Abstract
The aim of this study was to determine the prevalence of Tritrichomonas foetus infection and associated clinical signs in purebred cats in Germany, to investigate the role of co-infection, and identify determinants of infection. Faecal specimens accompanied by epidemiological questionnaires were scored and collected from 230 purebred cats. Faeces were examined for trichomonads and other enteroparasites. The prevalence of T foetus was 15.7% among cats and 18.5% among catteries. An abnormal faecal score and history of diarrhoea were observed in 64% and 61% of T foetus-positive cats, respectively, and correlated significantly with infection. Co-infection, observed in 36% of T foetus-infected cats, was not associated with diarrhoea. Norwegian Forest cats were infected significantly more often than other breeds. No association was found with any environmental factors. This study demonstrated a high prevalence of symptomatic T foetus infections in purebred cats in Germany. Co-infection with other enteroparasites did not worsen clinical signs of trichomonosis.
The single-celled flagellate Tritrichomonas foetus has recently been recognised as an enteropathogen in domestic cats. 1–4 T foetus predominantly colonises the distal ileum and colon, and infection may manifest as chronic or recurrent large-bowel diarrhoea that is unresponsive to commonly administered antimicrobial drugs. 1,2,5–9
Enteric trichomonads were long considered naturally-occurring organisms within the feline intestine. 10,11 However, experimental and field studies over the past 10 years have conclusively identified T foetus as a non-commensal obligate pathogen in cats, 1–3 and many publications have suggested a strong association between feline T foetus infection and chronic diarrhoea. 12–18 Yet, it still remains unclear whether T foetus alone is sufficient to cause clinical signs or whether T foetus-associated diarrhoea is primarily a multifactorial disease process involving concurrent infection with other enteropathogens, host and environmental factors. 1,2,14,19
The importance of multi-cat environments not only in catteries but also in shelters in the epidemiology of feline trichomonosis is supported by the high prevalence of T foetus (32%) observed among 74 diarrhoeic domestic crossbred cats living in a rescue colony in Italy. 18 However, as the exact transmission mode of feline T foetus has yet to be determined and only one epidemiological study to date has examined at-risk housing facilities, 12 other environmental factors cannot be ruled out as potential influences on prevalence and should be further investigated.
Studies in Europe investigating feline T foetus infection have focused primarily on cats with chronic diarrhoea, and the pathogen has been detected in the faeces of 2–32% of cats in the UK, Italy, Switzerland and the Netherlands. 13,16–18,20,21 T foetus was first found in the faeces of several pedigreed cats from Germany attending an international cat show in the USA in 2001. 12 More recently, the protozoan was found in the faeces of 6/31 (19%) cats with chronic diarrhoea in Germany and Austria. 22 To date, however, no published data exists on the prevalence of T foetus infection in cat populations in Germany.
The purpose of this study was (1) to determine the prevalence of T foetus in the faeces of purebred cats in Germany, (2) to evaluate the association of infection with overt enteric disease and determine whether concurrent infection with other intestinal parasites exacerbates clinical signs of feline trichomonosis, and (3) to identify factors associated with T foetus infection.
Materials and methods
Data collection
Freshly voided faecal specimens and epidemiological questionnaires were collected from 230 purebred cats representing 124 catteries at five regional cat shows throughout Germany between April and August 2008. Faeces were collected from cats that were present at the cat show as well as other cats of the participating catteries not attending the cat show. Cats with a completed survey and an unrefrigerated fresh faecal specimen less than 4 h old were included in the study.
Epidemiological survey
The cats' owners were asked to fill out an epidemiological questionnaire providing detailed information on signalment, diet, water source, housing situation, direct and indirect contact with livestock and other pets, as well as medical history (Appendix I). Specifically, survey questions were designed to determine the onset, severity, and frequency of gastrointestinal signs and record any medications administered within the preceding 6 months.
