Abstract
Cats are the key species in the epidemiology of Toxoplasma gondii infection, even if the proportion of subjects excreting oocysts is low. The aim of the present paper was to obtain information about seroprevalence, oocyst shedding rate and presence of T gondii DNA in faeces collected from an urban population of colony cats in Florence (Tuscany). Fifty European shorthair feral cats were examined for anti-T gondii specific antibodies by a modified agglutination test (MAT), and for oocysts by microscopic examination and for faecal protozoal DNA, by means of a nested polymerase chain reaction (n-PCR) protocol. Twenty-two out of 50 serum samples (44%) were MAT positive. T gondii oocysts were not detected in any of the examined faecal samples. Eight out of 50 faecal specimens (16%) were n-PCR positive and sequencing of the bands was specific for T gondii. Detection by combination of the two methods was higher than single techniques and enhanced the detection of T gondii up to 48%. Our results suggest that the use of MAT plus PCR in faeces may be the best choice for diagnosis of feline toxoplasmosis. Further studies to ascertain the real infectivity of the copro-PCR positive subjects are required.
The life cycle of Toxoplasma gondii involves the domestic cat (Felis catus) as definitive host, being the sole domestic species able to shed environmentally resistant oocysts. For this reason, cats are considered the key species in the epidemiology of T gondii infection, despite the low prevalence and the short and discontinuous oocysts' excretion pattern.
Oocysts shedding makes the cat a health hazard for both humans and animals, given that oocysts can persist for up 2 years in the environment. 1 The proportion of cats excreting oocysts is low, ranging from 0.11% in Germany and other Central European countries 2 to 0.9% in California. 3 In addition oocysts' shedding does not last for more than 1–3 weeks in an animal's lifespan, 4 even if reshedding occurs. 5 Microscopic examination to detect T gondii oocysts is neither sensitive nor specific because morphological and morphometric features are very close to Hammondia hammondi oocysts. Recently, a molecular approach for coprodiagnosis to identify parasite DNA in host faeces has been proven to be much more sensitive than microscopic parasites' detection. 6
Seroprevalence has been estimated in a number of studies, using different tests and thresholds. Reported prevalence rates in feral and stray cats from Europe range from 18.27% 7 to 40.7%, 8 varying with the age and lifestyle of the cat. 9 The kinetics of anti-T gondii antibodies appear to be variable both in experimentally and naturally infected cats, with individual subjects showing different serological responses. 10 Serological diagnosis for toxoplasma infection in cats relies on IgM and IgG detection. The presence of detectable IgM titres indicates an active (or reactivated) infection, while a single specific IgG titre is difficult to interpret showing a prior exposure to toxoplasma antigens only, without a further test demonstrating the occurrence of seroconversion.
The aim of the present paper was to obtain information about seroprevalence, oocyst shedding rate and presence of T gondii DNA in faeces collected contemporaneously with blood drawn from an urban population of colony cats in Florence (Tuscany).
Fifty European shorthair feral cats from urban colonies in Florence (Tuscany, Italy) were examined for anti-T gondii specific antibodies, faecal oocysts and faecal protozoal DNA. The cats were fed leftovers from canteens and restaurants, supplemented with commercial canned or dried food. At physical examination all the animals appeared in a good health status. The cats, of both genders (42 females and eight males), with ages ranging from 6 months to 3 years (mean age 12.6 σ=±7.1 months) were trapped during a routine population control procedure for urban colonies. Following sterilisation procedures male cats were hospitalised for 24 h, and non-nursing females were hospitalised for 24–48 h before release. Sera were drawn during anaesthesia and faeces were collected as long as the animals were hospitalised. All faecal specimens were formed, with the exception of two loose samples.
Serum samples were screened for T gondii specific antibodies, both for IgM and IgG with a modified agglutination test (MAT), performed using a direct agglutination commercial kit (Toxo-Screen DA; bioMérieux, Rome, Italy). Sera were tested starting from a 1:20 dilution, with formalin-fixed whole tachyzoites as antigen and the addition of 2-β-mercaptoethanol to evaluate the presence of IgM. Twofold dilutions were achieved and cut-off titre was 1/20 as recommended by Dubey et al. 10
Faecal specimens were submitted for microscopic examination. Samples were processed by a conventional flotation method using 262 mg/ml ZnCl2 and 275 mg/ml NaCl, as described by Schares et al. 11 Microscopic examination was carried out at a 400× magnification.
