De Liberato2018) to 41% (Agresti et al., Reference Agresti, Berrilli, Maestrini, Procesi, Loretti, Vonci and Perrucci2022)]. Generally, such a high prevalence is most likely due to the simple and direct life cycle of Giardia, with easily spread dispersive forms excreted in feces, which facilitates transmission in highly dense populations, such as those found in animal shelters. Enterocytozoon bieneusi was also found to be a common pathogen in dogs in the present study, with an overall prevalence of 10.2%, which agrees with previous reports considering European dogs, with infection rates ranging between 4.9 and 11.7% (Mathis et al., Reference Mathis, Weber and Deplazes2005; Santín and Fayer, Reference Santín and Fayer2011; Piekarska et al., Reference Piekarska, Kicia, Wesołowska, Kopacz, Gorczykowski, Szczepankiewicz, Kváč and Sak2017). In turn, considerably low prevalences were observed for Encephalitozoon ssp. and Cryptosporidium spp. (~1% for both), comparably to previous reports regarding canine populations: 0–2.4% for Encephalitozoon spp. (Piekarska et al., Reference Piekarska, Kicia, Wesołowska, Kopacz, Gorczykowski, Szczepankiewicz, Kváč and Sak2017; Delrobaei et al., Reference Delrobaei, Jamshidi, Shayan, Ebrahimzade, Ashrafi Tamai, Rezaeian and Mirjalali2019) and 0.6–4.9% for Cryptosporidium spp. (Giangaspero et al., Reference Giangaspero, Iorio, Paoletti, Traversa and Capelli2006; Simonato et al., Reference Simonato, Frangipane di Regalbono, Cassini, Traversa, Tessarin, Di Cesare and Pietrobelli2017; Yu et al., Reference Yu, Ruan, Zhou, Chen, Zhang, Wang, Zhu and Yu2018; Piekara-Stępińska et al., Reference Piekara-Stępińska, Piekarska and Gorczykowski2021a), although in 1 German study the Cryptosporidium prevalence was as high as 10% (Murnik et al., Reference Murnik, Daugschies and Delling2022). Notwithstanding, the true prevalence of these pathogens among healthy hosts may, in fact, be higher as their forms are excreted periodically and irregularly, which may be overlooked with a single sampling (Sak et al., Reference Sak, Kašičková, Kváč, Květoňová and Ditrich2010). It would therefore be recommended to collect samples several times from the same animals, which may prove difficult due to the conditions specific to the shelters, such as the rotation of animals or irregular hours of cleaning the excrement. Nevertheless, the fact that the demonstrated prevalence of pathogens such as G. intestinalis was high even with only 1 sampling underscores the importance of their likely distribution in the population and the wide reservoir of the pathogen. Differences in the frequencies of the studied species between Polish and Czech dogs, as well as the higher prevalence of specific pathogens in individual facilities, may be related to some specific conditions typical of a particular location.