Faecal scoring
Faecal consistency was evaluated at the time of collection by the primary investigator (KK) and scored based on a modified continuous faecal scoring system for dogs and cats (Purina Fecal Scoring System for Dogs and Cats, Nestle-Purina) with a score of 1, representing liquid diarrhoea; 2, pudding consistency diarrhoea; 3, loose but formed faeces; and 4, firm faeces. Faecal specimens with a score of 1 and 2 were considered diarrhoeic, faecal specimens with a score of 3 and 4 were considered non-diarrhoeic.
T foetus faecal culture
An aliquot of fresh faeces (≤0.5 g) from each cat was inoculated into InPouch TF culture medium (Biomed Diagnostics). Pouches were stored vertically in the dark at room temperature and examined for motile trophozoites using light microscopy starting 48 h post inoculation. Microscopic evaluation was performed every day from day 2 through day 6 and every other day from day 6 through day 12. Each pouch was examined under low power (100× magnification) for ≥ 5 min, concentrating on the bottom portion and edges of the pouch. Upon identification of trophozoites, 400× magnification was used for confirmation of trichomonads. Observation of ≥ 1 motile trichomonad was considered a positive result. 23
Immediately following processing for faecal culture, the remaining faeces were placed in a cooler and refrigerated at 7°C within 6 h of collection.
Giardia and Cryptosporidium immunoassays
Fresh, refrigerated faecal specimens were examined for the presence of Giardia and Cryptosporidium species antigens by monoclonal microplate immunoassays (ProSpecT Giardia Microplate Assay, ProSpecT Cryptosporidium Microplate Assay, Remel) within 36 h. All tests were performed and visually interpreted according to manufacturers' instructions.
Faecal flotation
A portion of refrigerated faeces was examined within 72 h of collection for the presence of nematode eggs, Giardia species cysts and coccidian oocysts using zinc sulfate centrifugation flotation as previously described. 24
T foetus polymerase chain reaction (PCR) and trichomonad typing and sequencing
Faecal specimens were frozen at −70°C for up to 1 month prior to DNA extraction. Faecal DNA was isolated from all 230 specimens using the QIAamp DNA Stool Mini kit (Qiagen) according to manufacturer's instructions with the following modifications: 20 μl of proteinase K was incubated with the extracted solution at 56°C for 1 h prior to adding the lysis solution AL, and two washes were performed with buffer AW1.
PCR amplification followed the conditions established by Grahn et al with minor modifications. 25 Three stool sample DNA template volumes were used (0.5 μl, 2.0 μl, and 8.0 μl), and water was decreased accordingly to maintain sample volume and appropriate reaction concentrations. Bovine serum albumin (BSA) was added to a final reaction concentration of 0.1% and thermal profile cycling was increased from 30 to 35. Products were size separated on an ABI 3730 DNA analyzer with GeneScan LIZ 500 size standard (Applied Biosystems). Exact sizes were determined with STRand analysis software. 26
To verify the identity of amplified trichomonads, representative PCR amplicons were directly sequenced. Products were amplified using unlabelled primers under reaction conditions listed above to maximise sequence read length and obtain double stranded sequence. Twenty microlitres of PCR product was prepared for sequencing using ExoSAP-IT (USB) to remove unincorporated primers and dNTPs. Sequencing was performed using the Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing products were cleaned over Centri-Sep Spin Columns (Princeton Separations) and separated on an ABI 3730 DNA Analyzer. Sequences were visualised using the Sequencher Software (Gene Codes Corp), and sequence identity was confirmed with a BLAST search. 27
Statistical analysis
Statistical analyses were conducted using commercial software (SPSS Version 17, SPSS; StatCalc 5.4.1, AcaStat Software; Stata Version 9.1, Stata Corp). Descriptive statistics were performed for all variables. Prevalence was determined with a 95% confidence interval (CI). Continuous data was examined for normality by the Kolmogorov–Smirnov test. For variables non-Gaussian in distribution, the Mann–Whitney test was used to compare between groups. Categorical data was analysed using a χ2 test. In 2×2 contingency tables with any expected cell values < 5, Fisher exact two-tailed results were used. Prevalence odds ratios (PORs) and 95% CI were calculated where appropriate. All variables with a P-value of < 0.2 were further analysed using a multivariate logistic regression model with robust variance estimation to account for the cluster effect of catteries. A P-value of < 0.05 was considered statistically significant.