Faecal samples were also used to detect parasite DNA, using a nested polymerase chain reaction(n-PCR) protocol. DNA purification from faecal samples was performed on a 200 mg sample using a DNA extraction kit (QIAamp DNA Stool Mini Kit, Qiagen, Germany), according to the manufacturer's instructions. After extraction, DNA was stored at −20°C until use. Two pairs of oligonucleotide primers directed against the B1gene of T gondii were used to perform a n-PCR, as described by Jones et al. 12 Amplification reactions were analysed by 1.5% agarose gel electrophoresis, and visualised under ultraviolet light. Samples were scored as positive when a n-PCR product of 96 bp was detected. Purified PCR products were directly sequenced using both directions on an ABI (Applied Biosystems, CA, USA) prism automated DNA sequencer (model 3730XL).
Twenty-two out of 50 serum samples (44%) were MAT positive. In particular, IgG was detected in 20 sera (40%), and IgM in 21 (42%) sera. Antibody titres ranged from 1:20 to 1:5120 for IgG and from 1:320 to 1:81920, for IgM.
T gondii oocysts were not detected in any of the examined faecal samples. Other intestinal parasites were observed in 19 specimens (38%). Two of these were loosely formed. Toxocara cati and Cystoisospora species were identified in 15 (30%) and eight (16%) cats, respectively. The parasites were found in association (n=4 cats) or alone. The rate of recovery of T cati and Cystoisospora species from faecal specimens was in agreement with other authors. 13,14
Eight out of 50 faecal specimens (16%) were n-PCR positive and the sequencing of these bands was specific for T gondii. Faeces of all these subjects were formed. Six n-PCR positive cats were seropositive for anti-T gondii IgM and IgG, with titres ranging from 1/320 to 1/5120 for IgM and from 1/20 to 1/2560, for IgG. Two cats were seronegative. Five animals scored positive for other intestinal parasites. More detailed results are summarised in Table 1.
Signalment of positive cats, with serological, parasitological and molecular results.
F=female, M=male.
Several reports on the seroprevalence of T gondii in cats are present in literature, but, to the best of the authors' knowledge, an epidemiological study dealing simultaneously with serological, microscopic and molecular findings has not been performed.
The serological data in this report were in agreement with those reported in urban population of cats from other authors in Europe, with seroprevalences higher than 30%. 15–17 In the present study, IgM titres were evaluated to investigate both the duration of infection and a possible relationship between seropositivity and faecal findings. All but two of the seropositive animals showed anti-toxoplasma IgM antibodies indicating a recent infection.
Microscopic examination was negative for Toxoplasma/Hammondia species oocysts in all examined subjects, confirming that its recovery is a rare finding, mainly in cross-sectional studies. For these reasons the sensitivity of these methods is low and detection is difficult when the shedding of oocysts is reduced as it occurs after infection with some strains or when cats are infected by non-bradyzoite stages. 18,19
The molecular investigation revealed the presence of parasite DNA in eight (16%) of the animals. The significance of the faecal-PCR positivity should be carefully evaluated. This finding could be related both to the presence of free Toxoplasma species DNA or to oocysts. The first possibility could be a consequence of an acute visceral infection, or of a re-infection in an immune subject, considering that asexual T gondii types are found in the intestine of immune cats after a challenge. 20 However, the young age of the animals in this study would seem to exclude this hypothesis.
If the parasite DNA was of oocystic origin, on the contrary, a positive n-PCR could indicate a low shedding rate, not detectable by conventional faecal examination techniques. Comparison of serological tests and faecal-PCR assays showed that the detection rate with MAT (44%) was considerably higher than with n-PCR on faeces (16%). Detection by combination of the two methods was higher than single techniques and enhanced the detection of T gondii up to 48%. The presence of IgG and/or IgM antibodies to T gondii in faecal-PCR negative cats suggests that cats were previously infected, became immune and were not excreting oocysts. Whereas, the presence of IgG and/or IgM antibodies to T gondii in faecal-PCR positive cats suggests that these cats were previously infected even if they were excreting oocysts. This can occur when cats are re-infected after a chronic primary Toxoplasma species infection with a different strain and after primary infection with Isospora felis in a previously T gondii infected cat. 5,21,22 I felis experimentally infected cats challenged with T gondii, will develop an antibody titre to T gondii and demonstrate an anamnestic response to I felis antigen. 23 Finally, cats found to be serologically negative but copro-PCR positive were likely to shed oocysts. In general, for assessing human health risk, interpretation of serological test results from healthy cats is performed, especially when a pregnant woman owns a cat. These results suggest that the use of MAT plus PCR in faeces may be the best choice for diagnosis of feline toxoplasmosis, as it can be expected to improve the value of diagnostic procedures determining if cats are currently shedding oocysts. However, only a bioassay in mice will determine if viable T gondii oocysts are excreted in faecal-PCR positive cats.