Sequence analyses of detected pathogens showed that most infections involved dog-specific genotypes or species, the transmission of which may be favoured by intensive contact among large numbers of dogs living together. In the case of 12 isolates, assignment to the appropriate G. intestinalis assemblage was difficult because subtyping results varied depending on the locus used. Similar discrepancies have been reported in previous studies (Read et al., Reference Read, Monis and Andrew Thompson2004; Traub et al., Reference Traub, Monis, Robertson, Irwin, Mencke and Thompson2004), emphasizing the importance of using multilocus genotyping in the molecular analysis of Giardia diversity. Nevertheless, all G. intestinalis-positive samples harboured assemblage C or D, which have a strong host specificity for dogs and other canines (Feng and Xiao, Reference Feng and Xiao2011). These assemblages were also found to be highly prevalent in different dog populations in Europe (Simonato et al., Reference Simonato, Frangipane di Regalbono, Cassini, Traversa, Beraldo, Tessarin and Pietrobelli2015; Adell-Aledón et al., Reference Adell-Aledón, Köster, de Lucio, Puente, Hernández-de-Mingo, Sánchez-Thevenet, Dea-Ayuela and Carmena2018; Pereira et al., Reference Pereira, Teixeira, Sousa, Parreira, Campino, Meireles and Maia2021) and although sporadically they have been reported in humans as well (Broglia et al., Reference Broglia, Weitzel, Harms, Cacció and Nöckler2013; Liu et al., Reference Liu, Shen, Yin, Yuan, Jiang, Xu, Pan, Hu and Cao2014; Villamizar et al., Reference Villamizar, Higuera, Herrera, Vasquez-A, Buitron, Muñoz, Gonzalez-C, Lopez, Giraldo and Ramírez2019), their zoonotic relevance seems to be low and limited to individuals at risk, for instance, children or immunocompromised persons. Likewise, cases of C. canis colonization in humans were described in individuals at increased risk (children, HIV-infected adults) and immunocompetent people as well (Learmonth et al., Reference Learmonth, Ionas, Ebbett and Kwan2004; Gatei et al., Reference Gatei, Das, Dutta, Sen, Cama, Lal and Xiao2007; Feng et al., Reference Feng, Wang, Duan, Gomez-Puerta, Zhang, Zhao, Hu, Zhang and Xiao2012; Liao et al., Reference Liao, Lin, Sun, Qi, Lv, Wu, Li, Hu, Yu, Cai, Xiao, Sun and Li2020). However, due to the relatively transient nature of these infections in humans, dogs do not seem to represent an important source of cryptosporidiosis for people (Villamizar et al., Reference Villamizar, Higuera, Herrera, Vasquez-A, Buitron, Muñoz, Gonzalez-C, Lopez, Giraldo and Ramírez2019; Liao et al., Reference Liao, Lin, Sun, Qi, Lv, Wu, Li, Hu, Yu, Cai, Xiao, Sun and Li2020). To date, 9 families of C. canis subtypes (XXa–XXi) have been identified based on gp60 locus subtyping, occurring not only in canids, but also in minks, foxes and humans (Jiang et al., Reference Jiang, Roellig, Guo, Li, Feng and Xiao2021; Murnik et al., Reference Murnik, Daugschies and Delling2022; Wang et al., Reference Wang, Wei, Cao, Wu, Zhao, Guo, Xiao, Feng and Li2022). According to the study of Jiang et al., the zoonotic potential may concern XXa family, detected in both dog and human samples (Jiang et al., Reference Jiang, Roellig, Guo, Li, Feng and Xiao2021). In our study, analysis of the C. canis gp60 locus in the 2806_P.1 isolate revealed that it belongs to the XXe family (Fig. 3), which was also the most prevalent among dogs in the report from Germany (Murnik et al., Reference Murnik, Daugschies and Delling2022). In turn, the newly detected Cryptosporidium sp. isolate 2735_P.2 also does not pose a significant risk to humans and probably not to dogs as well. An incidental infection/contamination was likely caused by rodent feces. Cryptosporidium sp. isolate 2735_P.2 is closely related to Cryptosporidium muris and C. proliferans, whose hosts are rodents. However, the phylogenetic position is not related to host specificity, and therefore another host cannot be excluded (Kváč et al., Reference Kváč, Vlnatá, Ježková, Horčičková, Konečný, Hlásková, McEvoy and Sak2018). To summarize the subtyping results, as in previous studies (de Lucio et al., Reference de Lucio, Bailo, Aguilera, Cardona, Fernández-Crespo and Carmena2017; Rehbein et al., Reference Rehbein, Klotz, Ignatius, Müller, Aebischer and Kohn2019), zoonotic transmission of giardiasis or cryptosporidiosis between dogs and humans is most likely a rare event.

All E. bieneusi isolates detected in the studied dogs, except for 1 clustering to genotype D, were identical to E. bieneusi genotype PtEbIX. This genotype appears to be specific to dogs; to date, it has been detected almost exclusively in dogs and sporadically in wolves, cats and swans (Santín et al., Reference Santín, Vecino and Fayer2008; Abe et al., Reference Abe, Kimata and Iseki2009; Santín and Fayer, Reference Santín and Fayer2011; Mori et al., Reference Mori, Mahittikorn, Thammasonthijarern, Chaisiri, Rojekittikhun and Sukthana2013; Karim et al., Reference Karim, Dong, Yu, Jian, Zhang, Wang, Zhang, Rume, Ning and Xiao2014a; Piekarska et al., Reference Piekarska, Kicia, Wesołowska, Kopacz, Gorczykowski, Szczepankiewicz, Kváč and Sak2017; Kváč et al., Reference Kváč, Myšková, Holubová, Kellnerová, Kicia, Rajský, McEvoy, Feng, Hanzal and Sak2021). In turn, both E. bieneusi genotype D and E. cuniculi genotype II detected in the present study have been reported in a broad range of hosts so far, including humans (Li et al., Reference Li, Xiao, Wang, Zhao, Zhao, Duan, Guo, Liu and Feng2012; Kváč et al., Reference Kváč, Hofmannová, Ortega, Holubová, Horčičková, Kicia, Hlásková, Květoňová, Sak and McEvoy2017; Piekarska et al., Reference Piekarska, Kicia, Wesołowska, Kopacz, Gorczykowski, Szczepankiewicz, Kváč and Sak2017; Delrobaei et al., Reference Delrobaei, Jamshidi, Shayan, Ebrahimzade, Ashrafi Tamai, Rezaeian and Mirjalali2019). Observation of microsporidian genotypes with a human-infection capacity in companion animals suggests that pets may be of importance as one of the potential sources of infection. However, the presented results do not indicate that dogs in shelters in Poland and the Czech Republic represent a significant source of zoonotic species and genotypes of the studied parasites for humans.

