Cross-sectional survey on Tritrichomonas foetus infection in Italian cats

Veterinary Parasitology: Regional Studies and Reports 6 (2016) 14–19

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Cross-sectional survey on Tritrichomonas foetus infection in Italian cats VeronesiF. a,1, GazzonisA.L. b,1, NapoliE. c, BriantiE. c, SantoroA. a, ZanzaniS.A. b, OlivieriE. a, DiaferiaM. a, GiannettoS. c, PennisiM.G. c, ManfrediM.T. b,⁎ a b c

Department of Veterinary Medicine, University of Perugia, Italy Department of Veterinary Medicine, Università degli Studi di Milano, Italy Department of Veterinary Sciences, University of Messina, Italy

a r t i c l e

i n f o

Article history: Received 29 April 2016 Received in revised form 1 August 2016 Accepted 28 November 2016 Available online 30 November 2016 Keywords: Cat Tritrichomonas foetus Italy Prevalence Risk factors

a b s t r a c t The feline genotype of Tritrichomonas foetus is a widespread cause of large-bowel diarrhoea in cats. The aim of this study was to determine the prevalence of the T. foetus infection in cat populations across Italy. Fresh, individual faecal samples were collected from 267 cats, kept in different environments (i.e., private households, breeding structures, municipal catteries and colonies) in three different sites across Italy. The faecal samples were tested by PCR to detect T. foetus. Moreover, the same samples were subjected to a concentrationflotation technique and a commercial direct fluorescent-antibody (DFA) test to detect additional enteric parasites, including Giardia duodenalis. The overall prevalence of T. foetus infection was 5.2%. All the infected cats showed diarrhoea at the time of sampling: 9 out of 14 positive cats were co-infected with G. duodenalis, 1 with Toxocara cati and 3 with Dipylidium caninum. The risk factor analysis showed that not only the breed, but also co-infections with G. duodenalis and Dipylidium caninum were significantly associated with the presence of T. foetus. This study confirms the presence of T. foetus in cats living in Italy, suggesting that this protozoan parasite should always be included in the differential diagnosis of patients referred with large-bowel disease symptoms, especially if they were purebred animals, or affected by other enteric protozoa, such as G. duodenalis. © 2016 Published by Elsevier B.V.

1. Introduction Tritrichomonas foetus (Trichomonadida, Tritrichomonadidae) is a flagellated protozoan parasite, commonly regarded as a worldwide venereal pathogen of cattle (BonDurant, 1997; Felleisen et al., 1998; Yao, 2013) and recently recognized as agent of feline diarrhoea (feline trichomoniasis) (Gookin et al., 1999; Gookin et al., 2001; Levy et al., 2003). Recent studies investigating the genetic relationship among T. foetus isolated from cattle and domestic cats support the existence of two different genotypes e.g. “cattle genotype” and “cat genotype”, host adapted, that differ each other further than for biological and pathogenic behaviours (Stockdale et al., 2008; Slapeta et al., 2010, 2012). Like other trichomonad protozoa, T. foetus only has the trophozoite stage and it is distinguished by a direct life cycle, as the oral-faecal route is the most common transmission pathway. However, it has been demonstrated that T. foetus trophozoite stages could survive in different substrates (i.e., diarrhoeic stool, feline urine and cat food) (Hale et al., 2009; Rosypal et al., 2012), suggesting that transmission is not ⁎ Corresponding author at: Department of Veterinary Medicine, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy. E-mail address: [email protected] (M.T. Manfredi). 1 These two Authors have contributed equally to the work.

http://dx.doi.org/10.1016/j.vprsr.2016.11.004 2405-9390/© 2016 Published by Elsevier B.V.