Results
Study population
Freshly voided faecal specimens and a completed epidemiological survey were collected from 230 purebred cats representing 124 catteries in Germany. The number of cats sampled per cattery ranged from one to 11 (median 1, mean 1.9). Surveyed catteries contained a median of nine cats with a range of one to 33 cats per household. Ninety of the cats were male (63 intact, 27 castrated) and 136 were female (108 intact, 27 spayed). The gender of four cats was not recorded. The age of sampled cats ranged from 3 weeks to 18 years (median of 1.1 years, mean of 2.3 years). The age of two cats was unknown. The study population was made up of 25 different breeds. Maine Coon cats (n=77), British Shorthairs (n=40), Birmans (n=18), and Norwegian Forest (NFO) cats (n=15) were over represented.
Prevalence of T foetus in Germany
The overall prevalence of T foetus infection was 15.7% (36/230 [95% CI 11.5–20.9]) among individual cats and 18.5% (23/124 [95% CI 12.7–26.3]) among catteries. Diagnosis of T foetus was based on either positive faecal culture results (29/230) or demonstration of T foetus DNA via PCR of faecal DNA (28/230). No significant difference was observed in the geographical distribution of T foetus infection throughout Germany.
Clinical signs
The prevalence of diarrhoea in this study was based on faecal score and data obtained from the epidemiological survey (Table 1). An abnormal faecal consistency (corresponding to a faecal score of 1 and 2) on the day of sampling at the cat show was observed in 64% (23/36) of T foetus-positive cats and correlated strongly with the detection of T foetus (P<0.001; POR 3.98; 95% CI 1.90–8.33). Firm faeces (corresponding to a faecal score of 4) were noted in only 11% (4/36) of infected cats. A history of diarrhoea in the past 6 months, documented in 61% (22/36) of T foetus-positive cats, was also significantly associated with T foetus infection (P=0.027; POR 3.15; 95% CI 1.14–8.74). Likewise, 70% (16/23) of T foetus-positive catteries reported a history of diarrhoea among cats within the cattery in the past 6 months (P=0.010; POR 3.22; 95% CI 1.23–9.87). T foetus-infected cats without a history of diarrhoea were significantly more likely to have diarrhoea on the day of the cat show than T foetus-negative cats without a history of diarrhoea (P=0.002). No correlation was observed between the age of T foetus-positive cats and either faecal score or a history of diarrhoea. Frequency of defecation, faecal incontinence, faeces containing mucus and blood, or weight loss were not associated with T foetus infection.
Table 1.
Association of feline T foetus infection with diarrhoea based on faecal score and a history of diarrhoea in the past 6 months.
| T foetus positive | T foetus negative | ||||
|---|---|---|---|---|---|
| Cats (n=36/230) | Catteries (n=23/124) | Cats (n=194/230) | Catteries (n=101/124) | ||
| Faecal score* | 1 | 1 (3%) | 6 (3%) | ||
| 2 | 22 (61%) | 45 (23%) | |||
| 3 | 9 (25%) | 56 (29%) | |||
| 4 | 4 (11%) | 87 (45%) | |||
| History of diarrhoea in past 6 months | Yes | 22 (61%) | 16 (70%) | 73 (38%) | 45 (45%) |
| No | 14 (39%) | 7 (30%) | 121 (62%) | 56 (55%) | |
Faecal score based on a modified continuous faecal scoring system: 1=liquid diarrhoea, 2=pudding consistency diarrhoea, 3=loose but formed faeces, 4=firm faeces.
