Novel gp60 subtypes and genetic diversity of Cryptosporidium felis in domestic and stray cats from Central Europe

TL;DR

This study identifies new genetic subtypes of Cryptosporidium felis in cats from Central Europe, revealing higher infection rates in stray cats and geographic patterns.

Contribution

The study reports five novel gp60 subtypes of C. felis in Central Europe, previously unreported globally.

Findings

Stray cats had a significantly higher infection rate (6.7%) compared to domestic cats (2.3%).

Five new C. felis subtypes were identified, all within the XIXa subtype family.

Subtypes showed distinct geographic distribution patterns among stray cats.

Abstract

Cryptosporidium felis, a host-specific protozoan with possible zoonotic potential, is a common parasite of cats (Felis catus). To date, there have been few studies on the molecular subtyping of C. felis worldwide, and no have been conducted in Central Europe. The aim of this study was to analyse the prevalence and genetic variability of C. felis in domestic and stray cats in Central Europe, particularly in the Czech Republic, Poland and Slovakia.

Faecal samples were collected from domestic and stray cats. The presence of Cryptosporidium spp. was analysed by light microscopy with aniline-carbol-methyl violet staining and polymerase chain reaction (PCR)/sequencing of the small subunit rRNA gene (18S rDNA). All PCR-positive samples were further subtyped using PCR and sequencing of the 60 kDa glycoprotein gene (gp60). A chi-square test and odds ratio (OR) analysis were used to compare infection rates and assess the risk of C. felis infection between domestic and stray cats.

A total of 711 faecal samples were collected – 350 from domestic cats and 361 from stray cats. The overall infection rate of Cryptosporidium spp. was 4.5% (32/711), with stray cats being significantly more frequently infected (6.7%) than domestic cats (2.3%). Oocysts of Cryptosporidium spp. were not detected microscopically in any of the samples. There were no significant differences between the infection rates in the three countries. All isolates were identified as C. felis, and analysis of the gp60 gene revealed five different subtypes, all belonging to the XIXa subtype family of C. felis. These subtypes formed five well-supported phylogenetic clusters, none of which had been previously reported worldwide. Only one subtype was found in domestic cats, whereas all five subtypes were found in stray cats. The subtypes identified in stray cats showed a clear geographical distribution in the study region.

The results of this study extend our knowledge of the genetic variability of C. felis and indicate a possible geographical distribution of the detected subtypes. The significance of the observed genetic variability in terms of geographical distribution, host specificity and zoonotic potential remains unclear and requires further investigation.

Figures

Map of the geographical distribution of phylogenetically identical *Cryptosporidium felis* subtypes (cluster) in domestic and stray cats in the Czech Republic, Slovakia and Poland, based on genotyping of the gene encoding the 60 kDa glycoprotein (*gp60*)

Phylogenetic relationships between *Cryptosporidium felis* isolates detected in this study and other *C*. *felis* subtypes, based on the partial sequence of the 60 kDa glycoprotein gene (*gp60*). Maximum likelihood analysis was performed using the General Time Reversible model. Numbers in the nodes represent bootstrap values for nodes that received more than 50% support in the bootstrap test after 1000 replicates. The scale bar is included in the tree. Isolates obtained in this study are highlighted in grey; red dots represent domestic and blue dots represent stray cats

Full text

Background

Cryptosporidium (Apicomplexa) is a genus of obligate parasitic protists that predominantly infect the gastrointestinal and respiratory tracts of vertebrates such as mammals, birds, reptiles, fish and amphibians [1–3]. A clinical manifestation of cryptosporidial infection, known as cryptosporidiosis, is characterised by watery diarrhoea, abdominal pain and nausea. The severity of infection depends on the Cryptosporidium species and the host species, including its age and immune status. In general, infection is more severe and sometimes fatal in juveniles, non-specific hosts and immunocompromised individuals [4–6].

The domestic cat (Felis catus) is one of the most widespread pet species and a favourite companion animal in many households worldwide. According to statistics, 350–370 million cats are kept as pets worldwide and the number of stray cats is estimated at 480 million [7]. Keeping cats has many benefits for humans – they improve mental well-being, reduce stress, promote socialisation and can have a positive effect on cardiovascular health [8]. However, the potential health risks must also be considered. Besides the usual injuries to the owner from scratching or biting and allergic reactions [9], cats can be carriers of various zoonotic parasite infections such as toxoplasmosis, giardiasis, or cryptosporidiosis [10–12].