Our study had some limitations. Firstly, due to its screening nature, a detailed analysis in the context of the demographic data of the tested dogs or the drugs used was not possible to conduct. Moreover, the study groups differed in size – in Poland it was possible to collect material from a larger number of animals than in the Czech Republic, which may have an impact on the differences in prevalence.

This study included clinically healthy animals without signs of intestinal infection, yet dispersive forms of potentially pathogenic and infectious organisms were observed. It should also be borne in mind that asymptomatic hosts could shed cysts, oocysts or spores occasionally and irregularly, and thus their screening with multiple sampling could increase the real observed prevalence (Sak et al., Reference Sak, Kašičková, Kváč, Květoňová and Ditrich2010). Nevertheless, since infections of the studied pathogens in dogs can often be asymptomatic, they may not be detected by routine veterinary examinations, and their occurrence in companion animals may be underestimated. On the contrary, the majority of species and genotypes observed in canine samples herein are not commonly associated with human infections, and aforesaid transmission routes seem to be rare. The exceptions are genotypes D (E. bieneusi) and II (E. cuniculi) observed in the present study, whose zoonotic potential should be emphasized due to their occurrence in a wide range of different hosts (Hinney et al., Reference Hinney, Sak, Joachim and Kváč2016; Li et al., Reference Li, Feng and Santin2019, Reference Li, Feng and Xiao2020). Despite the low likelihood of transmission of the studied pathogens and due to the fact that they mainly affect immunosuppressed individuals, in whom the consequences of opportunistic infections may be life-threatening, the awareness among new dog owners is recommended, especially those with various levels of immunosuppression, on the relevance of diagnosing and treating zoonotic diseases.

Supplementary material

The supplementary material for this article can be found at https://doi.org/10.1017/S003118202400009X

Data availability statement

The data that support the findings of this study are available from the corresponding author, MS, upon reasonable request.

Acknowledgements

The authors acknowledge the staff of dog shelters for their assistance in sample collection and the technician staff of the Laboratory of Veterinary and Medical Protistology, Biology Centre CAS, for sample processing during this study.

Author's contribution

M. S., M. Ki. and Ż. Z. conceived and designed the study. M. S., M. Ki., Ż. Z., M. Ka., M. Kv., B. S. and N. H. conducted data gathering. M. S., M. Ki., Ż. Z., M. Ka., B. Ł. and A. L. prepared the samples and conducted molecular analysis. M. S. conducted statistical analyses. M. Kv. prepared phylogenetic analyses. M. S., M. Ki. and M. Kv. wrote the article.

Financial support

This work was supported with a subvention by the Polish Ministry of Health (M. S., grant number SUBK.A060.23.027 from the IT Simple system of Wroclaw Medical University) and the Grant Agency of the Czech Republic (M. Kv., GACR 21-23773S and B. S. 23-06571S).

Competing interests

None.

Ethical standards

The collection of samples carried out in a non-invasive way, without interfering with the organism of the animals included in the study.