Other enteric parasites
Enteric parasites other than T foetus were identified in 49/230 faecal specimens (21.3%). Of the 36 cats that tested positive for T foetus, 13 (36.1%) were co-infected with other parasites. No other enteric parasite other than T foetus was associated with an abnormal faecal score and a history of diarrhoea. Co-infection with Giardia species and T foetus was observed in 10/230 cats from 6/124 catteries, representing 27.7% (10/36) of T foetus-positive cats. Six cats from four catteries were positive for T foetus and Isospora species, representing 16.7% (6/36) of T foetus-positive cats. Of these cats, three were co-infected with T foetus, Giardia species and Isospora species. Faecal consistency did not differ in T foetus-positive cats co-infected with Giardia species and/or Isospora species as compared to cats infected solely with T foetus. The small number of faecal specimens positive for both T foetus and Cryptosporidium species (n=1) precluded determination of any association between co-infection with this parasite and faecal consistency.
Factors associated with T foetus infection prevalence
No association was found between gender and T foetus infection. The age of T foetus-positive cats ranged from 3 weeks to 7.2 years (median age: 1.0 year). Nearly 70% (25/36) of T foetus-positive cats were ≤1 year old (P=0.034; POR 0.85; 95% CI 0.73–0.99), and the prevalence of T foetus infection decreased with age (POR=0.83; 95% CI 0.70–0.98). T foetus infection was identified in cats of 11/25 breeds (Table 2). Approximately 67% (10/15) of NFO cats and 88% (7/8) of NFO catteries throughout Germany tested positive for T foetus. NFO cats had a significantly higher prevalence of T foetus infection than other breeds (P<0.001, POR 25.89; 95% CI 7.63–87.72). No significant association was observed between T foetus infection and any other breed.
Table 2.
Breed distribution of T foetus-positive cats and catteries (values are expressed as a fraction and percentage [%] of the total number of cats and catteries of each breed).
| Breeds (n=25) | T foetus positive | |||||
|---|---|---|---|---|---|---|
| Cats (n=36) | Catteries (n=23) | |||||
| Maine Coon | 5/77 | (6.5%) | P=0.018* | 4/45 | (8.9%) | |
| British Shorthair | 7/41 | (17.1%) | 2/18 | (11.1%) | ||
| Birman | 0/18 | (0.0%) | 0/10 | (0.0%) | ||
| NFO cat | 10/15 | (66.7%) | P<0.001* | 7/8 | (87.5%) | P<0.001* |
| Burmese | 4/9 | (44.4%) | 2/4 | (50.0%) | ||
| Balinese | 2/8 | (25.0%) | 1/2 | (50.0%) | ||
| Persian | 1/8 | (12.5%) | 1/8 | (12.5%) | ||
| Bengal | 2/7 | (28.6%) | 1/3 | (33.3%) | ||
| Thai | 1/7 | (14.3%) | 1/2 | (50.0%) | ||
| Somali | 2/6 | (33.3%) | 2/3 | (66.6%) | ||
| Siberian cat | 1/6 | (16.7%) | 1/3 | (33.3%) | ||
| Devon Rex | 0/5 | (0.0%) | 0/2 | (0.0%) | ||
| Oriental Shorthair | 1/3 | (33.3%) | 1/2 | (50.0%) | ||
| Abyssinian | 0/3 | (0.0%) | 0/2 | (0.0%) | ||
| Highlander | 0/3 | (0.0%) | 0/2 | (0.0%) | ||
| Siamese | 0/3 | (0.0%) | 0/1 | (0.0%) | ||
| Savannah | 0/2 | (0.0%) | 0/1 | (0.0%) | ||
| Tonkinese | 0/2 | (0.0%) | 0/2 | (0.0%) | ||
| Neva Masquerade | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
| Sphinx | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
| Russian Blue | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
| Oriental Longhair | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
| Selkirk Rex | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
| Mandarin Oriental | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
| Ragdoll | 0/1 | (0.0%) | 0/1 | (0.0%) | ||
P-values<0.05 are shown.
