Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in Northern Spain

Infection, Genetics and Evolution 50 (2017) 62–69

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Research paper

Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in Northern Spain Horacio Gil a,b,1, Lourdes Cano a,1, Aida de Lucio a, Begoña Bailo a, Marta Hernández de Mingo a, Guillermo A. Cardona c, José A. Fernández-Basterra d, Juan Aramburu-Aguirre d, Nuria López-Molina d, David Carmena a,⁎ a

Parasitology Service, National Centre for Microbiology, Health Institute Carlos III, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain European Program for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control (ECDC), Granits väg 8, 171 65 Solna, Sweden c Livestock Laboratory, Regional Government of Álava, Ctra. de Azua 4, 01520 Vitoria-Gasteiz, Spain d Department of Social Policy and Public Health, City Council of Vitoria-Gasteiz, Pintor Teodoro Dublang, 25, 01008 Vitoria-Gasteiz, Spain b

a r t i c l e

i n f o

Article history: Received 16 January 2017 Received in revised form 14 February 2017 Accepted 15 February 2017 Available online 17 February 2017 Keywords: Giardia duodenalis Cryptosporidium Dogs Cats Molecular epidemiology Spain

a b s t r a c t Domestic dogs and cats may act as natural reservoirs of a large number of zoonotic pathogens, including the enteric parasites Giardia duodenalis and Cryptosporidium spp., the most relevant protozoan species causing gastrointestinal disease worldwide. A cross-sectional epidemiological study aiming to assess the prevalence and molecular diversity of G. duodenalis and Cryptosporidium spp. was conducted in an animal rescue centre in the province of Álava (Northern Spain). A total of 194 and 65 faecal dropping samples from individual dogs and cats, respectively, were collected between November 2013 and June 2016. G. duodenalis cysts and Cryptosporidium spp. oocysts were detected by direct fluorescence microscopy and PCR-based methods targeting the small subunit ribosomal RNA gene of these parasites. Overall, G. duodenalis and Cryptosporidium spp. were detected in 33% (63/194) and 4.1% (8/194) of dogs, and 9.2% (6/65) and 4.6% (3/65) of cats, respectively. G. duodenalis and Cryptosporidium co-infections were observed in 1.5% (3/194) of dogs, but not in cats. No significant differences in infection rates could be demonstrated among dogs or cats according to their sex, age group, status, or geographical origin. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of G. duodenalis allowed the characterization of 19 canine isolates that were unambiguously assigned to sub-assemblages AII (n = 7), BIII (n = 1), and BIV (n = 7), and assemblages C (n = 3) and D (n = 1). Two feline isolates were genotyped as assemblages A and F, respectively. No mixed assemblage or sub-assemblage infections were identified. C. canis (n = 5) and C. hominis (n = 1) were the Cryptosporidium species found in dogs, whereas C. felis (n = 1) was identified in cats. The finding of G. duodenalis sub-assemblages AII, BIII, and BIV circulating in dogs (but not cats) may have zoonotic potential, although most of the AII and BIV isolates sub-genotyped corresponded to genetic variants not previously found in Spanish human populations. Dogs may also act as novel suitable hosts for C. hominis. We recommend to considerer companion animals as sentinel surveillance system for zoonotic giardiasis and cryptosporidiosis in order to minimize the risk of spreading of these parasitic diseases among the human population. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Cats, dogs, and humans have shared a close relationship due to companionship, recreation, protection, and occupational reasons, for over 10,000 years. Pet ownership has been recognizably proven to exert beneficial effects on human health by improving physical condition and mental and emotional well-being (Hodgson et al., 2015). Consequently, ⁎ Corresponding author. E-mail address: [email protected] (D. Carmena). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.meegid.2017.02.013 1567-1348/© 2017 Elsevier B.V. All rights reserved.

pets are being increasingly used as an effective therapeutic option in healthcare facilities (Wells, 2007). However, cats and dogs may also act as natural reservoirs of human infections by pathogenic bacteria, viruses, and parasites (Chomel, 2014), including the enteric protozoan Giardia duodenalis and Cryptosporidium spp. (Esch and Petersen, 2013). G. duodenalis is the only Giardia species infective to dogs, cats, and humans. Of the eight genetic variants (assemblages A–H) forming part of G. duodenalis, dogs and cats are predominantly infected by caninespecific (C–D) or feline-specific (F) assemblages. Zoonotic assemblages A and B (particularly the former) are responsible for a lower proportion of cases in pets (Ryan and Cacciò, 2013). The genus Cryptosporidium

H. Gil et al. / Infection, Genetics and Evolution 50 (2017) 62–69

encompasses at least 26 valid species, of which C. canis and C. felis cause the vast majority of infections in dogs and cats, respectively (Ryan et al., 2014). Both species are considered of low zoonotic risk to humans. Additionally, novel Cryptosporidium species and genotypes have been proposed in recent years (Jezkova et al., 2016; Kváč et al., 2016). Although the zoonotic potential of G. duodenalis and Cryptosporidium spp. is not questioned, the extent and frequency of such transmission events remain a topic of intense debate (Xiao and Feng, 2008; Ballweber et al., 2010; Lucio-Forster et al., 2010). Large household- or community based molecular epidemiological surveys coincide in concluding that domestic cats and dogs play a minor role as source of human giardiasis or cryptosporidiosis (Cooper et al., 2010; Inpankaew et al., 2014). A similar conclusion has been reached by seasonal models analysing large time series of human and canine Giardia cases in USA (Mohamed et al., 2014). However, other studies suggest that zoonotic transmission may occur sporadically (Xiao et al., 2007; Beser et al., 2015) or under certain epidemiological conditions (Inpankaew et al., 2007; Volotão et al., 2007). Both G. duodenalis and Cryptosporidium spp. have been reported in European cats and dogs with prevalence rates ranging from b1–15% in asymptomatic animals (Overgaauw et al., 2009; Osman et al., 2015; Paoletti et al., 2015) to up to 25% in symptomatic populations (Batchelor et al., 2008; Epe et al., 2010). In Spain, G. duodenalis has been detected in 15% and 1–38% of the studied feline and canine populations, respectively (reviewed in Carmena et al., 2012 and Bouzid et al., 2015). Infection by Cryptosporidium spp. has only been documented in 7–15% of the dogs investigated (reviewed in Navarro-i-Martinez et al., 2011). Genotyping data are only available from few molecular studies carried out in the Autonomous Region of Madrid (Dado et al., 2012b) and Catalonia (Ortuño et al., 2014). In recent years, our research group has conducted a series of community-based and field studies aiming to characterize the transmission dynamics of G. duodenalis and Cryptosporidium spp. in the province of Álava, Northern Spain. Molecular data was, therefore, gathered from human (Cardona et al., 2011), livestock (Cardona et al., 2011, 2015), and wild animal (Cano et al., 2016) populations, and from environmental water samples (Carmena et al., 2007). Additionally, these studies also evidenced that ownership of domestic dogs or cats tended to increase the prevalence odds of human giardiasis and cryptosporidiosis (Cardona et al., 2011). In an attempt to complete our understanding of the epidemiology of these parasitic diseases in Álava, we report here the prevalence, molecular diversity and frequency of G. duodenalis and Cryptosporidium species in sheltered cats and dogs from this region. Additionally, generated genotyping information were used to evaluate the potential role of pet animals as suitable reservoirs of human disease. 2. Material and methods