References

Abe, N, Kimata, I and Iseki, M (2009) Molecular evidence of Enterocytozoon bieneusi in Japan. Journal of Veterinary Medical Science 71, 217219.CrossRefGoogle ScholarPubMed
Adell-Aledón, M, Köster, PC, de Lucio, A, Puente, P, Hernández-de-Mingo, M, Sánchez-Thevenet, P, Dea-Ayuela, MA and Carmena, D (2018) Occurrence and molecular epidemiology of Giardia duodenalis infection in dog populations in eastern Spain. BMC Veterinary Research 14, 111.10.1186/s12917-018-1353-zCrossRefGoogle ScholarPubMed
Agresti, A, Berrilli, F, Maestrini, M, Procesi, IG, Loretti, E, Vonci, N and Perrucci, S (2022) Prevalence, risk factors and genotypes of Giardia duodenalis in sheltered dogs in Tuscany (Central Italy). Pathogens (Basel, Switzerland) 11, 19.Google Scholar
Alderisio, KA, Mergen, K, Moessner, H and Madison-Antenucci, S (2023) Identification and evaluation of Cryptosporidium species from New York City cases of cryptosporidiosis (2015 to 2018): a watershed perspective. Microbiology Spectrum 11, 18.CrossRefGoogle ScholarPubMed
Bajer, A and Bednarska, M (2007) Cryptosporidium spp. and Giardia spp. infections in sled dogs. Medycyna Weterynaryjna 63, 681687.Google Scholar
Bouzid, M, Halai, K, Jeffreys, D and Hunter, PR (2015) The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples. Veterinary Parasitology 207, 181202.CrossRefGoogle Scholar
Broglia, A, Weitzel, T, Harms, G, Cacció, SM and Nöckler, K (2013) Molecular typing of Giardia duodenalis isolates from German travellers. Parasitology Research 112, 34493456.10.1007/s00436-013-3524-yCrossRefGoogle ScholarPubMed
Buckholt, MA, Lee, JH and Tzipori, S (2002) Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. Applied and Environmental Microbiology 68, 25952599.CrossRefGoogle Scholar
Cacciò, SM, De Giacomo, M and Pozio, E (2002) Sequence analysis of the β-giardin gene and development of a polymerase chain reaction–restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. International Journal for Parasitology 32, 10231030.CrossRefGoogle ScholarPubMed
Cacciò, SM, Beck, R, Lalle, M, Marinculic, A and Pozio, E (2008) Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. International Journal for Parasitology 38, 15231531.CrossRefGoogle ScholarPubMed
Delrobaei, M, Jamshidi, S, Shayan, P, Ebrahimzade, E, Ashrafi Tamai, I, Rezaeian, M and Mirjalali, H (2019) Molecular detection and genotyping of intestinal microsporidia from stray dogs in Iran. Iranian Journal of Parasitology 14, 159166.Google ScholarPubMed
de Lucio, A, Bailo, B, Aguilera, M, Cardona, GA, Fernández-Crespo, JC and Carmena, D (2017) No molecular epidemiological evidence supporting household transmission of zoonotic Giardia duodenalis and Cryptosporidium spp. from pet dogs and cats in the province of Álava, northern Spain. Acta Tropica 170, 4856.CrossRefGoogle ScholarPubMed
Dengjel, B, Zahler, M, Hermanns, W, Heinritzi, K, Spillmann, T, Thomschke, A, Löscher, T, Gothe, R and Rinder, H (2001) Zoonotic potential of Enterocytozoon bieneusi. Journal of Clinical Microbiology 39, 44954499.CrossRefGoogle ScholarPubMed
Didier, ES, Vossbrinck, CR, Baker, MD, Rogers, LB, Bertucci, DC and Shadduck, JA (1995) Identification and characterization of three Encephalitozoon cuniculi strains. Parasitology 111, 411421.CrossRefGoogle ScholarPubMed
Didier, ES, Didier, PJ, Snowden, KF and Shadduck, JA (2000) Microsporidiosis in mammals. Microbes and Infection 2, 709720.CrossRefGoogle ScholarPubMed
Dubná, S, Langrová, I, Nápravník, J, Jankovská, I, Vadlejch, J, Pekár, S and Fechtner, J (2007) The prevalence of intestinal parasites in dogs from Prague, rural areas, and shelters of the Czech Republic. Veterinary Parasitology 145, 120128.CrossRefGoogle ScholarPubMed
European Scientific Counsel Companion Animal Parasites (ESCCAP) (2018) Control of intestinal protozoa in dogs and cats. Available at https://www.esccap.org/guidelines/gl6/ (accessed 13 January 2024).Google Scholar
Feng, Y and Xiao, L (2011) Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clinical Microbiology Reviews 24, 110140.CrossRefGoogle ScholarPubMed
Feng, Y, Wang, L, Duan, L, Gomez-Puerta, LA, Zhang, L, Zhao, X, Hu, J, Zhang, N and Xiao, L (2012) Extended outbreak of cryptosporidiosis in a pediatric hospital, China. Emerging Infectious Diseases 18, 312314.CrossRefGoogle Scholar
Gatei, W, Das, P, Dutta, P, Sen, A, Cama, V, Lal, AA and Xiao, L (2007) Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata, India. Infection, Genetics and Evolution 7, 197205.CrossRefGoogle ScholarPubMed
General Veterinary Inspectorate (2023) Annual report on visits to animal shelters for 2022. Available at https://www.wetgiw.gov.pl/nadzor-weterynaryjny/schroniska-dla-bezdomnych-zwierzat (accessed 13 January 2024).Google Scholar