only through close contact between cats, but also contaminated food or water may be sources of infection. Moreover, some Authors speculated that slugs may facilitate the passive transmission of T. foetus between cats, since the trophozoite stages can survive as they pass through the alimentary tract of common slug species (Van der Saag et al., 2011). T. foetus infection may develop from an asymptomatic form to severe large-bowel syndromes, distinguished by malodorous, pasty, yellowgreen faeces (Xenoulis et al., 2013), mucous and fresh blood in the faeces, flatulence, tenesmus, anus inflammation and faecal incontinence (Gookin et al., 1999; Mardell and Sparkes, 2006). Infected cats may have persistent diarrhoea for up to 2 years and can remain infected for life (Foster et al., 2004; Stockdale et al., 2009), acting as asymptomatic spreaders. Systemic signs, including anorexia, depression, vomiting and weight loss, can also be observed (Yao and Köster, 2015). T. foetus infection in cats has been reported in several countries, including the USA (Gookin et al., 2004), Australia (Bissett et al., 2008; Bell et al., 2010), New Zealand (Kingsbury et al., 2010), South Korea (Lim et al., 2010) and Japan (Doi et al., 2013). In Europe, surveys have been conducted in the UK (Gunn-Moore et al., 2007), Switzerland (Burgener et al., 2009; Frey et al., 2009), Greece (Xenoulis et al., 2010), Spain (Mirò et al., 2011) and France (Profizi et al., 2013). In Italy, only a few studies have investigated T. foetus infection in cat populations (Holliday et al., 2009; Mancianti et al., 2015) and no large-scale,

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epidemiological studies have been conducted in the country so far. Therefore, the aim of this study was to carry out a large-scale, epidemiologic survey across Italy in order to: i) investigate the prevalence of T. foetus in cats; ii) determine the feline genotype of T. foetus occurring in Italian cats; iii) elucidate the risk factors associated with feline trichomoniasis. 2. Materials and methods 2.1. Study population A multi-centric, cross-sectional survey was conducted from 2012 to 2014 in three different Italian sites. A total of 267 cats were included in the study, selected among patients referred to the Veterinary Teaching Hospitals of the Università degli Studi di Milano, north-eastern Italy (Site 1, No = 114 cats), the University of Perugia, central Italy (Site 2, No = 90 cats), and the University of Messina, southern Italy (Site 3, No = 63 cats). Animals were registered in the survey regardless of their age, sex, breed, or presence of gastro-intestinal symptoms. Faecal samples were collected at the time of consultation or by the owner within 24 h, in most cases directly from litters; subsamples used for DNA isolation were then collected superficially in order to take a sample of mucus layer. Each sample was stored at +4 °C until further examination. Individual, anamnestic data (e.g. age, gender, reproductive status, breed and presence of diarrhoea at sampling time), and also housing environment (e.g. breeding structures, municipal catteries, colonies, private households) were recorded for each cat. 2.2. DNA extraction and PCR amplification Two hundred micrograms of each faecal sample were submitted to genomic DNA extraction using a QIAamp® Fast DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany), according to the modified protocol described by Gookin et al. (2002). These modifications included the prolonged incubation of each sample in a Proteinase K solution (20 μl) for 1 h at 56 °C (manufacturer step 10), washing the extracted DNA twice with 500 μl of buffer AW1 (manufacturer step 15), and adding an extra centrifugation step after the final wash in buffer AW2 (manufacturer step 16). A faecal sample that had scored positive for T. foetus in a previous study (Zanzani et al., 2016) was submitted to DNA extraction in parallel to be used as a positive control in each PCR run. The DNA quantity and quality was measured by reading the absorption spectrum (220–750 nm) and calculating the absorbance ratio at 260/280 nm with NanoDrop (Biophotometer, Eppendorf AG, Hamburg, Germany). The genomic DNA was tested by means of a nested PCR protocol described by Gookin et al. (2002) with some modifications in the thermic conditions. The 1st reaction amplified a 348 bp fragment of the 5.8S ribosomal DNA (rDNA) and the flanking internal transcribed spacer regions (ITS-1, ITS-2) of the T. foetus genome and the subsequent reaction amplified an internal 208 bp fragment including part of the 5.8S rDNA and the ITS-1. Each reaction mixture consisted of 10 μl aliquots of DNA template, 25 μl of NZYTaq 2× Green Master Mix (NZYTech Lda, Lisbon, Portugal), 0.2 μM of each primer (TFR4/TFR3 for the 1st reaction and TFITS-F/ TFITS-R for the 2nd reaction) and RNase-free water to make a final volume of 50 μl. The DNA amplifications were carried out in a thermal cycler StepOnePlus™ (Applied Biosystems, Foster City, CA). The following cyclic profile was used in the 1st reaction: initial denaturation at 95 °C for 5 min, followed by 40 cycles of either denaturation at 95 °C for 30 s, annealing at 67 °C for 30 s and an extension at 72 °C for 30 s, followed by a final extension for 7 min at 72 °C. For the subsequent PCR the following temperature profiles was applied: initial denaturation at 95 °C for 5 min, followed by 50 cycles of either denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s and an extension at 72 °C for 30 s, followed by a final extension for 7 min at 72 °C. To check for the presence of contamination, a negative control sample containing all of the reaction