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were placed in screw-topped specimen containers and uniquely labelled indicating identification number and date of collection. Information regarding sex, age, breed, and geographical origin of the animal was also consigned. Faecal samples were stored at − 20 °C and shipped to the Spanish National Centre for Microbiology for further diagnostic and molecular analyses. 2.2. Direct fluorescent antibody test A direct fluorescent antibody test (DFAT) was used to detect Giardia cysts and Cryptosporidium oocysts by fluorescence microscopy. Briefly, ~ 1 g of faecal material was processed using the concentration system PARASEP Midi® (Grifols Movaco, Barcelona, Spain) according to the manufacturer's instructions. Five microliter of concentrated faecal material were placed on welled slides. Smears were air-dried, methanol fixed, stained with fluorescein-labelled mouse monoclonal antibodies (Crypto/Giardia Cel, Cellabs, Sydney, Australia), and examined at 400× magnification. The burden of the infection was estimated by counting the number of (oo)cysts per well. 2.3. DNA extraction and purification Total DNA was extracted from a new aliquot (~200 mg) of each faecal sample using the QIAamp® DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Purified DNA samples (200 μL) were stored at −20 °C for further downstream molecular analysis. A water extraction control was routinely included in each sample batch processed. 2.4. Molecular detection of Giardia duodenalis Detection of G. duodenalis DNA was achieved using a real-time PCR method targeting a 62-bp region of the small subunit ribosomal RNA (ssu rRNA) gene of the parasite (Verweij et al., 2003). Amplification reactions were conducted in a volume of 25 μL containing 3 μL of template DNA, 12.5 pmol of primers Gd-80F and Gd-127R, 10 pmol of probe (Supplemental content 1), and 12.5 μL TaqMan® Gene Expression Master Mix (Applied Biosystems, CA, USA). Detection of parasitic DNA was performed on a Corbett Rotor-Gene 6000 real-time PCR cycler (Qiagen Corbett, Hilden, Germany) using an amplification protocol consisting on an initial hold step of 2 min at 55 °C and 15 min at 95 °C followed by 45 cycles of 15 s at 95 °C and 1 min at 60 °C. The ramping of the machine was 10 °C/s in every step. Ten-fold dilutions of a DNA isolate obtained from a human stool sample with a known number of G. duodenalis cysts were included in each experiment for sensitivity and quantification purposes (see below). No-template water (negative) and DNA (positive) controls of genomic DNA were included in each PCR run.

2.1. Study area and faecal sample collection 2.5. Molecular characterization of Giardia duodenalis isolates The province of Álava (Northern Spain) numbers 51 municipalities distributed in seven administrative regions. Although no official pet census is currently available, it is estimated that N7000 dogs and 5000 cats are kept as companion animals only in the capital city Vitoria-Gasteiz. Stray, abandoned, or surrendered animals in the province are sent and held at the Armentia Municipal Animal Shelter (AMAS). In average, the AMAS provides care for 1250 dogs and 350 cats every year, and its adoption program allows finding a new home for 76%–94% of these animals. A total of 194 and 65 faecal dropping samples from individual dogs and cats, respectively, were regularly collected as soon as practicably possible after defecation between November 2013 and June 2016 by the AMAS personnel. Faecal material was obtained within 24 h after the animals entered the AMAS in order to prevent acquired infections in the centre through continuous exposure to already infected animals or potential high environmental contamination. Faecal specimens

G. duodenalis isolates that tested positive by real-time PCR were subsequently assessed at the glutamate dehydrogenase (gdh) and β-giardin (bg) loci. The semi-nested-PCR protocol proposed by Read et al. (2004) with minor modifications was used to amplify a ~ 432-bp fragment of the gdh gene. PCR reaction mixtures (25 μL) consisted of 5 μL of template DNA, 0.5 μM of each primer (GDHeF/GDHiR in the primary reaction and GDHiF/GDHiR in the secondary reaction, respectively, Supplemental content 1), 2.5 units of MyTAQ™ DNA polymerase (Bioline GmbH, Luckenwalde, Germany), and 5 μL of MyTAQ™ Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl2. Both amplification protocols consisted of an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, with a final extension of 72 °C. Similarly, a ~ 511-bp fragment of the bg gene of G. duodenalis was amplified using the nested-PCR protocol described by Lalle et al.

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H. Gil et al. / Infection, Genetics and Evolution 50 (2017) 62–69