Giangaspero, A, Iorio, R, Paoletti, B, Traversa, D and Capelli, G (2006) Molecular evidence for Cryptosporidium infection in dogs in central Italy. Parasitology Research 99, 297299.CrossRefGoogle ScholarPubMed
Gil, H, Cano, L, de Lucio, A, Bailo, B, de Mingo, MH, Cardona, GA, Fernández-Basterra, JA, Aramburu-Aguirre, J, López-Molina, N and Carmena, D (2017) Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in northern Spain. Infection, Genetics and Evolution 50, 6269.CrossRefGoogle ScholarPubMed
Hall, T A (1999) BioEdit: a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 95–98.Google Scholar
Hinney, B, Sak, B, Joachim, A and Kváč, M (2016) More than a rabbit's tale – Encephalitozoon spp. in wild mammals and birds. International Journal for Parasitology: Parasites and Wildlife 5, 7687.Google ScholarPubMed
Jiang, W, Roellig, DM, Guo, Y, Li, N, Feng, Y and Xiao, L (2021) Development of a subtyping tool for zoonotic pathogen Cryptosporidium canis. Journal of Clinical Microbiology 59, 18.CrossRefGoogle ScholarPubMed
Karim, MR, Dong, H, Yu, F, Jian, F, Zhang, L, Wang, R, Zhang, S, Rume, FI, Ning, C and Xiao, L (2014a) Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications. Journal of Clinical Microbiology 52, 32973302.CrossRefGoogle Scholar
Karim, MR, Wang, R, Dong, H, Zhang, L, Li, J, Zhang, S, Rume, FI, Qi, M, Jian, F, Sun, M, Yang, G, Zou, F, Ning, C and Xiao, L (2014b) Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Applied and Environmental Microbiology 80, 18931898.CrossRefGoogle ScholarPubMed
Katzwinkel-Wladarsch, S, Lieb, M, Helse, W, Löscher, T and Rinder, H (1996) Direct amplification and species determination of microsporidian DNA from stool specimens. Tropical Medicine & International Health 1, 373378.CrossRefGoogle ScholarPubMed
Kicia, M, Wesolowska, M, Jakuszko, K, Kopacz, Z, Sak, B, Květonova, D, Krajewska, M and Kváč, M (2014) Concurrent infection of the urinary tract with Encephalitozoon cuniculi and Enterocytozoon bieneusi in a renal transplant recipient. Journal of Clinical Microbiology 52, 17801782.CrossRefGoogle Scholar
Kicia, M, Wesolowska, M, Kopacz, Z, Jakuszko, K, Sak, B, Květonová, D, Krajewska, M and Kváč, M (2016) Prevalence and molecular characteristics of urinary and intestinal microsporidia infections in renal transplant recipients. Clinical Microbiology and Infection 22, 462.e5462.e9.CrossRefGoogle ScholarPubMed
Kicia, M, Szydłowicz, M, Cebulski, K, Jakuszko, K, Piesiak, P, Kowal, A, Sak, B, Krajewska, M, Hendrich, AB, Kváč, M and Kopacz, Ż (2019) Symptomatic respiratory Encephalitozoon cuniculi infection in renal transplant recipients. International Journal of Infectious Diseases 79, 2125.CrossRefGoogle ScholarPubMed
Kozisek, F, Craun, GF, Cerovska, L, Pumann, P, Frost, F and Muller, T (2008) Serological responses to Cryptosporidium-specific antigens in Czech populations with different water sources. Epidemiology and Infection 136, 279286.CrossRefGoogle ScholarPubMed
Kumar, S, Stecher, G and Tamura, K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33, 18701874.CrossRefGoogle ScholarPubMed
Kváč, M, Hofmannová, L, Ortega, Y, Holubová, N, Horčičková, M, Kicia, M, Hlásková, L, Květoňová, D, Sak, B and McEvoy, J (2017) Stray cats are more frequently infected with zoonotic protists than pet cats. Folia Parasitologica 64, 034. doi: 10.14411/fp.2017.034CrossRefGoogle ScholarPubMed
Kváč, M, Vlnatá, G, Ježková, J, Horčičková, M, Konečný, R, Hlásková, L, McEvoy, J and Sak, B (2018) Cryptosporidium occultus sp. n. (Apicomplexa: Cryptosporidiidae) in rats. European Journal of Protistology 63, 96104.CrossRefGoogle Scholar
Kváč, M, Myšková, E, Holubová, N, Kellnerová, K, Kicia, M, Rajský, D, McEvoy, J, Feng, Y, Hanzal, V and Sak, B (2021) Occurrence and genetic diversity of Cryptosporidium spp. in wild foxes, wolves, jackals, and bears in central Europe. Folia Parasitologica 68, 18.CrossRefGoogle ScholarPubMed
Lalle, M, Jimenez-Cardosa, E, Cacciò, SM and Pozio, E (2005) Genotyping of Giardia duodenalis from humans and dogs from Mexico using a β-giardin nested polymerase chain reaction assay. Journal of Parasitology 91, 203205.CrossRefGoogle ScholarPubMed
Learmonth, JJ, Ionas, G, Ebbett, KA and Kwan, ES (2004) Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand. Applied and Environmental Microbiology 70, 39733978.CrossRefGoogle ScholarPubMed
Li, N, Xiao, L, Wang, L, Zhao, S, Zhao, X, Duan, L, Guo, M, Liu, L and Feng, Y (2012) Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Neglected Tropical Diseases 6, e1809.CrossRefGoogle ScholarPubMed
Li, W, Feng, Y and Santin, M (2019) Host specificity of Enterocytozoon bieneusi and public health implications. Trends in Parasitology 35, 436451.CrossRefGoogle ScholarPubMed