Cats are primarily parasitised by the host-specific species Cryptosporidium felis, but other species and genotypes of Cryptosporidium have also been detected in cats, namely Cryptosporidium muris, Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium ryanae, Cryptosporidium hominis, Cryptosporidium sp. rat genotype III and IV [10, 13–16]. Cryptosporidium felis is classified as a species with zoonotic potential, with low to moderate prevalence in immunocompetent individuals or in populations with limited access to sanitation and clean water [17].

In 2020, Rojas-Lopez et al. [18] developed a molecular method for subtyping C. felis on the basis of the analysis of the gene encoding the 60 kDa glycoprotein (gp60). Phylogenetic analyses of this gene revealed the existence of five subtypes of C. felis (designated XIXa–XIXe), which differ both in their host specificity and geographical distribution [18–20]. Nevertheless, the amount of available data is still quite limited. Especially in the Europe, the genetic variation of C. felis is poorly documented, with few published data [10, 18–21].

The aim of the present study is to cover the knowledge gaps on the geographical distribution and genetic variability of C. felis in Central Europe by molecular subtyping based on the gp60 gene.

Methods

Area and specimens studied

For this work, faecal samples were collected from domestic cats kept indoors, and from stray cats in animal shelters or from cats near farm buildings, especially cattle and pig stables. The samples were collected in the Czech Republic, Slovakia and the southern part of Poland (Fig. 1). To avoid re-sampling the same stray cat, samples were collected from the ground after the animal had defecated, within 1 day/location. No domestic cat was sampled twice. Each faecal sample was placed in a sterile plastic vial labelled with the sample ID and stored at 4–8 °C without fixative until laboratory processing. A smear was taken from each faecal sample, stained with aniline-carbol-methyl violet and examined using a light microscope to detect Cryptosporidium spp. oocysts [22].Fig. 1. Map of the geographical distribution of phylogenetically identical Cryptosporidium felis subtypes (cluster) in domestic and stray cats in the Czech Republic, Slovakia and Poland, based on genotyping of the gene encoding the 60 kDa glycoprotein (gp60)

Molecular analyses

Total genomic DNA (gDNA) was extracted from 200 mg of faeces by bead disruption at 5.5 m/s for 60 s, using 0.5 mm glass beads in a FastPrepR24 instrument (MP Biomedicals, CA, USA), followed by DNA isolation using a commercial kit according to the manufacturer’s instructions (Exgene™ Stool DNA mini, Genially Biotechnology Co. Ltd., Seoul, Korea). The purified DNA was stored at −20°C before being used for PCR. A nested PCR approach was used to amplify a partial region of the small subunit rRNA gene (18S rDNA) [23] and the gp60 gene [18]. The PCR reaction was carried out in a total volume of 20 µl. The reaction mixture contained 2 µl of gDNA as template, or 2 µl of the primary PCR product in case of a secondary reaction, 10 µl of 2 × AmpONE™ HS-Taq premix (GeneAll), 200 nM of each primer (forward and reverse) and molecular grade water to the total volume. Cryptosporidium serpentis (for 18S rDNA) and C. felis (for gp60) DNA and molecular grade water were used as positive and negative controls, respectively. The secondary PCR products were detected by agarose gel electrophoresis, stained with ethidium bromide and isolated using the GenElute Gel Extraction Kit (Sigma, St. Louis, MO, USA). The purified secondary products were sequenced bidirectionally at a commercial company (SeqMe s.r.o, Dobříš, Czech Republic) using Sanger sequencing. Each positive sample was independently sequenced twice.