(2005). PCR reaction mixtures (25 μL) consisted of 3 μL of template DNA, 0.4 μM of each primer (G7_F/G759_R in the primary reaction and G99_F/ G609_R in the secondary reaction, respectively, Supplemental content 1), 2.5 units of MyTAQ™ DNA polymerase (Bioline GmbH), and 5 μL of MyTAQ™ Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl2. The primary PCR reaction was carried out with the following amplification conditions: 1 cycle of 95 °C for 7 min, followed by 35 cycles of 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 1 min with a final extension of 72 °C for 7 min. The conditions for the secondary PCR were identical to the primary PCR except that the annealing temperature was 55 °C. PCR reactions were carried out on a 2720 thermal cycler (Applied Biosystems). Laboratory-confirmed positive and negative DNA samples were routinely used as controls and included in each round of PCR. PCR amplicons were visualized on 2% D5 agarose gels (Conda, Madrid, Spain) stained with Pronasafe nucleic acid staining solution (Conda). Positive-PCR products were directly sequenced in both directions using the internal primer set described above. DNA sequencing was conducted by capillary electrophoresis using the BigDye® Terminator chemistry (Applied Biosystems). 2.6. Molecular detection and characterization of Cryptosporidium spp. isolates The presence of Cryptosporidium spp. was assessed using a nested-PCR protocol to amplify a 587-bp fragment of the ssu rRNA gene of the parasite (Tiangtip and Jongwutiwes, 2002). Amplification reactions were conducted in a volume of 50 μL consisting of 3 μL of DNA sample, 0.3 μM of each primer (CR-P1/CR-P2 in the primary reaction and CR-P3/CPB-DIAGR in the secondary reaction, respectively, Supplemental content 1), 2.5 units of MyTAQ™ DNA polymerase (Bioline GmbH), and 5 μL of MyTAQ™ Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl2. Both PCR reactions were carried out as follows: one cycle of 94 °C for 3 min, followed by 35 cycles of 94 °C for 40 s, 50 °C for 40 s and 72 °C for 1 min, concluding with a final extension of 72 °C for 10 min. Additionally, sub-typing of C. hominis isolates of canine origin was attempted at the 60-kDa glycoprotein (gp60) locus following the nested-PCR protocol proposed by Feltus et al. (2006). Agarose gel electrophoresis and DNA sequencing reagents were performed as described above. 2.7. Sensitivity analyses of the molecular methods Two human stool specimens of laboratory-confirmed clinical cases of giardiasis and cryptosporidiosis, respectively, were processed using the concentration system PARASEP Midi® (Grifols Movaco) following the manufacturer's instructions, except that formalin was replaced by saline solution. Five microliter of the obtained concentrate were fixed on welled slides per duplicate and prepared for immunofluorescence staining as described above. The Giardia cysts and Cryptosporidium oocysts were counted and their average concentrations per gram of faecal material calculated. A 200 μL volume of the faecal concentrate was subsequently extracted with the QIAamp® DNA Stool Mini Kit (QIAGEN) as described above. The relative sensitivity of the molecular methods used for the detection of G. duodenalis and Cryptosporidium spp. was assessed using 10-fold serial dilutions of the corresponding purified DNAs in experiments performed in two different days. G. duodenalis cyst concentrations in field faecal specimens that tested positive by real-time PCR were estimated by regressing cycle threshold (Ct) values of the unknown samples with log-transformed cyst numbers in the generated standard curve.

Raw sequencing data in both forward and reverse directions were viewed using the Chromas Lite version 2.1 sequence analysis program (http://chromaslite.software.informer.com/2.1/). The BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare nucleotide sequences with sequences retrieved from the National Center for Biotechnology Information (NCBI) database. Generated DNA consensus sequences were aligned to appropriate reference sequences using the MEGA 6 free software (http://www.megasoftware.net/) to identify Giardia species and assemblages/sub-assemblages and Cryptosporidium species (Tamura et al., 2013). For the identification of the phylogenetic inferences among the identified positive samples, a phylogenetic tree was inferred using the Neighbor-Joining method in MEGA 6. The evolutionary distances were computed using the Kimura 2-parameter method, and modelled with a gamma distribution. The reliability of the phylogenetic analyses at each branch node was estimated by the bootstrap method using 1000 replications. Representative reference sequences of the different G. duodenalis sub-assemblages taken from the NCBI database and sequences of human origin from a previous molecular epidemiological study conducted in Madrid, Spain (de Lucio et al., 2015) were also included in the phylogenetic analysis for comparative purposes. The sequences obtained in this study have been deposited in GenBank under accession numbers KX757733 to KX757755 (G. duodenalis) and KX774311 to KX774314 (Cryptosporidium spp.). 3. Results 3.1. Detection of G. duodenalis and Cryptosporidium spp. in canine and feline faecal samples DFAT- and/or PCR-positive results for G. duodenalis were obtained in 33% [95% Confident Interval (CI): 26–40%] and 9.2% (95% CI: 2.2–16%) of the canine (n = 194) and feline (n = 65) faecal samples tested, respectively. Cryptosporidium spp. was detected at lower infection rates of 4.1% (95% CI: 1.3–6.9%) and 4.6% (95% CI: −0.5–9.7%) in dog and cat populations, respectively (Table 1). Co-infections by both parasitic species were found in dogs (n = 3), but not in cats. G. duodenalis and Cryptosporidium spp. infections were generally detected by DFAT at low intensity in both canine and feline populations, ranging from 1 to N100 (oo)cysts per examined slide. Overall, 48% of the Giardia- and Cryptosporidiumpositive samples contained less than three (oo)cysts per slide. Similarly, Giardia real-time PCR-positive results had Ct values ranging from 24 to 40 [mean: 33; Standard Deviation (SD): 3.8] in dogs and from 26 to 36 (mean: 31; SD: 5.5) in cats. Coincidental DFAT and PCR-based results were obtained for 80% and 96% of the faecal samples tested for G. duodenalis and Cryptosporidium, respectively (Table 2). Both parasitic species (particularly the former) were more frequently detected by PCR than by DFAT. Three and four Giardia- and Cryptosporidium-positive samples by DFAT, respectively, could not be confirmed by molecular methods (Table 2). 3.2. Epidemiological analyses G. duodenalis and Cryptosporidium spp. infections were equally present in the canine and feline populations studied, independently of the sex, age group, status, and geographical origin of the animal. Although without statistical significance, both parasites tended to be more frequently found in dogs and cats younger than one year-old. Similarly, G. duodenalis was more common in stray/abandoned dogs than in surrendered dogs (P = 0.082).

2.8. Data analyses 3.3. Molecular characterization of G. duodenalis isolates The chi-square test was used to compare parasite infection rates in the canine and feline population under study by sex, age group, status (stray, abandoned or surrendered) and origin of the animals. A probability (P) value b 0.05 was considered evidence of statistical significance.

Among the 66 canine and feline DNA isolates that tested positive for G. duodenalis 30% (20/66) and 4.5% (3/66) were successfully amplified at the gdh and bg markers, respectively. A total of 18 (16

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Table 1 Prevalence of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats, as determined by direct fluorescent antibody and PCR-based methods, according to their sex, age group, status, and geographical region of origin. Variable

Sex Male Female No data Age (years) ≤1 N1 to ≤5 N5 No data Status Stray/abandoned Surrendered Origin Añana Ayala Campezo-Montaña Alavesa Gorbeialdea Laguardia-Rioja Alavesa Salvatierra Vitoria No data Total

Dogs

Cats

No.

G. duodenalis

%

Cryptosporidium

%

No.