Li, W, Feng, Y and Xiao, L (2020) Diagnosis and molecular typing of Enterocytozoon bieneusi: the significant role of domestic animals in transmission of human microsporidiosis. Research in Veterinary Science 133, 251261.CrossRefGoogle ScholarPubMed
Li, J, Ryan, U, Guo, Y, Feng, Y and Xiao, L (2021) Advances in molecular epidemiology of cryptosporidiosis in dogs and cats. International Journal for Parasitology 51, 787795.CrossRefGoogle ScholarPubMed
Liao, S, Lin, X, Sun, Y, Qi, N, Lv, M, Wu, C, Li, J, Hu, J, Yu, L, Cai, H, Xiao, W, Sun, M and Li, G (2020) Occurrence and genotypes of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in household, shelter, breeding, and pet market dogs in Guangzhou, southern China. Scientific Reports 10, 111.CrossRefGoogle ScholarPubMed
Liu, H, Shen, Y, Yin, J, Yuan, Z, Jiang, Y, Xu, Y, Pan, W, Hu, Y and Cao, J (2014) Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China. BMC Infectious Diseases 14, 16.CrossRefGoogle ScholarPubMed
Mathis, A, Weber, R and Deplazes, P (2005) Zoonotic potential of the microsporidia. Clinical Microbiology Reviews 18, 423445.CrossRefGoogle ScholarPubMed
Mori, H, Mahittikorn, A, Thammasonthijarern, N, Chaisiri, K, Rojekittikhun, W and Sukthana, Y (2013) Presence of zoonotic Enterocytozoon bieneusi in cats in a temple in central Thailand. Veterinary Parasitology 197, 696701.CrossRefGoogle Scholar
Murnik, L-C, Daugschies, A and Delling, C (2022) Cryptosporidium infection in young dogs from Germany. Parasitology Research 121, 29852993.CrossRefGoogle ScholarPubMed
Pereira, A, Teixeira, J, Sousa, S, Parreira, R, Campino, L, Meireles, J and Maia, C (2021) Giardia duodenalis infection in dogs from the metropolitan area of Lisbon, Portugal: prevalence, genotyping and associated risk factors. Journal of Parasitic Diseases 45, 372379.CrossRefGoogle ScholarPubMed
Piekara-Stępińska, A, Piekarska, J and Gorczykowski, M (2021a) Cryptosporidium spp. in dogs and cats in Poland. Annals of Agricultural and Environmental Medicine 28, 345347.CrossRefGoogle ScholarPubMed
Piekara-Stępińska, A, Piekarska, J, Gorczykowski, M and Bania, J (2021b) Genotypes of Giardia duodenalis in household dogs and cats from Poland. Acta Parasitologica 66, 428435.CrossRefGoogle ScholarPubMed
Piekarska, J, Bajzert, J, Gorczykowski, M, Kantyka, M and Podkowik, M (2016) Molecular identification of Giardia duodenalis isolates from domestic dogs and cats in Wroclaw, Poland. Annals of Agricultural and Environmental Medicine 23, 410415.CrossRefGoogle ScholarPubMed
Piekarska, J, Kicia, M, Wesołowska, M, Kopacz, Ż, Gorczykowski, M, Szczepankiewicz, B, Kváč, M and Sak, B (2017) Zoonotic microsporidia in dogs and cats in Poland. Veterinary Parasitology 246, 108111.CrossRefGoogle ScholarPubMed
Plutzer, J, Lassen, B, Jokelainen, P, Djurković-Djaković, O, Kucsera, I, Dorbek-Kolin, E, Šoba, B, Sréter, T, Imre, K, Omeragić, J, Nikolić, A, Bobić, B, Živičnjak, T, Lučinger, S, Stefanović, LL, Kučinar, J, Sroka, J, Deksne, G, Keidāne, D, Kváč, M, Hůzová, Z and Karanis, P (2018) Review of Cryptosporidium and Giardia in the eastern part of Europe, 2016. Eurosurveillance 23, 123.CrossRefGoogle ScholarPubMed
Raza, A, Rand, J, Qamar, AG, Jabbar, A and Kopp, S (2018) Gastrointestinal parasites in shelter dogs: occurrence, pathology, treatment and risk to shelter workers. Animals 8, 108. doi: 10.3390/ani8070108CrossRefGoogle ScholarPubMed
Read, CM, Monis, PT and Andrew Thompson, RC (2004) Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infection, Genetics and Evolution 4, 125130.CrossRefGoogle ScholarPubMed
Rehbein, S, Klotz, C, Ignatius, R, Müller, E, Aebischer, A and Kohn, B (2019) Giardia duodenalis in small animals and their owners in Germany: a pilot study. Zoonoses and Public Health 66, 117124.CrossRefGoogle ScholarPubMed
Remesar, S, García-Dios, D, Calabuig, N, Prieto, A, Díaz-Cao, JM, López-Lorenzo, G, López, C, Fernández, G, Morrondo, P, Panadero, R and Díaz, P (2022) Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain. Transboundary and Emerging Diseases 69, e3141e3153.CrossRefGoogle ScholarPubMed
Ryan, U and Zahedi, A (2019) Molecular epidemiology of giardiasis from a veterinary perspective. Advances in Parasitology 106, 209254.CrossRefGoogle ScholarPubMed
Sak, B, Kašičková, D, Kváč, M, Květoňová, D and Ditrich, O (2010) Microsporidia in exotic birds: intermittent spore excretion of Encephalitozoon spp. in naturally infected budgerigars (Melopsittacus undulatus). Veterinary Parasitology 168, 196200.CrossRefGoogle ScholarPubMed
Sak, B, Brady, D, Pelikánová, M, Květoňová, D, Rost, M, Kostka, M, Tolarová, V, Hůzová, Z and Kváč, M (2011) Unapparent microsporidial infection among immunocompetent humans in the Czech Republic. Journal of Clinical Microbiology 49, 10641070.CrossRefGoogle ScholarPubMed
