Abstract
The feline gut can harbour a number of protozoan parasites. Recent genetic studies have highlighted new epidemiological findings about species of Cryptosporidium, assemblages of Giardia duodenalis and Toxoplasma gondii. Furthermore, epidemiological studies suggest the occurrence of Tritrichomonas foetus in cats is on the increase worldwide. The prevalence of selected intestinal protozoa was determined by PCR using DNA previously extracted from the faeces of 146 privately owned healthy cats from Italy. Molecular genotyping on T gondii, G duodenalis and Cryptosporidium DNA was achieved. PCR assays were positive in 32 (22.9%) samples. Three animals (2.0%) were positive for T foetus and Cryptosporidium DNA, 15 specimens (10.3%) were positive for T gondii and 11 (7.5%) for G duodenalis. Co-infections were never observed. Results of the typing analysis allowed the identification of Cryptosporidium felis in all cases. The specimens positive for T gondii hinted at clonal genotype I (n = 7), genotype II (n = 1) and genotype III (n = 7). The G duodenalis isolates were referable to assemblages F (n = 9) and C (n = 2). In conclusion, the results obtained in this study add to the literature regarding the epidemiology of these parasites by confirming their presence in the faeces of healthy pet cats.
Short Communication
Toxoplasma gondii represents a public health hazard. Cats are the only domestic felids to shed T gondii oocysts, with prevalences of 0.1–0.4% in European countries.1–13 In North America and Europe three T gondii clonal lineages – designated clonal types I, II and III – have predominantly been described, as analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and microsatellite typing.14,15 Atypical genetically different genotypes have been described to be prevalent in South America and Asia. 16 Feline genotypes of T gondii have never been described in Italy.
The genus Cryptosporidium currently contains at least 20 valid species and over 40 genotypes, with different levels of animal host adaptation. 17 Feline infection is reported worldwide, and is demonstrated to be caused by feline-specific Cryptosporidium felis. Cryptosporidium parvum and Cryptosporidium muris have occasionally been reported in naturally infected cats, probably owing to the broader host range of these species. The published studies on this infection in cats in Europe give prevalence rates ranging from 0% to 24.5%.18,19 The health status of investigated animals (normal or diarrhoeic), different age groups and diagnostic techniques used probably contribute to the variations in infection rates in different studies.
Giardia duodenalis is genetically composed of assemblages named A–G. Assemblages A–F were all detected in cats in a multicountry study, 20 although assemblage A and the cat-specific assemblage F were the dominant ones. This finding was also corroborated by other studies.21,22 The overall prevalence of Giardia infection in cats in Europe ranges from 4.4% to 37.0%.18,23
The aim of this retrospective study was to determine the prevalence of selected intestinal protozoa by PCR using DNA previously extracted from the faeces of privately owned healthy cats in Italy. Considering the low amount of information reported from this country, further molecular genotyping was achieved using T gondii, G duodenalis and Cryptosporidium DNA in order to add to the available information about types occurring in healthy pet cats.
One hundred and forty-six stool samples were collected from cats living in Tuscany (provinces of Pisa and Florence) and Liguria (province of Genoa) immediately after voiding. All samples were obtained from privately owned, healthy European shorthair cats, of both genders, aged between 1 and 3 years, referred to private veterinary clinics for neutering. The samples had been previously collected for an epidemiological survey to ascertain the presence of T gondii in healthy adult pet cats. Inclusion criteria were that animals were allowed to roam outdoors, that they had no diarrhoea in the preceding 3 months and that they had not received any drug in the previous 30 days.
Faecal specimens were maintained at 4°C and then subjected to copromicroscopic examination and DNA extraction within 24 h of collection. Samples were divided into two aliquots; 0.04 g was processed by a conventional flotation method using 262 mg/ml ZnCl2 and 275 mg/ml NaCl, as described by Schares et al. 24 Copromicroscopic examination was carried out at × 400 magnification.
Faecal DNA was extracted from samples using a ZR Fecal DNA MiniPrep kit (Zymo Research).
All primers and restriction enzymes were provided by Eurofins MGW (M-Medical).
The DNA of Tritrichomonas foetus was detected by using a single-tube nested PCR that amplified the T foetus internal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene, as described by Gookin et al. 25
Nested PCR (nPCR) for T gondii DNA was performed following the procedure reported by Jones et al, 26 using two pairs of oligonucleotide primers to amplify regions of the B1 gene. Genotypes were determined via multiplex multilocus PCR-RFLP for 12 genetic markers (SAG1, 3’-SAG2, 5’-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8,C29-2, L358, PK1 and Apico). 27
G duodenalis was detected by amplifying the glutamate dehydrogenase (gdh) gene, and genotyping was performed by PCR-RFLP using the restriction enzymes NlaIV and RsaI. 28
Cryptosporidium species DNA was detected by nPCR that amplified the 18s small subunit rRNA gene locus; genotyping with PCR-RFLP was performed by the restriction enzymes SspI and VspI. 29
Furthermore, to confirm the results obtained by PCR-RFLP, the PCR products of T foetus, G duodenalis and Cryptosporidium species were purified using a QIAquick PCR purification kit (Qiagen), according to the manufacturer’s instructions, and then sequenced. All sequencing procedures were performed by a commercial laboratory (BMR-Genomics, Padova, Italy). Sequences were assembled and corrected by visual analysis of the electropherogram using Bioedit v.7.0.2 30 and then compared with those available in GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST).