Phylogenetic analyses

The nucleotide sequences obtained for each gene in this study were processed and edited using ChromasPro v2.4.1 (Technelysium Pty Ltd., South Brisbane, Australia). Sequence alignments were performed both between samples and with reference sequences from GenBank, using the MAFFT online platform (version 7) with automatic alignment mode selection (http://mafft.cbrc.jp/alignment/software/). Phylogenetic analyses were performed using the MEGA X software [24, 25], which was also used to determine the best-fitting DNA/protein evolutionary models based on the Bayesian information criterion (BIC). The maximum likelihood (ML) method was applied to generate phylogenetic tree. The General Time Reversible model [26] was used for gp60 tree. Branch support was evaluated using 1,000 bootstrap repetitions. The resulting phylograms were generated in MEGAX and then manually refined in CorelDraw X7. All newly generated sequences of gp60 genes were deposited in GenBank under the following accession numbers [PV817847–PV817854].

Statistical analysis

Statistical analysis was performed using chi-square tests with Yates correction in EpiInfo™ 7.2.7 (CDC, Atlanta, USA), to compare the frequency of C. felis occurrence between domestic and stray cats, and the distribution in each country (Czech Republic, Slovakia and Poland). A P-value < 0.05 was considered statistically significant.

Results

Occurrence and prevalence of Cryptosporidium spp. in cats

A total of 711 cat samples were collected – 350 from domestic cats and 361 from stray cats (Table 1). Of the 711 samples analysed, no oocysts of Cryptosporidium spp. were detected microscopically in any sample. However, the results of the molecular analyses for amplification of 18S rDNA showed the presence of specific Cryptosporidium spp. DNA in 32 samples (Table 1). Table 1. Numbers of tested and positive domestic and stray cats (Felis catus) for the presence of Cryptosporidium felis in the Czech Republic, Slovakia and PolandCountryCat typeExaminedPositivityMicroscopy**PCRCzech RepublicDomestic14103Stray156012Subtotal297015SlovakiaDomestic8902Stray10206Subtotal19108PolandDomestic12003Stray10306Subtotal22309Total711032

All PCR products were successfully sequenced, and the results confirmed the presence of C. felis in all samples. The overall prevalence of C. felis in the analysed regions was 4.5%. The highest prevalence was found in the Czech Republic (5.1%), followed by Slovakia (4.2%) and Poland (4.0%). Of the 350 domestic and 361 stray cats, 8 (2.3%) and 24 animals (6.7%), respectively, were positive for C. felis. Domestic cats were 3.04 times less likely to be parasitised by C. felis than stray cats (P = 0.0086, χ^2^ = 6.8863, df. = 1, OR = 3.04).

Molecular subtyping of Cryptosporidium felis

All 32 PCR-positive samples were successfully genotyped for the gp60 locus. Analysis of the gp60 gene identified five subtypes of C. felis, which were assigned to subtype family XIXa (Fig. 2). When analysed phylogenetically, the subtypes formed five separate clusters with high bootstrap support. None of the subtypes detected in this study were identical to subtypes previously identified in cats or humans worldwide (Fig. 2). All isolates from domestic cats were identical to each other and belonged exclusively to cluster no. 1 (Fig. 2), which also included isolates from stray cats (Fig. 2). These isolates were phylogenetically related to isolate C. felis 7378 (GenBank MT458675), which originated from a cat in Slovakia (the type of cat is unknown), and to six other isolates obtained in this study from stray cats from Poland and the Czech Republic, which formed a separate cluster (no. 2). The analysis of the occurrence of the isolates in the study area indicates a geographical distribution pattern (Fig. 1). While isolates belonging to cluster no. 1 were detected in all three countries, the Czech Republic, Slovakia and Poland, isolates belonging to cluster no. 2 were found only in the Czech Republic and Poland. Cluster no. 3 was found exclusively in Slovakia, and clusters no. 4 and 5 only in the Czech Republic (Fig. 1 and 2).Fig. 2. Phylogenetic relationships between Cryptosporidium felis isolates detected in this study and other C. felis subtypes, based on the partial sequence of the 60 kDa glycoprotein gene (gp60). Maximum likelihood analysis was performed using the General Time Reversible model. Numbers in the nodes represent bootstrap values for nodes that received more than 50% support in the bootstrap test after 1000 replicates. The scale bar is included in the tree. Isolates obtained in this study are highlighted in grey; red dots represent domestic and blue dots represent stray cats

