Conjunctivitis is a common condition of cats and is usually

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1 J Vet Intern Med 2003;17: Prevalence of Chlamydophila felis and Feline Herpesvirus 1 in Cats with Conjunctivitis in Northern Italy A. Rampazzo, S. Appino, P. Pregel, A. Tarducci, E. Zini, and B. Biolatti The prevalence of Chlamydophila felis and feline herpesvirus 1 (FHV-1) infection in cats with conjunctivitis in northern Italy was investigated by conventional polymerase chain reaction (PCR) testing. In cats with conjunctivitis, C felis and FHV-1 were detected in 14 of 70 (20%) and in 23 of 70 (33%) animals, respectively. None of the 35 control cats were positive for C felis, whereas 7 (20%) of these cats were positive for FHV-1. Mixed infections were present in 5 of 70 cats (7%). Cats positive for C felis were significantly younger than control animals (P.02), whereas no significant age differences were observed between FHV-1 positive cats and control cats (P.41) or between FHV-1 positive animals and C felis positive animals (P.16). Cats sampled during acute-phase conjunctivitis were also investigated for the presence of C felis by conjunctival scrapings. In this acute phase, substantial agreement was found when comparing the results of the 2 methods (.80). The association between PCR results and conjunctivitis was evaluated for the 2 pathogens. The presence of C felis was significantly associated with conjunctivitis (P.004), whereas the detection of FHV-1 did not significantly correlate with the clinical sign (P.25), suggesting that, by itself, PCR is not suitable for the diagnosis of FHV-1 related conjunctivitis. Key words: Chlamydial inclusions; Conjunctival scrapings; Polymerase chain reaction; feline. Conjunctivitis is a common condition of cats and is usually caused by viral and bacterial infections, although allergies, parasite infestations, foreign bodies, chemical irritants, entropion, trichiasis, and congenital eyelid malformations may also cause conjunctivitis. The most common microorganisms involved are feline herpesvirus 1 (FHV-1) and Chlamydophila felis. 1 Feline calicivirus (FCV) has also been reported to cause conjunctivitis, but it is generally associated with other clinical signs attributable to FCV infection. Furthermore, FCV-associated conjunctivitis is typically milder than that observed with either FHV-1 or C felis. 2 5 Of lesser importance is Mycoplasma felis, which has not been definitively shown to play a primary role in feline conjunctivitis. 6,7 C felis is a primary conjunctival pathogen, 8 10 and concurrent upper respiratory tract signs are infrequent in infected cats. 1,11 Young animals are particularly susceptible to infection. The highest prevalence of chlamydial infection has been reported to be between 9 weeks and 6 months in one study 12 and between 5 weeks and 9 months in another report. 13 In the early stages of infection, cats develop conjunctivitis with serous ocular discharge and blepharospasm. 1,11 The conjunctiva is hyperemic and markedly chemotic but smooth and shiny. 14 The involvement may be more prominent in one eye than in the other, or it may be unilateral at initial examination and progress to bilateral involvement within a few days. 11 The only features of upper respiratory tract involvement may be mild rhinitis and intermittent sneezing. 11 If left untreated, chlamydial conjunctivitis becomes chronic. 9,15 18 Hyperemia, chemosis, and serous ocular discharge decrease, whereas the conjunctiva be- From the College of Veterinary Medicine, University of Torino, Grugliasco, Italy. Reprint requests: Dr A. Rampazzo, Università di Medicina Veterinaria, Dipartimento Patologia Animale, Via Leonardo da Vinci 44, Grugliasco (TO), Italy; rampella@tiscali.it. Submitted December 17, 2002; Revised February 12 and March 26, 2003; Accepted May 6, Copyright 2003 by the American College of Veterinary Internal Medicine /03/ /$3.00/0 comes thicker and may develop small follicles. 14 A mucous or mucopurulent discharge may persist for months. 1 FHV-1 has a tissue tropism for the epithelium of the conjunctiva, cornea, and upper respiratory tract Primary infection usually occurs in kittens and young adults, and an estimated 80% of cats will become latently infected. 22,23 Acute infection is characterized by conjunctivitis, which is usually bilateral and accompanied by copious serous or mucopurulent discharge as well as upper respiratory tract involvement with nasal discharge, sneezing, and coughing. In severe cases, the conjunctival epithelium becomes necrotic, and symblepharon may occur. Corneal dendritic lesions may be present as the result of corneal epithelium damage. 20,21,24,25 Older cats are more likely to experience persistent low-grade infections or recrudescent disease caused by virus reactivation. Ocular signs of FHV-1 chronic infections are usually unilateral and range from mild conjunctivitis to corneal ulcers, stromal keratitis, keratoconjunctivitis sicca, corneal sequestra, and eosinophilic keratitis ,24 However, the role of FHV-1 in these 2 latter conditions has not been completely elucidated. 