T foetus infection was not correlated with any environmental factor, including diet, water source, direct and indirect contact to livestock or other pets, proximity to agricultural facilities, ratio of cats to litter boxes, and housing density as determined by number of cats per household and square metres per cat.
Discussion
In this study, 15.7% (36/230) of examined purebred cats and 18.5% (23/124) of surveyed catteries in Germany tested positive for T foetus. While T foetus has previously been identified in German cats, no published studies exist on the prevalence of T foetus in Germany. Furthermore, only two other epidemiological studies examining the overall prevalence of T foetus in a large population of both diarrhoeic and non-diarrhoeic cats, both conducted in the USA, have been published to date. 12,14 T foetus was identified in 31% of 117 purebred cats from 89 catteries at an international cat show in the USA. 12 Although the current study investigated a similar cat population using similar methodologies, the prevalence of T foetus observed in purebred cats in Germany was much lower. Inherent variations between the two populations might explain some of the differences in the results between the two studies. However, it is also possible that the prevalence of T foetus may have been underestimated in our study. Because the present study relied on testing of a single faecal specimen and did not exclude cats that received antibiotics within 2 weeks of faecal testing, the prevalence of T foetus in purebred cats in Germany may actually be higher than our results indicate.
In a recent investigation of 61 purebred cats in 36 catteries in the USA, a history of diarrhoea was reported by owners in only 25% of the study population and was not significantly associated with trichomonosis. 28 While it has been postulated that chronic asymptomatic T foetus infections may be quite common, 6 the current study identified very few animals with subclinical trichomonosis. T foetus infection correlated strongly with an abnormal faecal score, and of the 36 cats that tested positive for T foetus, only four cats (11%) had firm faeces as determined by faecal score on the day of the survey. A larger percentage of T foetus-positive cats (14/36, 40%), on the other hand, had no history of diarrhoea in the past 6 months. This suggests that in asymptomatic cats, bouts of diarrhoea may readily be triggered by environmental stress, such as a cat show, explaining the waxing and waning of clinical signs commonly seen in feline T foetus infections.
Although enteric co-infections have frequently been documented in T foetus-infected cats, 1,2,12,14,16 their association with clinical signs has not been well established. A study of experimental T foetus infection observed more severe diarrhoea and increased shedding of trichomonads in four cats concurrently infected with Cryptosporidium species. 1 In contrast, the current study of naturally infected cats found no association between co-infection with other enteric parasites and T foetus. Furthermore, T foetus-associated diarrhoea was not exacerbated by co-infection with Giardia species or coccidia, and T foetus infection alone was sufficient to cause significant clinical signs.
Several studies have suggested that T foetus infection is primarily a disease of young cats and kittens. 2,13,14,16 In agreement with current views, the present study was able to show a significant association of T foetus infection with young age. Nearly 70% of infected cats were ≤ 1 year, and the prevalence of disease decreased with advancing age. Of the 28 cats older than 5 years sampled in this study, only one cat tested positive for T foetus. In contrast, a recent investigation in a rescue colony in Italy found that 67% of 24 infected cats were over 1 year of age. 18 While these results clearly indicate that older cats are also at risk for disease, younger cats may be more vulnerable to disease due to an immature immune system. 2 Alternatively, the high prevalence of T foetus infection among kittens and young cats may reflect the time point at which transmission of T foetus within a cattery is most likely to occur. It is plausible that the risk of faecal–oral transfer of T foetus is greatest between an infected queen and her kittens and, subsequently, among kittens within that litter. Interestingly, severity of T foetus-associated diarrhoea was not age-related, and older infected cats in this study were just as likely to have an abnormal faecal score or history of diarrhoea as young animals.