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regents with sterile distilled water to substitute for the DNA template was added at each PCR run as well as the positive control. The PCR products of both the 1st and 2nd reactions were run separately in stained (SafeView NBS biologicals, Cambridgeshire, England) 1.2% agarose gel electrophoresis at 130 V for 30 min. The bands of the expected size were separated and visualized on a UV transilluminator (EuroClone S.p.a., Milan, Italy). When bands of an appropriate size for each primer set used were examined, they were excised and extracted using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's recommendations. All the amplified products obtained from PCR were bidirectionally sequenced using a 16-capillary ABI PRISM 3130 × l Genetic Analyzer, assembled and edited with Chromas software version 2.33 (Technelysium Pty. Ltd., Australia). The sequences were then compared with representative T. foetus sequences available in GenBank using Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1990). 2.3. Copro-microscopic examinations Faecal samples were subjected to a concentration-flotation technique. Briefly, 5 g of each faecal sample was processed by homogenization with phosphate buffered saline solution (PBS, pH 7.4), filtered through a 250 μm-mesh filter and subjected to one cycle of centrifugation (4400g for 10 min). The supernatant was discarded and the sediment was re-suspended with zinc sulphate (ZnSO4) solution (1180 specific gravity) and centrifuged a second time at 4400g for 10 min. Then, the flotation solution was added until a rounded meniscus was formed on the surface of the mixture; the wet mount was microscopically observed at 100× and 400× magnification to detect pre-imaginal form of gastro-intestinal parasites. In Site 3 for the detection of G. duodenalis cyst for each animal three different samples collected in one week were analysed as describe above. In site 1 and 2 a commercially available Direct Immunofluorescence Assay (DFA) (MeriFluor®, Meridian, Bioscience, Cincinnati, OH, USA) was performed and interpreted according to the manufacturer's instructions to detect G. duodenalis. 2.4. Statistical analysis A univariate, binary, logistic regression analysis was performed to explore the relation between T. foetus infection and associated risk factors, including sampling site, age (young: ≤1 year; adult: N1 year), gender, reproductive status (neutered or intact), breed (cross-breed or pure-breed), presence of diarrhoea, housing environment (private household, breeding structures, municipal catteries, colonies), infections with other parasites. All variables showing a p-value b 0.1 were subsequently entered into a multivariate model, developed by backward elimination until all remaining variables were significant (pvalue b 0.05). Goodness-of-fit of the model was assessed by the Hosmer-Lemeshow statistic. Results were presented as adjusted odds ratios (OR) with 95% confidence intervals (CI). Statistical analysis was performed using a commercial software (SPSS, Version 22.0; Chicago, IL). 3. Results The studied population consisted of 140 owned cats (53 cats from breeding structures and 87 from private households) and 127 unowned cats (63 from colonies and 64 from municipal catteries). According to age, 127 adults and 140 young cats were included in the study. There were 142 females and 125 males, which included 176 neutered cats. Two hundred and one animals were common European short hair, whereas 66 cats belonged to 13 different purebreds (Table 1). Eighty-five cats showed diarrhoea at the time of sampling. The 1st PCR amplification using the set primers TFR4/TFR3 did not yield any