G. duodenalis

%

Cryptosporidium

%

124 65 5

38 23 2

31 35 40

5 2 1

4.0 3.1 20

29 35 1

3 3 0

10 8.6 0.0

1 2 0

3.4 5.7 0.0

40 94 32 28

16 33 7 7

40 35 22 25

3 3 1 1

7.5 3.2 3.1 3.6

16 41 8 0

2 4 0 0

13 10 0.0 0.0

2 1 0 0

13 2.4 0.0 0.0

159 35

56 7

35 20

8 0

5.0 0.0

54 11

6 0

11 0.0

3 0

5.6 0.0

27 21 7 25 21 16 76 1 194

5 7 2 10 6 7 26 0 63

19 33 29 40 29 44 34 0.0 33

1 0 1 0 1 2 3 0 8

3.7 0.0 14 0.0 4.8 13 3.9 0.0 4.1

2 0 0 5 1 0 57 0 65

0 0 0 0 0 0 6 0 6

0.0 0.0 0.0 0.0 0.0 0.0 11 0.0 9.2

0 0 0 0 0 0 3 0 3

0.0 0.0 0.0 0.0 0.0 0.0 5.3 0.0 4.6

canine and two feline) isolates were characterized at the gdh gene only, and an additional canine isolate at the bg gene only. Multilocus genotyping data were available for two canine isolates (Table 3). None of the three (one canine and two feline) samples that tested positive by DFAT only could be amplified at the gdh and/or bg loci (see Table 2). In dogs, a total of 19 isolates were sub-genotyped at the gdh and/or bg loci, revealing the presence of assemblages A (37%), B (42%), C (16%), and D (5%). All seven assemblage A sequences belonged to the sub-assemblage AII and varied among them by 1–2 single-nucleotide polymorphisms (SNPs) including a number of heterozygous positions in the form of double peaks in the electropherograms. Four AII sequences corresponded to novel genotypes not previously reported in public databases (Table 3). A much higher genetic variability at the nucleotide level was observed among the assemblage B sequences, particularly those assigned to the sub-assemblage BIV at the gdh locus. All seven BIV (two reported in public databases, five novel) sequences varied among them by 3–8 SNPs, although polymorphic positions were only detected in a single isolate (Table 3). There was far less nucleotide variability in the few G. duodenalis isolates genotyped as assemblage C (n = 3) or D (n = 1) at both gdh and bg genes (Table 3). Only two feline isolates could be molecularly characterized at the gdh (but not the bg) marker, being genotyped as sub-assemblage AII and assemblage F, respectively. Both sequences were identical to the reference sequences used in this study (Table 3). As a consequence of the extensive nucleotide sequence heterogeneity observed at the gdh gene, particularly among the AII and BIV isolates, a number of polymorphic positions were demonstrated to cause amino-

acid substitutions in the deduced protein sequence (Supplemental content 2). As expected, our phylogenetic analysis revealed that the unambiguous (homozygous) sequences obtained in the present study clustered together in well-supported clades with the corresponding sub-assemblage reference sequences from NCBI (Fig. 1). Interestingly, the AII and BIV sequences of human origin from the Madrid area included in the analysis also fell within these clades (Fig. 1), although they tended to group into independent sub-groups reflecting noticeable changes at the nucleotide level with the canine and feline isolates. 3.4. Molecular characterization of Cryptosporidium spp. isolates A total of seven Cryptosporidium isolates were successfully genotyped at the ssu rRNA locus. Out of the six canine isolates obtained, five (83%) were assigned to host-specific C. canis and an additional one (17%) was unmistakably genotyped as human-specific C. hominis. Attempts to amplify this C. hominis isolate at the gp60 locus failed, so the sub-genotype of the parasite remains unknown. The only feline isolate characterized belonged to C. felis. Multiple sequence alignment analyses of all C. canis and C. felis sequences generated in this study produced perfect matches with their corresponding reference sequences, excepting a novel C. canis sequence not previously reported (Table 4). 3.5. Sensitivity analyses of the molecular methods Mean concentration of G. duodenalis cysts and Cryptosporidium oocysts in the two human stool specimens used as reference material

Table 2 Detection of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats, as determined by direct fluorescent antibody and PCR-based methods targeting the small subunit ribosomal RNA genes of these parasites. Test combinations

DFAT (+) and PCR (+) DFAT (+) and PCR (−) DFAT (−) and PCR (+) DFAT (−) and PCR (−) Total a b

Molecular detection by real-time PCR. Molecular detection by nested PCR.

Giardia duodenalisa

Cryptosporidium spp.b

Number of samples

Percentage

Number of samples

Percentage

17 3 49 190 259

6.6 1.1 19 73 100

1 4 6 248 259

0.4 1.5 2.3 95 100

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H. Gil et al. / Infection, Genetics and Evolution 50 (2017) 62–69

Table 3 Diversity, frequency, and molecular features of Giardia duodenalis isolates in sheltered dogs and cats in Álava, Spain. GenBank accession numbers are provided. Novel genotypes were shown underlined. Host Assemblage Sub-assemblage Isolate

Locus Reference sequence

Stretch

Dog

48_16 251_15 4_16 56_16 47_16 65_16 252_15 181_14 11_16 273_14 10_16 263_14 244_15 8_16 33_16

gdh gdh gdh gdh gdh gdh gdh gdh gdh gdh gdh gdh gdh gdh gdh

L40510 L40510 L40510 L40510 L40510 L40510 L40510 AF069059 L40508 L40508 L40508 L40508 L40508 L40508 L40508

84–476 84–476 84–476 84–476 84–476 84–476 84–476 48–440 84–476 84–476 84–476 84–476 84–476 84–476 84–476

9_14 240_15 142_15 9_14 3_14 3_14 259_15 28_14

gdh gdh bg bg gdh bg gdh gdh

U60984 U60984 AY545646 AY545646 U60986 AY545647 L40510 AF069057

A

AII

B

BIII BIV

C

D Cat

A F

AII

SNPsa

None T143Cb A164Gb C258Mb G118Ab, A400Cb G191Ab, A436Gb C263Mb, A367Rb C309T A122Gb, T387C, C423T T183C, T387C, C396T, C423T T169Wb, T183Y, A242Rb, T387C, C423T T183C, G202Ab, C273T, T387C, C432T T183C, T387C, C396T, C423T, C440Tb T183C, C184Tb, C345T, T387C, C396T, C423T A175Tb, T183C, A236Gb, G371Tb, T387C, C396T, C423T, G457Ab 84–476 None 84–476 T364A 11–495 C217T 11–495 C217T, C451T 84–476 None 128–238 A201G 200–461 None 84–476 None

GenBank accession no. KX757733 KX757734 KX757735 KX757736 KX757737 KX757738 KX757739 KX757740 KX757741 KX757742 KX757743 KX757744 KX757745 KX757746 KX757747 KX757748 KX757749 KX757753 KX757754 KX757750 KX757755 KX757751 KX757752

M: A/C; R: A/G; W: A/T; Y: C/T. a Single nucleotide polymorphism. b Point mutations inducing amino-acid substitutions at the protein level (see Supplemental content 2).

