Introduction

Chlamydophila felis was first feline respiratory pathogen to be identified, and was thought to be responsible for most cases of respiratory tract disease, mainly in young animals, because they are particularly susceptible to infection. Highest prevalence of chlamydial infection has been reported to be between 5 weeks and 6 months in on study (Sykes et al. 1999) and between 5 weeks and 9 months in another report (Wills et al. 1988). According to Wills et al. (1988), the prevalence of Chlamydia infection was higher in males than female cats. The prevalence of chlamydiosis ranges from 0 to 32% of cases of upper respiratory tract disease (URTD), determined using a variety of different diagnostic assays (Studdert & Martin 1970; Fukushi et al. 1985; Wills et al. 1988; Nassise et al. 1993; Gruffydd-Jones et al. 1995; McDonald et al. 1998, Sykes et al. 1999). In the early stages of infection, cats develop conjunctivitis with serous ocular discharge and blepharospasm (Hoover et al. 1978). Laboratory diagnosis of C. felis infection can be performed using different diagnostic procedures, based on either isolation of the infectious organism, amplification of chlamydial DNA by polymerase chain reaction (PCR), or detection of antibodies by immunofluorescence assay and enzyme-linked immunosorbent assay (ELISA). Seropositivity for C. felis, even with high antibody titers, may indicate exposure to the organism, rather than active infection, especially in large cat populations. In household cats, serology correlates with chlamydial isolation and clinical disease (Wills et al. 1988). Our study was the first to characterize Brazilian cats positive by IFI and by PCR to Chlamydophila felis according to sex, age, breed, neutered or intact cats, vaccination and clinical signs or clinical historic of URTD. The aims of this study were to identify the risk factors associated with presence of respiratory tract disease caused by C. felis. The inferences drawn may be useful in devising control schemes for intensively housed cats.

Materials and Methods

Samples were collected from 151 domestic cats with or without clinical signs or clinical history compatible with feline chlamydiosis and previous vaccination regimen. The samples were obtained at catteries, veterinary clinical and public animal shelters in Jaboticabal, São José do Rio Preto, Sertãozinho, Ribeirão Preto and Descalvado cities in São Paulo State, Brazil. Data on individual cat were obtained by questionnaire for each animal and analyzed using univariable analysis. The specific questions about clinical historic, local, breed, sex, age and record whether the cat was vaccinated was collected at the time of the sample collection. Two outcome variables were used: the presence of antibody anti-C. felis by IFI and detection of C. felis from conjunctival swab by PCR. Serum samples were obtained from the cats by jugular venepuncture, and stored at -20°C until tested for antibodies against C. felis using IFI (Bion® Chlamydia-G Antibody Test System, Bion Enterprises, USA) with goat-anti-cat IgG conjugated with fluorescein isothiocyanate (FITC-Sigma, USA). Conjunctival swabs were obtained by rolling a dry sterile cotton-tipped swab firmly over the conjunctival sacs of both eyes and placed in a tube containing 1mL of ethanol (100%). DNA extraction was performed from sample using GFX Genomic Blood DNA Purification Kit (GE Healthcare, USA), according to manufacturer's instructions. PCR for the detection of Chlamydophila spp was performed with primers which corresponded to the conserved regions in the upstream non-coding region and 5' coding region of the chlamydial major outer membrane protein gene (Buxton et al. 1996). Comparison of prevalence of C. felis positive cats in was performed using Fisher's exact test. Differences were considered significant when P < 0.05.

Results

Cats were classified by their age, breed, sex, neutered or entire cats, vaccination status, clinical signs or clinical history. The breed classification consisted of mixed breed and pedigree. The cats were placed into one of three age groups; zero to six weeks, six weeks to six year and more than six years. Regarding the questionnaires of each cat, from 151 cats studied, 58 (38.4%) are males and 93 (61.6%) are female; 58 (38.4%) cats were less than six months of age, 81 (53.7%) were between one and six years of age and 12 (7.9%) had more than six years of age. The large number of cats in this study were mixed breeds (91.4%), 25.2% were neutered cats and 33.8% of cats were vaccinated for Chlamydophila felis. Between the cats studied, 29 (19.2%) cats have clinical signs compatible with feline chlamydiosis and 43 (28.5%) had historic of respiratory clinical signs related to URTD. Of the 151 serum samples, 106 (70.19%) were positive by IFI. Six of seven predictor variables were found to be significantly associated with the presence of antibody anti-C. felis by IFI using univariable analysis (Table 1). The prevalence rate was significantly affected by the cat´s age, breed, desexing, presence of clinical signs, clinical history and vaccination status. Of the 151 samples of conjunctival swabs, 9 (6.21%) were positive for Chlamydiaceae by PCR. There was a not significant difference between sex, age group, breed, desexing, clinical signs and history (Table 2). Vaccination was shown not to have a significant effect on the detection of C. felis by PCR: 7.8% of vaccinated cats were shedding C. felis compared to 5% of unvaccinated cats.

