M.C. Seki; A.O.T. Carrasco; T.F. Raso; R.L.M. Sousa; A.A. Pinto
Depto de Patologia Veterinária, FCAV-UNESP-Jaboticabal, Jaboticabal, SP, Brazil
Chlamydophila felis (C. felis), formerly known as Chlamydia psittaci, is a member of the Chlamydiaceae family and an obligate intracellular bacteria with cell walls resembling those of gram-negative bacteria (Everett 2000). It was first isolated in the United States in 1942 from cats with respiratory disease (Baker et al. 1942). C. felis is primarily a conjunctival pathogen, capable of causing acute to chronic conjunctivitis, with blepharospasm, chemosis, congestion and serous to mucopurulent ocular discharge in cats (Hoover et al. 1978). Nasal discharge, sneezing and other signs of upper respiratory tract disease (URTD) may occur in some cats (O'Dair et al. 1994). Clinical signs are influenced by the age of the cat, immunocompetence, affected tissues and pathogenicity of inoculum (Ramsey 2000). Transmission is thought to occur mainly by direct contact with infection ocular secretion. Laboratory diagnosis can be difficult owing to the small number of organisms present in conjunctival swabs. Until the advent of the polymerase chain reaction (PCR), the diagnostic technique of choice was isolation in cell culture. However, isolation depends on the survival of this rather fragile organism while it is transferred to the laboratory. The PCR has the major advantage of detecting the DNA even from dead organisms, and because DNA remains viable in the environment for long periods, veterinarians do not have to ensure that their samples reach the laboratory within a few days of being taken. Detection of C. felis genome in cats using PCR as the diagnostic test ranges from 1.1 to 26.9% (McDonald et al. 1998, Sykes et al. 1997, Sykes et al. 1999, Mochizuchi et al. 2000, Iwamoto et al. 2001, Rampazzo et al. 2003, Von Bomhard et al. 2003, Marsílio et al. 2004). It is important to detect whether a cat is shedding C. felis for several reasons. For example, it may aid appropriate antibiotic choices for diseased animals and even support directions for proper disinfection programs. Moreover, precise diagnostic techniques ensure that cats shedding the organism will not be introduced into Chlamydophila-free households. Although a vaccine for feline chlamydiosis is available in Brazil, there are no studies that demonstrate the occurrence of feline chlamydiosis in this country. In this context, this is the first study undertaken in order to determine the occurrence of C. felis infections among Brazilian cats, through direct Chlamydial DNA detection.
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 from catteries (n = 73), veterinary clinics (n = 18) and public animal shelters (n = 60) in Jaboticabal, São José do Rio Preto, Sertãozinho, Ribeirão Preto and Descalvado cities in São Paulo State, Brazil. Of the 151 cats assessed in three groups, 51 (33.77%) were vaccinated against C. felis (Fel-O-Vax LVK-IV®, Fort Dodge, EUA) and 100 were unvaccinated. The cats with clinical signs and/or historic compatible with feline chlamydiosis at the moment of sample collection were 43.7% (66/151), while 57.3% (85/145) were healthy. 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%). After sample collection, tubes were frozen at -20°C until testing. Swab samples were submitted to vortex for 2 min and then centrifuged at 20000 x g for 30 min at 4°C. The pellet was resuspended into 40 μL of buffer (0.1 M NaCl, 10m M TRIS, 1 mM EDTA; 5% Triton X-100) and 9 U proteinase K (Invitrogen, USA), incubated at 56°C for 90 min and then centrifuged at 2000 x g for 2 min. DNA extraction was performed from supernatant using the GFX Genomic Blood DNA Purification Kit (GE Healthcare Life Science, USA), according to manufacturer's instructions. PCR detection of Chlamydophila spp was performed with primers designed by Buxton et al. (1996), modified by Raso et.al (2006), which corresponded to the conserved regions in the upstream non-coding region and 5' coding region of the chlamydial major outer membrane protein gene. The primer sequences were synthesized as follows: oligo 420 (5'-CAGGATATCTTGTCTGGCTTTAA-3') and oligo 423 (5'-GCAAG-GATCGCAAGGATC-3'), which amplify a 260-base pair (bp) DNA fragment. The reaction was performed in a 25 μL amplification mix composed of 10 mM Tris-HCl (pH 8.8) (Buffer 10X Biotools, Spain); 0.2mM of DNTPs (Biotools, Spain), 0.2 M of each primer (Invitrogen, EUA); 1.25U of Taq DNA polymerase (Biotools, Spain), and 5 μL of the extracted DNA. PCR cycling conditions were 10 min at 94°C; 34 cycles at 94°C for 1 min, 54°C for 1 min and 72°C for 1 min; and a final extension at 72°C for 4 min. Both positive (vaccine of C. felis -Fel-O-Vax LVK-IV®, Fort Dodge, EUA) and negative (autoclaved ultrapure water) controls were routinely included. A 10-μL aliquot of each reaction was subjected to 1.5 per cent agarose gel electrophoresis. Samples were considered positive when a clearly visible band of the expected size was apparent after staining with ethidium bromide and visualization with ultraviolet transilluminator.