Copromicroscopic examination was negative for T gondii, as well as for Giardia cysts, in all samples examined.
PCR assays were positive for 32 (22.9%) feline stools. Three animals (2.0%) were positive for T foetus and Cryptosporidium DNA, 15 specimens (10.3%) were positive for T gondii and 11 (7.5%) for G duodenalis. Co-infections were not observed.
The results of the typing analysis allowed the identification of C felis in all cases. The specimens positive for T gondii were not fully genotyped at all 12 loci, but hints for clonal genotypes I (n = 7), II (n = 1) and III (n = 7) were obtained. More detailed results are shown in Table 1. G duodenalis assemblages F (n = 9) and C (n = 2) were detected.
Genotyping results of Toxoplasma gondii DNA isolated from feline faeces
I = genotype I; II = genotype II; III = genotype III; NA = not amplified
The results obtained from sequenced DNA confirmed the identification obtained by PCR in all specimens
The prevalence registered for T foetus was 2%, which is in full agreement with Xenoulis et al. 12 In other molecular analyses of pet cats, positive subjects had diarrhoea.6,31 This parasite is considered to be significantly associated with diarrhoea, even if a recent report 32 suggested the possibility of isolating this agent from clinically normal cats, which is in agreement with the results obtained by us.
Fifteen cats were positive for T gondii DNA, with an overall prevalence of 10.3%. This findings is in agreement with a previous report carried out on colony stray cats from Tuscany. 33
Our results cannot be compared with any other similar study. Data from European literature refer to PCR carried out on oocysts revealed by copromicroscopy. Clonal type II, with the Apico I allele, was the most recurrent genotype in Germany and Switzerland.13,34 Furthermore, in Germany, a clonal genotype III and 10 mixed genotypes were found. 34 These findings do not agree completely with the results of our study: in this study all apparently clonal and unmixed genotypes were identified, and genotypes suggestive of I and III were prevalent. To the best of our knowledge, this is the first report of the genotyping of T gondii DNA from feline faeces in Italy. Genotyping has previously been carried out in Italy on T gondii DNA from both domestic and sylvatic animals, indicating the occurrence of strictly clonal genotypes in goats 35 and cats, and mixed types detected in free-ranging waterfowl 36 and foxes. 37 These results are in agreement with the hypothesis of two distinct cycles of T gondii in domestic and sylvatic environments. 38
Eleven cats were positive for G duodenalis DNA, with an overall prevalence of 7.5%, which is in agreement with Paoletti et al. 18 Genotyping yielded nine assemblages, with F the most frequent in cats and C the most common in dogs. Recovery of canine assemblages in cat faeces has been previously reported,39,40 and it could be owing to the occurrence of Giardia cysts on feline coats that are ingested during grooming. These assemblages have never been demonstrated in humans from the USA with G duodenalis infection; 39 even if in one study assemblage F was reported in humans, this finding remains to be confirmed at additional loci. 41 C felis was the sole Cryptosporidium species isolated in this study; it was isolated from three cats, with a prevalence of 2%. Despite the very close association between humans and cats, C felis infections are infrequently reported in humans. 42
Conclusions
The results obtained in our study add to the information available on the epidemiology of these parasites, and confirm their occurrence in faeces from healthy pet cats. Many cats share the same households, as shown in a questionnaire survey carried out in the USA. 43 Given the large number of multi-cat households, understanding and preventing the diseases that cats may transmit to each other are of paramount importance. Intestinal protozoan parasites, including T gondii, T foetus, C felis and G duodenalis, can cause insidious infections, with asymptomatic cats being capable of transmitting toxoplasmosis, trichomoniasis, cryptosporidiosis and giardiasis to other cats. These infections may be significant sources of disease in feline populations. Healthy cats are protected by effective immune systems and bolstered by good nutritional, sanitary and hygienic conditions. Immunocompromised cats, including animals with deficient nutrition, feline immunodeficiency virus and/or feline leukaemia virus infection, systemic and chronic diseases, concurrent administration of immunosuppressive drugs and environmental stress, are at a much higher risk of developing these protozoan diseases. Awareness of unapparent infections in healthy feline populations may be important to avoid exposure to these agents and thus diminish the likelihood of transmission to cats with compromised immune function.
Footnotes
Acknowledgements
We wish to thank Dr Nadia Ferrini for her excellent editing of the text.
Conflict of interest
The authors do not have any potential conflicts of interest to declare.
Funding
The authors received no specific grant from any funding agency in the public, commercial or not-for-profit sectors for the preparation of this short communication.