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F. Veronesi et al. / Veterinary Parasitology: Regional Studies and Reports 6 (2016) 14–19 Table 1 Breeds and number of cats enrolled in the study. Breed

N

Common European shorthair Balinese Bengal Birman British shorthair Chartreux Main Coon Norwegian Forest cat Persian Ragdoll Russian Blue Siamese Turkish Angora Turkish Van

201 1 3 10 2 1 22 7 7 7 3 1 1 1

visible product at the gel agarose run, however 14 out of the 267 faecal samples (5.2%) scored positive for T. foetus specific DNA at the subsequent amplification using the primer combination TFITS-F/TFITS-R. All the 14 PCR amplicons were successfully sequenced yielding sequences of 202 bp length, that tested identical each other. Comparative analysis with sequences currently available in Genbank showed a 99–100% sequence homology with previously published T. foetus isolates obtained from domestic cats from different European countries and also from America (e.g. KJ439572.1, AF466749.1, JX960422.1). The individual and anamnestic data on cats positive for T. foetus is summarized in Table 2. Positive cases were distributed over the northern (site 1, No = 7, P = 6.1%) and southern (site 3, No = 7, p = 11.1%) investigated areas (Fig. 1). On the contrary, no positivity was detected in central Italy (site 2). All the T. foetus-positive cats from the southern region were adult animals (No = 7) originating from the same municipal catteries with the exception of two subject they were housed all in different pens, and for one young cat kept from a colony. Conversely, positive animals from northern regions were purebred cats aged under one year; three of them were housed in breeding structures, whereas the other four were owned pets kept in two private households (Table 3). All cats infected by T. foetus showed diarrhoea at the time of sample collection. The results of coprological investigations are reported in Table 2. The higher prevalence rates were observed for G. duodenalis (19.5%), Cystoisospora spp. (11.6%) and Toxocara cati (11.9%). In particular, the highest prevalence for G. duodenalis was recorded in site 3 (34.9%), compared to sites 1 and 2 (16.4% and 12.2%, respectively). Giardia was detected by DFA in 30 samples (out of 204 examined, p = 14.7%) or by concentration-flotation method in 22 samples (out of 63 examined, p = 34.9%).

Table 2 Prevalence rates of enteric parasitic infections detected in the sampled cat populations with focus on Tritrichomonas foetus infection and co-infection rates.

Tritrichomonas foetus Giardia duodenalis Cystoisospora spp. Toxocara cati Toxascaris leonina Spirometra spp. Dipylidium caninum Ancylostoma spp. T. foetus + G. duodenalis T. foetus + Cystoisospora spp. T. foetus + Toxocara cati T. foetus + Toxascaris leonina T. foetus + Spirometra spp. T. foetus + Dipylidium caninum T. foetus + Ancylostoma spp.

N. positive

Prevalence % (95% CI)

14 52 31 32 1 1 5 4 9 0 1 0 0 3 0

5.2 (3–8.8) 19.5 (15.2–24.6) 11.6 (8.1–16.2) 11.9 (8.6–16.4) 0.4 (0.06–2.1) 0.4 (0.06–2.1) 1.8 (0.8–4.3) 1.5 (0.6–3.8) 3.4 (1.2–5.6) 0 (0–1.8) 0.4 (0.06–2.1) 0 (0–1.8) 0 (0–1.8) 1.1 (0.4–3.2) 0 (0–1.8)