Of the 151 samples of conjunctival swabs, Chlamydiaceae DNA was detected in 9 (6%) cats by PCR. In catteries, 5.5% (4/73) were shown to be DNA positive. Among the positive samples, four were from cats without clinical signs and/or history of feline chlamydiosis and all of them were vaccinated. This four positive sample were from cats with clinical signs/historic and all of them were vaccinated. Of the 60 swab samples from public animal shelters, four (6.6%) were PCR positive from which two positive cats had ocular discharge at the time of conjunctival swab collection. Of the 18 samples obtained from veterinary clinics, only one (5.5%) sample was positive by PCR and this cat had signs of feline chlamydiosis as conjunctivitis, blepharospasm, chemosis, congestion and ocular discharge. There was no significantly statistic difference among catteries, veterinary clinics and public animal shelters groups.
Discussion and Conclusions
PCR for the detection of C. felis performed with primers corresponded to the conserved regions in the upstream noncoding region and 5´coding region of the chlamydial major outer membrane gene, encodes an outer membrane protein which is considered to be a major protective antigen targeted by the host immune system (Sandbulte et al., 1998 apud Mochizuki et al., 2000. Of the 151 samples of swabs of conjunctiva, Chlamydiaceae DNA was detected in 9 (6%) cats. Among the positive samples, four were from animals without clinical signs and/or history of feline chlamydiosis. The direct diagnosis of C. felis in cats, even in the absence of clinical signs of disease, has been reported (Gruffydd-Jones et al. 1995, McDonald et al. 1998), and may occur due to subclinical infections, as already disclosed by McDonald et al. (1998). Among the cats with clinical signs, 22.2 per cent of PCR positive animals had ocular discharge while 11.1 per cent had conjunctivitis and ocular discharge and 11.1 per cent had URTD. These results agree with those reported by McDonalds et al. (1998) who found cats with ocular discharge (25%), recurrent or chronic conjunctivitis (18%), conjunctivitis and upper respiratory signs (18%), first signs of conjunctivitis (9.5%) and URTD (7%). These authors described that the healthy cats were positive but they were from the same household and in contact with a cat which was positive for Chlamydophila. In our study, out of nine positive samples obtained, four (44.4%) were from vaccinated cats; this result argues against a previously published report (Rampazzo et al., 2003), where none of the vaccinated cats were PCR positive for Chlamydophila. Thus, vaccination reduced the acute disease but did not prevent shedding of the organism (Wills et al., 1987). Nonetheless, the ability of the vaccine to control clinical disease but not infection may be explained by postulating that the acute eye disease is largely caused by release of soluble chlamydial toxic products into the eye rather than as a direct cytopathic effect of chlamydial replication within the conjunctival mucosa, and vaccine-induced antibodies neutralize these toxins while having no effect on infectivity or chlamydial replication. This study emphasizes that exposure to C. felis is frequent in Brazilian cats, once that C. felis is present in conjunctival swabs collected from cats with and without chlamydiosis signs. Additionally, vaccine does not seem to prevent the infection by the etiological agent, once that vaccinated animals presented positive DNA detection.
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