Santín, M and Fayer, R (2011) Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Research in Veterinary Science 90, 363371.CrossRefGoogle ScholarPubMed
Santín, M, Vecino, JAC and Fayer, R (2008) Enterocytozoon bieneusi genotypes in dogs in Bogota, Colombia. The American Journal of Tropical Medicine and Hygiene 79, 215217.CrossRefGoogle ScholarPubMed
Scaramozzino, P, Carvelli, A, Iacoponi, F and De Liberato, C (2018) Endoparasites in household and shelter dogs from central Italy. International Journal of Veterinary Science and Medicine 6, 4547.CrossRefGoogle ScholarPubMed
Simonato, G, Frangipane di Regalbono, A, Cassini, R, Traversa, D, Beraldo, P, Tessarin, C and Pietrobelli, M (2015) Copromicroscopic and molecular investigations on intestinal parasites in kenneled dogs. Parasitology Research 114, 19631970.CrossRefGoogle ScholarPubMed
Simonato, G, Frangipane di Regalbono, A, Cassini, R, Traversa, D, Tessarin, C, Di Cesare, A and Pietrobelli, M (2017) Molecular detection of Giardia duodenalis and Cryptosporidium spp. in canine faecal samples contaminating public areas in northern Italy. Parasitology Research 116, 34113418.CrossRefGoogle ScholarPubMed
Soliman, RH, Fuentes, I and Rubio, JM (2011) Identification of a novel assemblage B subgenotype and a zoonotic assemblage C in human isolates of Giardia intestinalis in Egypt. Parasitology International 60, 507511.CrossRefGoogle Scholar
Sommer, MF, Zdravković, N, Vasić, A, Grimm, F and Silaghi, C (2017) Gastrointestinal parasites in shelter dogs from Belgrade, Serbia. Veterinary Parasitology: Regional Studies and Reports 7, 5457.Google ScholarPubMed
State Veterinary Administration (2024) Registered animal shelters. Available at https://www.svscr.cz/registrovane-subjekty-svs/registrovane-utulky-pro-zvirata/ (accessed 13 January 2024).Google Scholar
Sulaiman, IM, Fayer, R, Bern, C, Gilman, RH, Trout, JM, Schantz, PM, Das, P, Lal, AA and Xiao, L (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerging Infectious Diseases 9, 14441452.CrossRefGoogle ScholarPubMed
Traub, RJ, Monis, PT, Robertson, I, Irwin, P, Mencke, N and Thompson, RCA (2004) Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128, 253262.CrossRefGoogle ScholarPubMed
Tůmová, L, Ježková, J, Prediger, J, Holubová, N, Sak, B, Konečný, R, Květoňová, D, Hlásková, L, Rost, M, McEvoy, J, Xiao, L, Santín, M and Kváč, M (2023) Cryptosporidium mortiferum n. sp. (Apicomplexa: Cryptosporidiidae), the species causing lethal cryptosporidiosis in Eurasian red squirrels (Sciurus vulgaris). Parasites & Vectors 16, 121.CrossRefGoogle Scholar
Villamizar, X, Higuera, A, Herrera, G, Vasquez-A, LR, Buitron, L, Muñoz, LM, Gonzalez-C, FE, Lopez, MC, Giraldo, JC and Ramírez, JD (2019) Molecular and descriptive epidemiology of intestinal protozoan parasites of children and their pets in Cauca, Colombia: a cross-sectional study. BMC Infectious Diseases 19, 111.CrossRefGoogle ScholarPubMed
Wang, W, Wei, Y, Cao, S, Wu, W, Zhao, W, Guo, Y, Xiao, L, Feng, Y and Li, N (2022) Divergent Cryptosporidium species and host-adapted Cryptosporidium canis subtypes in farmed minks, raccoon dogs and foxes in Shandong, China. Frontiers in Cellular and Infection Microbiology 12, 19.Google ScholarPubMed
Xiao, L (2010) Molecular epidemiology of cryptosporidiosis: an update. Experimental Parasitology 124, 8089.CrossRefGoogle ScholarPubMed
Xiao, L, Morgan, UM, Limor, J, Escalante, A, Arrowood, M, Shulaw, W, Thompson, RC, Fayer, R and Lal, AA (1999) Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Applied and Environmental Microbiology 65, 33863391.CrossRefGoogle ScholarPubMed
Xiao, L, Cama, VA, Cabrera, L, Ortega, Y, Pearson, J and Gilman, RH (2007) Possible transmission of Cryptosporidium canis among children and a dog in a household. Journal of Clinical Microbiology 45, 20142016.CrossRefGoogle Scholar
Xu, H, Jin, Y, Wu, W, Li, P, Wang, L, Li, N, Feng, Y and Xiao, L (2016) Genotypes of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in dogs and cats in Shanghai, China. Parasites & Vectors 9, 19.CrossRefGoogle ScholarPubMed
Yu, Z, Ruan, Y, Zhou, M, Chen, S, Zhang, Y, Wang, L, Zhu, G and Yu, Y (2018) Prevalence of intestinal parasites in companion dogs with diarrhea in Beijing, China, and genetic characteristics of Giardia and Cryptosporidium species. Parasitology Research 117, 3543.CrossRefGoogle ScholarPubMed
Zemanová, I, Husník, R and Svobodová, V (2005) Giardia intestinalis u psů – výskyt, zoonotický potenciál a využití endoskopické diagnostiky. [Giardia intestinalis in dogs – occurrence, zoonotic potential and use of endoscopic diagnostic]. Veterinarstvi 55, 319325.Google Scholar
Figure 0