Sequence analysis of the individual isolates revealed differences in sequence length, mainly due to the presence of repetitive sequences (Table 2). The sequence designated R1, which contains a 33-bp repetitive stretch of 5′ CCA CCT AGT GGC GGT GGC GTG TCC CCT GCT 3′ located approximately between nucleotides 450–530 in the sequence alignment, was present in only one copy in the isolates belonging to cluster no. 1 (Table 2; Fig. 2). A repetitive sequence designated R2, containing a 39-bp stretch of the tandem repeat 5′ AGC ACA ACT GCG GCT ACA GCG AGC ACT GCG AGT TCG ACA 3′ at a position between nucleotides 770–910, was detected in the sequences of eight isolates in repeats 1–3. Isolates with one tandem repeat belonged exclusively to cluster no. 3, those with two repeats to cluster no. 4 and those with three repeats to cluster no. 5 (Fig. 2; Table 2). The number of GTT triplet repeats at the end of the sequence, between nucleotide positions 1143–1154, was between two and four in the isolates analysed. Isolates belonging to clusters nos. 1 and 2 contained two GTT repeats, whereas isolates from clusters nos. 3–5 always had four repeats (Fig. 2; Table 2). Table 2. The Cryptosporidium felis isolates detected in this study, their subtype identity and the copy numbers of major tandem repeats in the gp60 geneCountryCat typeIsolate IDGenotyping at gp60ClusterSubtypeR1R2GGTCzech RepublicDomesticCZEcat3XIXa1–21DomesticCZEcat150XIXa1–21DomesticCZEcat177XIXa1–21StrayCZEcat22XIXa–244StrayCZEcat89XIXa–244StrayCZEcat96XIXa–345StrayCZEcat104XIXa––22StrayCZEcat119XIXa––22StrayCZEcat121XIXa–345StrayCZEcat157XIXa––22StrayCZEcat198XIXa1–21StrayCZEcat212XIXa1–21StrayCZEcat222XIXa1–21StrayCZEcat230XIXa1–21StrayCZEcat327XIXa1–21SlovakiaDomesticSVKcat75XIXa1–21DomesticSVKcat215XIXa1–21StraySVKcat26XIXa–143StraySVKcat54XIXa–143StraySVKcat69XIXa–143StraySVKcat110XIXa–143StraySVKcat112XIXa1–21StraySVKcat129XIXa1–21PolandDomesticPLNcat38XIXa1–21DomesticPLNcat40XIXa1–21DomesticPLNcat53XIXa1–21StrayPLNcat6XIXa1–21StrayPLNcat17XIXa1–21StrayPLNcat46XIXa1–21StrayPLNcat68XIXa––22StrayPLNcat71XIXa––22StrayPLNcat74XIXa––22Numbers of the clusters correspond to the clusters in Figs. 1 and 2

Discussion

This study deals with the occurrence of C. felis in cats in the Czech Republic, Slovakia and Poland, focussing on the intraspecific variation within the gp60 gene. Cryptosporidium spp. infections in cats are relatively common, and their prevalence in the population varies between different studies and countries, ranging from 0.6% to 40.8% [16]. In the present study, 4.5% of the 711 cats examined were classified as positive. This prevalence represents a relatively low incidence. Previous studies conducted in the Czech Republic, Slovakia and Poland found a similar prevalence of 4.4% [27]. Comparable prevalence rates were found in other European countries, with the exception of Germany, where an overall prevalence of 21.2% was observed [16].

The phylogenetic analyses showed that the cats included in the study were infected only with C. felis. These results are in agreement with those of other authors, who also either exclusively detected this species in their studies or dominated the occurrence of C. felis [10, 14, 19, 21, 28–33]. Sequence analysis of all 32 samples positive for Cryptosporidium-specific DNA showed no evidence of mixed infections, and all phylograms were clean. In a previous study conducted in the same area, no other Cryptosporidium species were detected in either stray or domestic cats [27]. The species C. muris, C. parvum, C. ryanae, Cryptosporidium sp. rat genotype III and Cryptosporidium sp. rat genotype IV, which have been detected together with C. felis in previous studies, mostly represent a group of species that are not host-specific to cats [2, 34]. The species and genotypes such as C. muris, Cryptosporidium sp. rat genotype III and Cryptosporidium sp. rat genotype IV are specific to mice and rats, which are a natural food source for cats [35, 36]. It is therefore not surprising that the specific DNA of these Cryptosporidium species is occasionally detected in cat faeces using sensitive molecular methods. Similarly, C. muris and C. tyzzeri have been detected in slurry and pig faeces; however, it was later experimentally confirmed that pigs are not susceptible to these Cryptosporidium species [37, 38]. The sporadic occurrence of C. ryanae in cats can be explained in a similar way. This Cryptosporidium species frequently infects cattle [39], it is likely that cats living on or near farms may become contaminated with cattle faeces.