Fig. 1. Evolutionary relationships among assemblages of G. duodenalis at the gdh locus inferred by a Neighbor-Joining analysis of the nucleotide sequence covering a 359-bp region (positions 103 to 461 of GenBank accession number L40508) of the gene. Bootstrap values lower than 50% were not displayed. Filled circles and triangles represent canine an feline sequences, respectively, from this study. Open squares indicate sequences previously reported in human isolates from Madrid, Spain (de Lucio et al., 2015). Spironucleus vortens was used as outgroup taxa.

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Table 4 Diversity, frequency, and molecular features of Cryptosporidium spp. isolates in sheltered dogs and cats in Álava, Spain. GenBank accession numbers are provided. Novel genotypes were shown underlined. Host

Species

No. of isolates

Locus

Reference sequence

Stretch

SNPsa

GenBank accession No.

Dog

C. canis

Cat

C. hominis C. felis

4 1 1 1

ssu rRNA ssu rRNA ssu rRNA ssu rRNA

AF112576 AF112576 AAF108865 AF108862

542–1016 542–1016 590–975 551–1021

None 687_688insTGb, T739A, T785C None None

KX774311 KX774312 KX774313 KX774314

a b

Single nucleotide polymorphism. ins: nucleotide insertions.

were calculated at 640,000 cysts·g−1 and 8000 oocysts·g−1 of faeces, respectively. Serial dilutions of extracted G. duodenalis DNA were tested by real-time PCR and the detection limit of the technique was set at 640 cysts·g−1 of faeces (Fig. 2, panel A). A standard curve was constructed by plotting obtained Ct values against log10 of G. duodenalis cyst numbers (Fig. 2, panel B). Estimated concentrations of G. duodenalis cysts in canine and feline faecal specimens that tested positive to the parasite by real-time PCR ranged from 53 to 4.8·106 cysts·g−1 of faeces (Supplemental content 3). Overall, 47% of the real-time PCR-positive samples had

b1000 cysts·g−1 of faeces. Using the serial dilutions of G. duodenalis DNA described above, the detection limit of the gdh-PCR was calculated at 64,000 cysts·g−1 of faecal material (Fig. 2, panel C), two order of magnitude more than that obtained by real-time PCR. No amplification product were obtained at the bg marker with any of the DNA dilutions tested (data not shown), demonstrating that the bg-PCR was at least two order of magnitude less sensitive than gdh-PCR. Similarly, the sensitivity of the ssu rRNA-PCR for the detection of Cryptosporidium was estimated at 800 oocysts·g−1 of faeces respectively (Fig. 2, panel D).

Fig. 2. Sensitivity analyses of the molecular methods used in this study for the detection, genotyping, and sub-genotyping of Giardia duodenalis and Cryptosporidium spp. Panel A: PCRbased results obtained with the serial dilutions of extracted DNA from known amounts of (oo)cysts tested by real-time PCR (G. duodenalis) or ssu-PCR (Cryptosporidium spp.). Ct values are shown for G. duodenalis. Panel B: Standard curve used to estimate G. duodenalis cyst concentrations in field specimens. Panel C: PCR detection limit of gdh amplicons on 2% agarose gel from the serial dilutions of G. duodenalis DNA. Lane 1: negative control; Lane 2: positive control; Lane 3: 100 bp DNA marker; Lane 4: 640 cysts·g−1 of faeces; Lane 5: 6400 cysts·g−1 of faeces; Lane 6: 64,000 cysts·g−1 of faeces; Lane 7: 640,000 cysts·g−1 of faeces. Panel D: PCR detection limit of ssu rRNA amplicons on 2% agarose gel from the serial dilutions of Cryptosporidium spp. DNA. Lane 1: 100 bp DNA marker; Lane 2: 8 oocysts·g−1 of faeces; Lane 3: 80 oocysts·g−1 of faeces; Lane 4: 800 oocysts·g−1 of faeces; Lane 5: 8000 oocysts·g−1 of faeces; Lane 6: negative control; Lane 7: positive control.