Discussion and Conclusions

In accordance with questionnaires of each cat and the results of laboratory diagnostic to C. felis, it was observed than the seroprevalence by IFI there was no significant difference between sex and was significantly higher in cats more one year. Mixed breed cats, neutered cats, cats without clinical signs and cats vaccinated was significantly higher than purebred cats, entire cats, cats with clinical signs and cats not vaccinated. Serological studies in which the prevalence of antibodies against C. felis was found to be highest in male cats and females cats aged from five weeks to nine months (Wills et al. 1988). In another study, there was no significant difference in seroprevalence for C. felis between cats younger than 1 year and older cats, between male cats and female cats or between neutered and entire cats (Holst et al. 2006). Presence of high antibody titres indicates exposure, do not necessarily mean that the cat is infectious (McDonalds et al. 1998). In experimental infection of cats with C. felis, high antibody titres can persist for over a year (Wills et al. 1988). Whether this represents antibodies with a long half-life, or persistent infection with the organism that is stimulating antibody production is not known. This way, the presence of antibodies against C. felis in Brazilian cats indicates that these animals had contact the agent in question in some stage of their lives. The PCR is therefore more effective than serology for demonstrating the infectious status of a cat (McDonalds et al. 1998). We did not detect significant differences between predictor variables with positive results by PCR detection. This also was observed by Von Bomhard et al. (2003) where they not detect significant differences between breed, age and sex distribution when comparing results for PCR detection. In studies from Australia, the median age of all Chlamydophila-negative cats was 24 months, suggesting that chlamydiosis is predominantly a cause of upper respiratory tract disease in younger cats. In this same study, the prevalence of chlamydiosis was highest in cats aged from 5 weeks to 9 months. However, there was no significant difference in the sex distribution and in the breed distribution between cats testing positive to C. felis and cats testing negative to C. felis (Sykes et al. 1997). Moreover, there are reports showing that cats aged between nine weeks and six months of age are more likely to chlamydiosis, while cats with conjunctivitis had a much greater chance of being infected with C. felis that cat without conjunctivitis (Sykes et al. 1999). It was observed that the vaccinated animals showed a higher prevalence of antibodies against C. felis that unvaccinated animals, suggesting an efficient seroconversion of animals Thus, vaccination reduced the acute disease but did not prevent shedding of the organism (Wills et al. 1987). According to Bannasch & Foley (2005), age was a significant risk factor, with increased risk in 0-to month-old and 7-to 11-month-old kittens and reduced risk in cats greater than 12 months. Cats housed in close proximity to dogs and the increasing population density has been previously shown to increase the prevalence of URTD likewise, shelters are environments in which cats are highly predisposed to URTD because of highly turnover in the cat population, density, stress and a proportion of the population with poor previous medical care, inadequate nutrition and concurrent medical problems. This, therefore, may restrict the generalizability of the study results. This study demonstrates that respiratory pathogens in cats are still widespread and it confirmed that exposure to C. felis is frequent in Brazilian cats. It therefore reinforces previous recommendations for disease control which have relied on a management procedure (Pedersen 1988) because the vaccination not prevents the infection by C. felis. So it is hoped that the findings of this study may be helpful in further investigation and in controlling risk factors for respiratory disease, mostly chlamydiosis, in Brazilian cats.

Table 1. Univariate analysis of risk factors associated with Chlamydophila felis infection and the presence of antibody anti-C. felis by IFI in cats with URTD.

Letters different in the same column indicate statistically significant difference.

Table 2. Univariate analysis of risk factors associated with Chlamydophila felis infection and positive PCR in cats with URTD.

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