NFO cats and catteries in Germany were at a significantly higher risk of T foetus infection than other breeds. Thus, T foetus was identified in nearly 67% (10/15) of NFO cats and 88% (7/8) of NFO catteries. Infected catteries were distributed throughout Germany and no association was found between NFO cats regarding recent ancestry or breeding programmes. Because of the high prevalence of T foetus among purebred cats, the possibility that some breeds may have a genetic predisposition for T foetus infection has been repeatedly discussed. 13,14 Although one study in the UK observed a high rate of T foetus infection among Siamese and Bengal cats, 13 this finding was not documented in other studies of purebred cats including the current investigation. 14,16,20 Two recent studies of diarrhoeic cats in Switzerland identified T foetus in NFO cats, but did not indicate that this breed was over represented. 16 Therefore, while a genetic predisposition of NFO cats in Germany cannot be ruled out at this time, other possible associations among the infected NFO cats in our study such as country of acquisition or management within these NFO catteries should be more closely investigated.
Indoor high density multi-cat housing, as commonly found in catteries and shelters, is regarded as a key risk factor for feline trichomonosis. Although the exact mode of transmission of T foetus is yet unknown, crowding may increase infection pressure by leading to an increased risk of oral–faecal contact via shared litter boxes. 12,29 While the square feet of facility per cat approached significance (P=0.056) in another study of T foetus in purebred cats living in catteries, 12 the current study was not able to identify an association between T foetus and housing density. Purebred catteries surveyed by Gookin et al (2004) contained a median number of 16 cats with a range of one to 59 cats per household and a median of 71.4 square feet of facility per cat in infected catteries. 12 In contrast, the housing density in German catteries documented in this study was much lower. Sampled catteries that tested positive for T foetus contained a median number of seven cats with a range of one to 33 cats per household and a median of 129 square feet of facility per cat. It is plausible that the housing density observed in this study was not high enough to influence disease prevalence. It is also possible that the questionnaire requesting information on the number of cats per household and square metres per facility was not answered honestly for fear of repercussions related to animal rights violations.
In addition, no correlation was observed between T foetus infection and the ratio of cats to litter boxes. Similarly, Gookin et al (2004) was not able to identify an association with litter box management or the type of litter used. 12 These results are unexpected considering the presumed environmental fragility of T foetus trophozoites. Because trichomonads do not form environmentally stable cysts, only freshly voided faeces are thought to contain infectious organisms, 12 and shared litter boxes have been implicated in the faecal–oral spread of T foetus. 29 Recently, however, an experimental study showed that T foetus is more resilient in cat faeces at room temperature than previously assumed. In moist faeces, the organism can survive for at least 7 days, 30 indicating that the faecal–oral spread of the organism may not require contact with a fresh faecal specimen. Thus, it is conceivable that transmission of T foetus is not limited to immediate oral contact with faeces in the litter box. Environmental contamination with faeces, especially diarrhoea, from infected cats may contribute to the spread of T foetus and may require more stringent sanitation measures than previously assumed.
In conclusion, this study found a high prevalence of T foetus infection in purebred cats throughout Germany. Clinical disease was very common, indicating that T foetus infection should be considered an important differential diagnosis for chronic diarrhoea in purebred cats in Germany, especially in young animals. Findings suggested that in asymptomatic cats, bouts of diarrhoea may potentially be brought on by stress. Co-infection with Giardia species or coccidia was not associated with the prevalence or severity of diarrhoea in T foetus-positive cats, indicating that T foetus infection alone is sufficient to cause clinical disease. No evidence was found to support the notion that clinical signs of T foetus infection are exacerbated by co-infections with other enteric parasites.
Acknowledgements
The authors gratefully thank Biomed Diagnostics, White City, OR, for donating the TF-Feline InPouches used in this study.
Appendix 1 Copy of survey used to obtain epidemiological information from participating cats and catteries
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