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4. Discussion In Spain canine giardiasis has been reported at 6–38% in Barcelona (Gracenea et al., 2009; Ortuño et al., 2014), 1% in Córdoba (MartínezMoreno et al., 2007), 10% in Murcia (Martínez-Carrasco et al., 2007), and 7–16% in Madrid (Miró et al., 2007; Dado et al., 2012b) using routine coprological examination in a variety of dog (owned, sheltered, stray) populations. A G. duodenalis infection rate of 4% has also been documented in sheltered cats in Madrid (Dado et al., 2012b). Additionally, a survey conducted in public parks of the later region revealed the presence of G. duodenalis cysts in 18% and 5% of the faecal and soil samples collected (Dado et al., 2012a). In our study, G. duodenalis has been found at considerable higher prevalences in the canine (33%) and feline (9%) populations surveyed, in agreement with the universal superior diagnostic sensitivity of PCR-based methods over conventional microscopy (Bouzid et al., 2015). Similarly, Cryptosporidium spp. was identified in 4% of dogs and 5% of cats, respectively. A higher prevalence of 7% has been previously found in dogs from Zaragoza (Causapé et al., 1996). Aside from geographical considerations, this difference in the prevalence may be explained because in the later study half of the Cryptosporidium-positive cases presented with diarrhoea, whereas in our survey most of the studied animals were asymptomatic. A recent meta-analysis work has demonstrated that symptomatic dogs and cats had higher prevalence rates ratios than animals with sub-clinical infections (Bouzid et al., 2015). Because of their immature immune system and poor care condition, dogs and cats younger than one year-old and animals in the stray/abandoned group harboured the highest infection rates by G. duodenalis and Cryptosporidium spp. Consistent with the generalized absence of clinical signs in our animal population, both G. duodenalis and Cryptosporidium spp. were identified causing light infections, as demonstrated by the relatively low (oo)cysts numbers detected by DFAT (or, in the case of G. duodenalis, estimated by real-time PCR) in the faecal specimens examined. This finding will certainly compromise the diagnostic performance of the molecular methods used in this study, mainly those based on the amplification of single-copy genes such as gdh and bg of G. duodenalis. The poor sensitivity of the gdh- and bg-PCR protocols (particularly the later) is further exacerbated by limitations in the PCR primer design, as recently demonstrated (de Lucio et al., 2016). Taken together, these facts help to explain the considerably lower PCR sensitivity obtained at the gdh and bg loci (between 2 and 4 order of magnitude) compared to that for the real-time PCR protocol targeting the multi-copy ssu rRNA gene. Surprisingly, our molecular analyses revealed that dogs were secondary infected (21%) by host-specific G. duodenalis assemblages C/D, whereas potential human-pathogenic assemblages A/B were found in 79% of the isolates genotyped. Very similar results (11% vs. 89%) have been previously reported in dogs from Madrid by PCR-RFLP (Dado et al., 2012b), whereas only assemblages C/D were identified in a limited number of isolates from sheltered and hunting dogs in Catalonia (Ortuño et al., 2014). These results seem to indicate that the diversity and frequency of G. duodenalis assemblages and sub-assemblages may vary depending on the geographical region and the dog population considered. Subsequent sub-genotyping studies at the gdh locus evidenced that AII and BIV were the predominant G. duodenalis sub-assemblages circulating in the studied canine population. Interestingly, an unexpected high degree of genetic variability was observed within assemblage A, with all the AII isolates analysed differing by at least one SNP and most of them representing novel genetic variants. This is in contrast with the findings reported in human molecular studies, where very little variability has been regularly detected within this G. duodenalis assemblage (Sprong et al., 2009; Cooper et al., 2010; de Lucio et al., 2015). As anticipated, canine BIV isolates exhibited a much greater degree of genetic diversity, resulting in the identification of five novel genotypes with up to eight polymorphic sites per sequence. Among the fifteen canine A/B isolates sub-genotyped at the gdh marker only single AII (KX757733) and BIV (KX757742) genotypes have been commonly

detected in Spanish human populations earlier (de Lucio et al., 2015). The very same AII genotype was also found infecting a cat in the present study (KX757751). The only canine isolate assigned to BIII represented a known genetic variant previously identified infecting humans in Australia (JQ700450), Japan (AB195224), and Palestine (AB295654), but not in Spain. G. duodenalis assemblages C and D have strong host specificities and narrow host ranges. Both are considered of limited zoonotic relevance, and the sporadic cases of human infections caused by these assemblages have been normally detected in immunocompromised individuals (Feng and Xiao, 2011; Ryan and Cacciò, 2013). Compared with assemblage A/B isolates, a much lower degree of genetic diversity at the nucleotide level was found in assemblage C/D isolates both at the gdh and bg genes. As a consequence, most of the genetic variants reported here represent known genotypes. Of particular interest is the finding that one of our C isolates characterized at the gdh marker (KX757748) has only been documented in a few dogs from Brazil (EF507623), India (KJ499990), and Thailand (e.g. KT634139). A similar situation occurs with our D isolate at the same locus (KX757750), only reported to date in dogs from Brazil (e.g. KT728539) and USA (JX448631). Taken together, and although limited by the low number of gdh and bg sequences available for comparative molecular studies and the potential bias associated to their different geographical origin, our data suggest that pet dogs and cats can harbour potentially zoonotic sub-assemblages AII, BIII, and BIV. Regarding Cryptosporidium, both dogs and cats were infected by the expected host-specific species C. canis and C. felis, respectively. Of relevance was the finding of a novel C. canis genotype (KX774312). Another interesting outcome was the identification of C. hominis in one of the canine isolates genotyped. C. hominis is well-known to infect exclusively humans, but this notion has been challenged by recent reports demonstrating the presence of the parasite in cattle in Brazil (Inácio et al., 2017), horses and donkeys in Algeria and China (Laatamna et al., 2015; Jian et al., 2016), a sheep and a goat in UK (Giles et al., 2009), wild foxes in Australia (Schiller et al., 2016), and a wild badger in Spain (Mateo et al., 2017). Although accidental acquisition and mechanical carriage of C. hominis oocysts of anthroponotic origin via environmental contamination may explain some of the above mentioned cases, at present there is growing molecular epidemiological evidence suggesting that C. hominis may indeed be able to infect, colonise, and be secreted by a number of non-human species. Because of its potential public and veterinary health impact, more research should be conducted to ascertain the actual extent of this possibility. A number of factors may limit the accuracy of our epidemiological and molecular results. For instance, G. duodenalis and Cryptosporidium spp. infection rates reported here are probably underestimations of the true prevalences, as diagnosis was based on the analysis of a single faecal sample per animal and both pathogens are known to be intermittently shedded. As discussed above, low parasitic burdens and inadequate sensitivities of the conventional and molecular diagnostic methods used will enhance this problem. More importantly, no sequences of G. duodenalis and Cryptosporidium spp. of human origin from the province of Álava were available for comparative analyses, so the assessment of the zoonotic potential of our canine and feline isolates was partial and need to be fully addressed in future molecular studies involving human isolates from this region. In summary, the epidemiological scenario depicted by our study indicate that pet dogs and cats can act as significant contributors to G. duodenalis cyst and Cryptosporidium spp. oocyst environmental contamination in the province of Álava. Although zoonotic transmission seems to be a rare event in normal circumstances, the actual extent of this statement need to be confirmed in larger molecular epidemiological surveys studying simultaneously isolates of human and animal origin. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2017.02.013.