T. foetus was found in association with G. duodenalis (3.4%) and T. cati (0.4%); in addition, three cats scored positive for T. foetus, G. duodenalis and Dipylidium caninum (Tables 3, 4). The results of univariate and multivariate logistic regression analysis are summarized in Tables 4 and 5, respectively. The variable “diarrhoea” was not submitted to statistical analysis, since all Tritrichomonas-positive cats showed diarrhoea. By analogy, the variables concerning the infection with Cystoisospora spp., Toxascaris leonina and Spirometra spp. were not considered in the statistical analysis, since the pathogens were not found in association with T. foetus. In the univariate analysis, the variables of “reproductive status”, “breed”, “housing environment” and the co-infections with G. duodenalis and D. caninum showed a p-value b0.1 and were, therefore, included in the multivariate analysis. In the final model, the variables of “reproductive status” and the “housing environment” were not maintained. The Hosmer-Lemeshow test confirmed the goodness-of-fit of the model (p = 0.339; Chi-square = 0.914). The model revealed that the purebred factor increased the risk of testing positive for T. foetus. Furthermore, co-infection with G. duodenalis and D. caninum influenced infection with T. foetus, as co-infected cats are at a higher risk of testing positive compared to uninfected cats (Table 5). 4. Discussion This study represents one of the larger surveys conducted on T. foetus in cats, and underlines the presence of this protozoan infection across feline populations in a large part of Italy. From Italy, only two studies addressing feline T. foetus infection have been published so far: a study has focused on a T. foetus “outbreak” in a rescue cat colony (Holliday et al., 2009), and another molecular study on intestinal protozoal infections in healthy pet cats (Mancianti et al., 2015). Not surprisingly, the prevalence of 5.2% found in our study including healthy and diarrhoeic cats laid in between the prevalence found in diseased cats (32%) and in healthy cats (2%). In the present survey all T. foetus-positive animals were tested or confirmed by means of a nested PCR protocol targeting the partial ITS1 and the 5.8S ribosomal rDNA of T. foetus, as this diagnostic tool is recognized as having greater sensitivity and specificity (Gookin et al., 2002, 2004; Tysnes et al., 2011). To better define the genotype of the feline isolates, the products obtained using the primer combination TFR3/TFR-4 were run separately from the final nested products. Unfortunately, no products of the predicted length size were detectable after the 1st reaction and thus no sequences comprising the ITS-2 region, useful to discriminate between cat and cattle genotypes on the basis of the nucleotide polymorphism (Slapeta et al., 2010), were obtained. These negative results might be an expression of a low load of parasitic DNA, not detectable by means of a single amplification. However, the sequencing of the nested products allowed demonstrating a strong sequence homology of the feline isolates here detected with those obtained from other worldwide domestic cats. The prevalence of T. foetus infection reported herein is also lower compared to those reported in other European and non-European countries, where prevalence ranges from 14% to 80% (Gookin et al., 2004; Gunn-Moore et al., 2007; Bissett et al., 2008; Burgener et al., 2009; Stockdale et al., 2009; Kingsbury et al., 2010; Kuehner et al., 2011; Mirò et al., 2011; Tysnes et al., 2011; Profizi et al., 2013). This discrepancy may have been induced by the differences in the populations under examination. Indeed, the previously cited papers focused on particular animal populations, such as purebred cats or symptomatic patients, thus resulting in an overestimation of the general rate of positivity. As a matter of fact, the prevalence reported in other surveys conducted on randomly sampled cats is significantly lower (8.8–10%) (Stockdale et al., 2009; Doi et al., 2012). The risk factors associated with T. foetus infection in cats are still poorly understood and the results obtained from the epidemiological surveys are often contradictory. Some authors have suggested, for instance, that feline trichomoniasis commonly occurs in animals under 12 months of age, and shows a natural tendency to self-limitation in

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Fig. 1. Map of Italy showing the provenience of the faecal samples tested for Tritrichomonas foetus. Sampled provinces are colored in red, while provenience of positive cats is represented by yellow points.

Table 3 Data on cats tested positive to Tritrichomonas foetus by PCR. (⁎, #, ±: cats showing the same symbol were housed together). Age

Gender Reproductive status Breed

Housing environment Site

Diarrhoea Co-infections

Adult Adult Adult Adult Adult Adult Young Young Young Young Young Young Young Young

Male Male Female Female Male Male male Female Female Female Female Female Female Female

Municipal cattery⁎ Municipal cattery⁎ Municipal cattery⁎ Municipal cattery⁎ Municipal cattery⁎ Municipal cattery⁎

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Giardia duodenalis Dipylidium caninum Toxocara cati 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Intact Intact Intact Neutered Intact Neutered Intact Intact Intact Intact Intact Intact Intact Intact