Figure 1. Schematic arrangement of shelters in Poland (P.1–P.5) and the Czech Republic (CZ.1–CZ.5) from which samples were collected.

Figure 1

Table 1. Occurrence of Cryptosporidium spp., Giardia intestinalis, Encephalitozoon cuniculi and Enterocytozoon bieneusi in individual dogs kept in animal shelters in Poland and the Czech Republic

Figure 2

Table 2. Results of genotyping of Cryptosporidium spp., G. intestinalis, E. cuniculi and E. bieneusi in all tested samples

Figure 3

Figure 2. Phylogenetic relationships between Cryptosporidium spp. detected in dogs in this study (highlighted in green) and other Cryptosporidium available in GenBank using an ML analysis of partial sequences of 18S rRNA (sequence alignment length: 820 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2806) followed by region and location (P.1, Poland location 1).

Figure 4

Figure 3. Phylogenetic relationship between Cryptosporidium canis detected in 1 dog in this study (highlighted in green) and other C. canis available in GenBank using an ML analysis of a region of gp60 gene (sequence alignment length: 540 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (2806) followed by region and location (P.1, Poland location 1).

Figure 5

Figure 4. Phylogenetic relationships between Giardia intestinalis assemblages detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other G. intestinalis assemblages available in GenBank using an ML analysis of a region of TPI gene (sequence alignment length: 467 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2966) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Figure 6

Figure 5. Phylogenetic relationships between G. intestinalis assemblages detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other G. intestinalis assemblages available in GenBank using an ML analysis of a region of BG gene (sequence alignment length: 820 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2966) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Figure 7

Figure 6. Phylogenetic relationships between G. intestinalis assemblages detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other G. intestinalis assemblages available in GenBank using an ML analysis of a region of GDH gene (sequence alignment length: 439 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2966) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

Figure 8

Figure 7. Phylogenetic relationships between Enterocytozoon bieneusi genotypes detected in dogs in this study (highlighted in green – Poland or in grey – Czech Republic) and other E. bieneusi genotypes available in GenBank using an ML analysis of ITS region of rRNA gene (sequence alignment length: 309 bp). Percentage supports (>50%) from 1000 pseudoreplicates are indicated next to the supported node. The branch length scale bar indicates the number of substitutions per site. Sequences from this study are identified by an isolate number (e.g. 2973) followed by region and location (P.1, Poland location 1, CZ.1, Czech Republic location 1).

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