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Acknowledgments This study was funded by the Health Institute Carlos III, Ministry of Economy and Competitiveness under project CP12/03081. The authors are grateful to Andrés Cabeza Vengoechea, Pablo Batanete Ginés, Rubén Fernández Martín, and Olga Ocio Lanz of the Department of Social Policy and Public Health, City Council of Vitoria-Gasteiz, for conducting the sampling of this study and their logistic assistance. References Ballweber, L.R., Xiao, L., Bowman, D.D., Kahn, G., Cama, V.A., 2010. Giardiasis in dogs and cats: update on epidemiology and public health significance. Trends Parasitol. 26, 180–189. Batchelor, D.J., Tzannes, S., Graham, P.A., Wastling, J.M., Pinchbeck, G.L., German, A.J., 2008. Detection of endoparasites with zoonotic potential in dogs with gastrointestinal disease in the UK. Transbound. Emerg. Dis. 55, 99–104. Beser, J., Toresson, L., Eitrem, R., Troell, K., Winiecka-Krusnell, J., Lebbad, M., 2015. Possible zoonotic transmission of Cryptosporidium felis in a household. Infect. Ecol. Epidemiol. 5, 28463. Bouzid, M., Halai, K., Jeffreys, D., Hunter, P.R., 2015. The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples. Vet. Parasitol. 207, 181–202. Cano, L., de Lucio, A., Bailo, B., Cardona, G.A., Muadica, A.S., Lobo, L., Carmena, D., 2016. Identification and genotyping of Giardia spp. and Cryptosporidium spp. isolates in aquatic birds in the Salburua wetlands, Álava, Northern Spain. Vet. Parasitol. 221, 144–148. Cardona, G.A., Carabin, H., Goñi, P., Arriola, L., Robinson, G., Fernández-Crespo, J.C., Clavel, A., Chalmers, R.M., Carmena, D., 2011. Identification and molecular characterization of Cryptosporidium and Giardia in children and cattle populations from the province of Álava, North of Spain. Sci. Total Environ. 412–413, 101–108. Cardona, G.A., de Lucio, A., Bailo, B., Cano, L., de Fuentes, I., Carmena, D., 2015. Unexpected finding of feline-specific Giardia duodenalis assemblage F and Cryptosporidium felis in asymptomatic adult cattle in Northern Spain. Vet. Parasitol. 30, 258–263. Carmena, D., Aguinagalde, X., Zigorraga, C., Fernández-Crespo, J.C., Ocio, J.A., 2007. Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in Northern Spain. J. Appl. Microbiol. 102, 619–629. Carmena, D., Cardona, G.A., Sánchez-Serrano, L.P., 2012. Current situation of Giardia infection in Spain: implications for public health. World J. Clin. Infect. Dis. 2, 1–12. Causapé, A.C., Quílez, J., Sánchez-Acedo, C., del Cacho, E., 1996. Prevalence of intestinal parasites, including Cryptosporidium parvum, in dogs in Zaragoza city, Spain. Vet. Parasitol. 67, 161–167. Chomel, B.B., 2014. Emerging and re-emerging zoonoses of dogs and cats. Animals 4, 434–435. Cooper, M.A., Sterling, C.R., Gilman, R.H., Cama, V., Ortega, Y., Adam, R.D., 2010. Molecular analysis of household transmission of Giardia lamblia in a region of high endemicity in Peru. J. Infect. Dis. 202, 1713–1721. Dado, D., Izquierdo, F., Vera, O., Montoya, A., Mateo, M., Fenoy, S., Galván, A.L., García, S., García, A., Aránguez, E., López, L., del Águila, C., Miró, G., 2012a. Detection of zoonotic intestinal parasites in public parks of Spain. Potential epidemiological role of microsporidia. Zoonoses Public Health 59, 23–28. Dado, D., Montoya, A., Blanco, M.A., Miró, G., Saugar, J.M., Bailo, B., Fuentes, I., 2012b. Prevalence and genotypes of Giardia duodenalis from dogs in Spain: possible zoonotic transmission and public health importance. Parasitol. Res. 111, 2419–2422. Epe, C., Rehkter, G., Schnieder, T., Lorentzen, L., Kreienbrock, L., 2010. Giardia in symptomatic dogs and cats in Europe—results of a European study. Vet. Parasitol. 173, 32–38. Esch, K.J., Petersen, C.A., 2013. Transmission and epidemiology of zoonotic protozoan diseases of companion animals. Clin. Microbiol. Rev. 26, 58–85. Feltus, D.C., Giddings, C.W., Schneck, B.L., Monson, T., Warshauer, D., McEvoy, J.M., 2006. Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin. J. Clin. Microbiol. 44, 4303–4308. Feng, Y., Xiao, L., 2011. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin. Microbiol. Rev. 24, 110–140. Giles, M., Chalmers, R., Pritchard, G., Elwin, K., Mueller-Doblies, D., Clifton-Hadley, F., 2009. Cryptosporidium hominis in a goat and a sheep in the UK. Vet. Rec. 164, 24–25. Gracenea, M., Gómez, M.S., Torres, J., 2009. Prevalence of intestinal parasites in shelter dogs and cats in the metropolitan area of Barcelona (Spain). Acta Parasitol. 54, 73–77. Hodgson, K., Barton, L., Darling, M., Antao, V., Kim, F.A., Monavvari, A., 2015. Pets' impact on your patients' health: leveraging benefits and mitigating risk. J. Am. Board Fam. Med. 28, 526–534. Inácio, S.V., Widmer, G., de Brito, R.L., Zucatto, A.S., de Aquino, M.C., Oliveira, B.C., Nakamura, A.A., Neto, L.D., Carvalho, J.G., Gomes, J.F., Meireles, M.V., Bresciani, K.D., 2017. First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil. Vet. Parasitol. 233, 48–51. Inpankaew, T., Traub, R., Thompson, R.C., Sukthana, Y., 2007. Canine parasitic zoonoses in Bangkok temples. Southeast Asian J. Trop. Med. Public Health. 38, 247–255. Inpankaew, T., Schär, F., Odermatt, P., Dalsgaard, A., Chimnoi, W., Khieu, V., Muth, S., Traub, R.J., 2014. Low risk for transmission of zoonotic Giardia duodenalis from dogs to humans in rural Cambodia. Parasit. Vectors 7, 412. Jezkova, J., Horcickova, M., Hlaskova, L., Sak, B., Kvetonova, D., Novak, J., Hofmannova, L., McEvoy, J., Kvac, M., 2016. Cryptosporidium testudinis sp. n., Cryptosporidium ducismarci Traversa, 2010 and Cryptosporidium tortoise genotype III (Apicomplexa: Cryptosporidiidae) in tortoises. Folia Parasitol. 14, 63.