Our findings, along with other studies, show that stray cats are more likely to be infected with Cryptosporidium spp. than domestic cats. In our dataset, the prevalence of C. felis infection was higher in stray cats (6.7%) than in domestic cats (2.3%). This is consistent with our previous study, in which 7.4% of stray cats were C. felis-positive, compared with only 0.8% in domestic cats [27]. Other studies investigating the influence of cat origin on the prevalence of Cryptosporidium spp. have reported similar trends. Yang et al. [15] showed that cats from refuge centres (13.4%) were more frequently infected than privately owned cats (7.1%). Similarly, Xu et al. [40] reported a higher prevalence in cats from animal shelters (7.5%) compared with cats from pet shops (3.8%).

Genotyping of Cryptosporidium spp. using the gene encoding gp60 is frequently used to study intraspecific variation, host specificity and pathogenicity of subtypes within individual taxa. The gp60 gene exhibits a high degree of polymorphism at both inter- and intraspecific levels, which enables the classification of individual isolates into specific families and subtypes within these families [18, 41–43].

Similar to other species, in particular C. parvum, C. hominis or Cryptosporidium meleagridis, a variation of the gp60 gene has been described in C. felis, comprising five allelic families with dozens of subtypes [19]. In our study, we detected only isolates belonging to allele family XIXa, the most widespread allele family of C. felis worldwide. Previous studies suggest a possible geographical splitting of certain C. felis subtypes based on the occurrence of genetically identical clusters in geographical areas [19].

In line with these findings, our study not only detected new subtypes not previously reported, but also identified three clusters that appear to be geographically specific. However, it should be emphasised that these findings are based on limited data, in contrast to the well-studied species C. parvum or C. hominis. Further studies with larger samples size and in other countries are needed to draw more robust conclusions.

The significance of the diversity of C. felis subtypes in relation to pathogenicity – as described for C. parvum or C. hominis, for example – remains unclear [44–47]. To date, no studies have demonstrated an influence of a specific subtype on the clinical course of C. felis infection in cats or humans. In our study, we observed no clinical signs of cryptosporidiosis in any of the positive cats. However, it should be noted that all cats involved in the study were adults, which is consistent with previous findings that adult and healthy cats shed only small amounts of oocysts into the environment after infection [15, 48–51]. This is also consistent with our microscopically negative results, as the low level of oocyst excretion is below the detection limit of conventional staining methods, whereas PCR analyses detected specific C. felis DNA in certain samples.

Previous studies have shown that the subtype families XIXb, XIXd and XIXe have been detected exclusively in humans, whereas some subtypes from the families XIXa and XIXc have zoonotic potential [18–21]. Similar differences in the host specificity among subtype families and their subtypes have also been described for C. parvum or Cryptosporidium equi. For example, the subtypes of subtype family IIc of C. parvum are mainly found in humans, with no known natural hosts other than hedgehogs [52, 53]. A similar situation was observed with C. equi. While the subtypes of subtype family VIa occur exclusively in horses, donkeys and other equids, infections with subtypes from families VIb and VIc are almost exclusively associated with humans [54]. Since new subtypes of subtype family XIXa were detected in the countries investigated, and no cases of C. felis infections in humans have been reported from these areas, the zoonotic potential of these subtypes of family XIXa cannot yet be assessed.

Conclusions

This study expands the current knowledge on the intraspecific diversity and distribution of C. felis in Central Europe. The detection of new subtypes of subtype family XIXa and evidence of a possible geographical clustering emphasise the need for further studies to clarify the zoonotic potential and biological significance. The higher prevalence in stray cats compared with domestic cats emphasises the role of lifestyle and environmental exposure in transmission dynamics. Finally, the discrepancy between microscopically negative and PCR-positive results emphasises the importance of molecular methods for reliable detection.