Common European shorthair Common European shorthair Common European shorthair Common European shorthair Common European shorthair Common European shorthair Common European shorthair Birman Maine Coon Persian Maine Coon Maine Coon Norwegian Forest Cat Bengal

Colony Breeding structure Breeding structure Breeding structure Private Household# Private Household# Private Household± Private Household±

Site 3 Site 3 Site 3 Site 3 Site 3 Site 3 Site 3 Site 1 Site 1 Site 1 Site 1 Site 1 Site 1 Site 1

Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes No No

No Yes Yes Yes No No No No No No No No No No

No No No No No No Yes No No No No No No No

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Table 4 Risk factors for Tritrichomonas foetus infection in cats by univariate analysis (statistically significant variables are indicated by bold typing). Variable

Category

N. positive/n. examined

Prevalence %

Odds ratio

95% CIa

p-Value

Site

Site. 1 Site. 2 Site. 3 (reference) Male (reference) Female Intact (reference) Neutered Adult (reference) Young Cross-breed (reference) Pure-breed Colony (reference) Breeding structure Private Household Municipal cattery No (reference) Yes No (reference) Yes No (reference) Yes

7/63 0/90 7/114 5/125 9/142 12/158 2/176 8/127 6/140 7/201 7/66 1/63 3/53 4/87 6/64 5/215 9/52 13/235 1/32 11/262 3/5

11.1 0 6.1 4.0 6.3 7.6 1.8 6.3% 4.3% 3.5 10.6 1.6 5.7 4.6 9.4 2.3 17.3 5.5% 3.1 4.2% 60%

0.523 nc 1 1 1.627 1 0.219 1 1.501 1 3.288 1 3.720 2.988 6.414 1 4.400 1 0.551 1 34.227

0.175–1.567 nc

0.247 0.998

0.530–4.999

0.395

0.047–1.010

0.052

0.506–4.452

0.464

1.108–9.755

0.032

0.375–36.868 0.326–27.393 0.749–54.900

0.262 0.333 0.090

1.379–14.035

0.012

0.700–4.358

0.572

5.179–226.182

0.0001

Gender Reproductive status Age Breed Housing/source

Giardia duodenalis co-infection Toxocara cati co-infection Dipylidium caninum co-infection nc= not calculated. a Confidence interval (CI)

cats above 2 years, due to the changes in the ecology of the gut and the maturing of the local immune system (Gookin et al., 1999; Foster et al., 2004; Stockdale et al., 2006; Gunn-Moore et al., 2007). Other authors showed that the infection may also occur in adult animals (Gookin et al., 2004; Frey et al., 2009; Klein et al., 2010; Gray et al., 2010; Xenoulis et al., 2010). In this study, no correlation between age and T. foetus infection was observed and, among the considered risk factors, only the breed and co-infections were found to be significantly associated with T. foetus infection. Purebred animals have been recognized as being more sensitive to T. foetus infection compared to the Common European shorthair cat (Gookin et al., 1999, 2004; Gunn-Moore et al., 2007; Stockdale et al., 2009; Kuehner et al., 2011; Queen et al., 2012). The higher prevalence in purebred cats may be influenced by the type of housing (e.g. multi-cat households or breeding structures), in which the high number of animals, the close contact between them and the sharing of litter boxes may increase the transmission of the protozoan parasite. However, our results also indicate that feline trichomoniasis is not merely a disease of purebred cats, as corroborated by previous findings (Gunn-Moore et al., 2007; Stockdale et al., 2007; Doi et al., 2012). In fact, seven out of the fourteen positive cats were common European shorthair cats living in catteries and colonies, contexts in which the high animal density could explain the spread of infection. Indeed, no significant association was found between the housing environment and the presence of T. foetus. In fact, the prevalence detected in cats living in private houses is very similar to that of cats living in catteries, colonies or breeding structures, in which transmission may be supposed to be favoured by close, direct contact among animals (Stockdale et al., 2009). However, it has to be considered that all the positive cats living in private housing were young purebred animals, recently introduced into the home, which had come from breeders. Thus, they may have acquired the infection in their original breeding structures, and then