69

Jian, F., Liu, A., Wang, R., Zhang, S., Qi, M., Zhao, W., Shi, Y., Wang, J., Wei, J., Zhang, L., Xiao, L., 2016. Common occurrence of Cryptosporidium hominis in horses and donkeys. Infect. Genet. Evol. 43, 261–266. Kváč, M., Havrdová, N., Hlásková, L., Daňková, T., Kanděra, J., Ježková, J., Vítovec, J., Sak, B., Ortega, Y., Xiao, L., Modrý, D., Chelladurai, J.R., Prantlová, V., McEvoy, J., 2016. Cryptosporidium proliferans n. sp. (Apicomplexa: Cryptosporidiidae): molecular and biological evidence of cryptic species within gastric Cryptosporidium of mammals. PLoS One 11, e0147090. Laatamna, A.E., Wagnerová, P., Sak, B., Květoňová, D., Xiao, L., Rost, M., McEvoy, J., Saadi, A.R., Aissi, M., Kváč, M., 2015. Microsporidia and Cryptosporidium in horses and donkeys in Algeria: detection of a novel Cryptosporidium hominis subtype family (Ik) in a horse. Vet. Parasitol. 208, 135–142. Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D., Cacciò, S.M., 2005. Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int. J. Parasitol. 35, 207–213. de Lucio, A., Martínez-Ruiz, R., Merino, F.J., Bailo, B., Aguilera, M., Fuentes, I., Carmena, D., 2015. Molecular genotyping of Giardia duodenalis isolates from symptomatic individuals attending two major public hospitals in Madrid, Spain. PLoS One 10, e0143981. de Lucio, A., Amor-Aramendía, A., Bailo, B., Saugar, J.M., Anegagrie, M., Arroyo, A., LópezQuintana, B., Zewdie, D., Ayehubizu, Z., Yizengaw, E., Abera, B., Yimer, M., Mulu, W., Hailu, T., Herrador, Z., Fuentes, I., Carmena, D., 2016. Prevalence and genetic diversity of Giardia duodenalis and Cryptosporidium spp. among school children in a rural area of the Amhara Region, North-West Ethiopia. PLoS One 11, e0159992. Lucio-Forster, A., Griffiths, J.K., Cama, V.A., Xiao, L., Bowman, D.D., 2010. Minimal zoonotic risk of cryptosporidiosis from pet dogs and cats. Trends Parasitol. 26, 174–179. Martínez-Carrasco, C., Berriatua, E., Garijo, M., Martínez, J., Alonso, F.D., de Ybáñez, R.R., 2007. Epidemiological study of non-systemic parasitism in dogs in southeast Mediterranean Spain assessed by coprological and post-mortem examination. Zoonoses Public Health 54, 195–203. Martínez-Moreno, F.J., Hernández, S., López-Cobos, E., Becerra, C., Acosta, I., MartínezMoreno, A., 2007. Estimation of canine intestinal parasites in Córdoba (Spain) and their risk to public health. Vet. Parasitol. 143, 7–13. Mateo, M., Hernández de Mingo, M., de Lucio, A., Morales, L., Balseiro, A., Espí, A., Barral, M., Lima-Barbero, J.F., Habela, M.A., Fernández-García, J.L., Calero-Bernal, R., Köster, P.C., Cardona, G.A., Carmena, D., 2017. Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain. Vet. Parasitol. 235, 86–93. Miró, G., Mateo, M., Montoya, A., Vela, E., Calonge, R., 2007. Survey of intestinal parasites in stray dogs in the Madrid area and comparison of the efficacy of three anthelmintics in naturally infected dogs. Parasitol. Res. 100, 317–320. Mohamed, A.S., Levine, M., Camp, J.W., Lund, E., Yoder, J.S., Glickman, L.T., Moore, G.E., 2014. Temporal patterns of human and canine Giardia infection in the United States: 2003–2009. Prev. Vet. Med. 113, 249–256. Navarro-i-Martinez, L., del Águila, C., Bornay-Llinares, F., 2011. Cryptosporidium: un género en revisión. Situación en España. Enferm. Infecc. Microbiol. Clin. 29, 135–143. Ortuño, A., Scorza, V., Castellà, J., Lappin, M., 2014. Prevalence of intestinal parasites in shelter and hunting dogs in Catalonia, Northeastern Spain. Vet. J. 199, 465–467. Osman, M., Bories, J., El Safadi, D., Poirel, M.T., Gantois, N., Benamrouz-Vanneste, S., Delhaes, L., Hugonnard, M., Certad, G., Zenner, L., Viscogliosi, E., 2015. Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk. Vet. Parasitol. 214, 167–170. Overgaauw, P.A., van Zutphen, L., Hoek, D., Yaya, F.O., Roelfsema, J., Pinelli, E., van Knapen, F., Kortbeek, L.M., 2009. Zoonotic parasites in fecal samples and fur from dogs and cats in The Netherlands. Vet. Parasitol. 163, 115–122. Paoletti, B., Traversa, D., Iorio, R., De Berardinis, A., Bartolini, R., Salini, R., Di Cesare, A., 2015. Zoonotic parasites in feces and fur of stray and private dogs from Italy. Parasitol. Res. 114, 2135–2141. Read, C.M., Monis, P.T., Thompson, R.C., 2004. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect. Genet. Evol. 4, 125–130. Ryan, U., Cacciò, S.M., 2013. Zoonotic potential of Giardia. Int. J. Parasitol. 43, 943–956. Ryan, U., Fayer, R., Xiao, L., 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141, 1667–1685. Schiller, S.E., Webster, K.N., Power, M., 2016. Detection of Cryptosporidium hominis and novel Cryptosporidium bat genotypes in wild and captive Pteropus hosts in Australia. Infect. Genet. Evol. 44, 254–260. Sprong, H., Cacciò, S.M., van der Giessen, J.W., 2009. Identification of zoonotic genotypes of Giardia duodenalis. PLoS Negl. Trop. Dis. 3, e558. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tiangtip, R., Jongwutiwes, S., 2002. Molecular analysis of Cryptosporidium species isolated from HIV-infected patients in Thailand. Tropical Med. Int. Health 7, 357–364. Verweij, J.J., Schinkel, J., Laeijendecker, D., van Rooyen, M.A., van Lieshout, L., Polderman, A.M., 2003. Real-time PCR for the detection of Giardia lamblia. Mol. Cell. Probes 17, 223–225. Volotão, A.C., Costa-Macedo, L.M., Haddad, F.S., Brandão, A., Peralta, J.M., Fernandes, O., 2007. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop. 102, 10–19. Wells, D.L., 2007. Domestic dogs and human health: an overview. Br. J. Health Psychol. 12, 145–156. Xiao, L., Feng, Y., 2008. Zoonotic cryptosporidiosis. FEMS Immunol. Med. Microbiol. 52, 309–323. Xiao, L., Cama, V.A., Cabrera, L., Ortega, Y., Pearson, J., Gilman, R.H., 2007. Possible transmission of Cryptosporidium canis among children and a dog in a household. J. Clin. Microbiol. 45, 2014–2016.