developed and carried a persistent infection into the home environment. In this study, the T. foetus infection was found in 14 out of the 72 animals (19.4%) that showed diarrhoeic symptoms at the moment of sampling. Several studies focused on the association between the T. foetus infection and the current or history of chronic diarrhoea, showing percentages of trichomoniasis almost similar to that discovered in this survey (Gunn-Moore et al., 2007; Holliday et al., 2009; Bell et al., 2010). Therefore, the findings of this study corroborate the hypothesis that the history of diarrhoea enhances the risk of having a T. foetus-positive status (Yao and Köster, 2015). Moreover, the results obtained suggest a marked predominance of symptomatic infections, since all T. foetus infected cats showed diarrhoea. This finding disagrees with the observation of the fairly common recording of sub-clinical feline trichomoniasis (Foster et al., 2004). Furthermore, a significant risk factor for feline trichomoniasis detected in this study was a concurrent infection with other enteric parasites i.e. G. duodenalis and D. caninum. Indeed, prevalence rates recorded in this study for intestinal helminthes and protozoa are in line with others studies carried out in Italy in owned and stray cats (Riggio et al., 2013; Spada et al., 2013; Zanzani et al., 2014). Particularly, a highest prevalence of Giardia was recorded in Site 3 (34.9%), according to a previous survey (Napoli et al., 2012); this could be related to the housing of the cats in municipal catteries, being the transmission of G. duodenalis infection favoured by close contact among animals. A significant association between T. foetus and G. duodenalis was recorded in several surveys (Gookin et al., 1999, 2004; Steiner et al., 2007; Burgener et al., 2009; Stockdale et al., 2009). It can be speculated that one infection may exacerbate the other, or it may be that T. foetus and G. duodenalis simply share common risk factors. It is also interesting to notice that three cats scoring positive for T. foetus and G. duodenalis also resulted infected by D. caninum, suggesting that a T. foetus infection should not be

Table 5 Potential risk factors for Tritrichomonas foetus infection in cats by multivariate analysis statistically (significant variables are indicated by bold typing). Variable

Category

n. positive/n. examined

Prevalence %

Odds ratio

Breed

Cross-breed (reference) Pure-breed No (reference) Yes No (reference) Yes

7/201 7/66 5/215 9/52 11/262 3/5

3.5 10.6 2.3 17.3 4.2% 60%

1 39.888 1 11.170 1 84.692

Giardia duodenalis co-infection Dipylidium caninum co-infection a

Confidence interval (CI).

95% CIa

p-Value

5.966–266.701

0.0001

1.917–65.073

0.007

5.173–1386.511

0.002

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disregarded when helminthic infections also occur. The high frequency of co-infections also suggests conducting extensive, prospective efficacy studies on active molecules against both G. duodenalis and T. foetus, e.g. tinidazole or ronidazole, to improve the therapeutic management of the two pathogens (Pennisi et al., 2012a, 2012b; Zanzani et al., 2016). In conclusion, the acquired data indicates that T. foetus infection is spread in cats living in Italy. Therefore trichomoniasis should always be included in the differential diagnosis of a feline patient referred for largebowel distress, especially if it is a purebred animal. Moreover, co-infections with other enteric parasites, such as G. duodenalis, should always be suspected and properly diagnosed in T. foetus-positive cats. Competing interests The Authors declare that they have no competing interest and that the conceptual design, the conduct, the interpretation of the results and all the scientific aspects of the study were not influenced by any third party. Acknowledgements The Authors would like to acknowledge the veterinarian Dr. Giulia Morganti (Department of Veterinary Medicine, University of Perugia, Italy) and Dr. Mattia Ridolfi (Veterinary Practitioner of Macerata, Italy) for their support in the laboratory activities. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Bell, E.T., Gowan, R., Lingard, A.E., McCoy, R.J., Lapeta, J., Malik, R., 2010. Naturally occurring Tritrichomonas foetus infections in Australian cats: 38 cases. J. Feline Med. Surg. 12, 889–898. Bissett, S.A., Gowan, R., O'Brien, C., Stone, M., Gooking, J.L., 2008. 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