19,20,25 Clinical differentiation between C felis related conjunctivitis and FHV-1 related conjunctivitis can be problematic, because the clinical signs may be similar. A variety of techniques have been used to detect C felis and FHV-1 in the conjunctival sac, whereas serologic tests are not routinely used for either of the 2 pathogens. Seropositivity for C felis, even with high antibody titers, may indicate exposure to the organism, rather than active infection, especially in large cat populations (ie, breeding colonies or farm cats). In household cats, serology correlates with chlamydial isolation and clinical disease. 13 However, by means of more sensitive polymerase chain reaction (PCR) techniques, C felis was found in only 41% of pet cats showing high antibody titers. 26 With FHV-1, no correlation has been shown between seropositivity or magnitude of titer and the likelihood of detecting viruses or the presence of clinical signs. 27 Diagnostic tests to identify C felis include isolation, conjunctival scrapings, immunofluorescent antibody (IFA) testing, antigen-detection enzyme-linked immunosorbent assay (ELISA), and PCR. Isolation of C felis from conjunctival

2 800 Rampazzo et al swabs by culture in cell monolayers or embryonated eggs has been the gold standard technique because of its high specificity. However, isolation is difficult to execute routinely because the microorganisms must be vital and present in sufficient numbers to grow in culture. In chronic infections or antibiotic-treated animals, these conditions may not be met, lowering the sensitivity of the assay. 26,28 Evaluation of conjunctival scrapings for cytoplasmic inclusions is a quick and valuable method to detect C felis, although its usefulness is primarily limited to acutely infected cats, because inclusions are infrequently seen in chronic conjunctivitis. 1,11 IFA and antigen-detection ELISA tests are less commonly used. The former assay has been shown to have low sensitivity and specificity, 1,29 whereas the latter test is prone to produce variable results when different commercially available kits are used. 28,30,31 PCR detection of C felis is far more sensitive than the other methods. This technique can detect small numbers of microorganisms, which, moreover, do not need to be viable. DNA amplification tests have become of great value, especially in chronically infected or treated cats in which traditional methods (culture or scrapings) might prove inefficient. 1,12,26,28,32 34 Assays for detecting FHV-1 include viral isolation, IFA, ELISA, and PCR. Viral isolation has been the gold standard but, as with C felis, it is problematic to perform on routine clinical specimens 35 and, additionally, has a lower sensitivity than PCR. 36,37 IFA has both low sensitivity and specificity, 27,35 38 and ELISA has not been extensively investigated since the development of DNA amplification assays. At present, PCR is a widely used technique because of its high sensitivity 25,33,39 42 and because immediate sample processing is unnecessary, making it highly favorable over isolation for routine use. Conflicting results have been reported regarding the prevalence of FHV-1 in cats with conjunctivitis, upper respiratory tract disease, or both and in clinically normal cats. These disparate results most likely reflect the large number of different assays used, sites sampled, and types of specimens collected for viral detection. In conjunctival swabs or biopsy specimens, several studies have found a similar prevalence of FHV-1 in cats with conjunctivitis compared to control animals. 27,36 In contrast, other studies have reported markedly higher detection rates of FHV-1 in cats with conjunctivitis than in healthy cats. 12,25 To correctly interpret the value of viral detection in conjunctival samples for diagnostic purposes, it is extremely important to determine whether the detection of the virus in the conjunctiva is associated with the presence of conjunctivitis. The goal of the present study was to assess the prevalence of C felis and FHV-1 in cats with conjunctivitis in northern Italy by conventional PCR performed on conjunctival swabs. Moreover, in C felis affected cats, we compared PCR results with the cytologic detection of chlamydial inclusions from conjunctival scrapings. Materials and Methods Animals Cats with conjunctivitis were examined at the Veterinary Teaching Hospital, University of Torino (Grugliasco, Italy), and conjunctival swabs and scrapings were collected for investigation. Veterinary practitioners with clinics located throughout northern Italy who had observed cats with conjunctivitis were asked to collaborate. Seventy cats with clinical signs of conjunctivitis were sampled for PCR assay between January 2001 and January Thirty-five cats examined during the same period for reasons other than ocular disease or respiratory tract disease were used as a control group. A total of 105 conjunctival swabs were tested for C felis and FHV-1 by PCR. Additionally, conjunctival scrapings were obtained from 52 of the 70 cats with conjunctivitis for detection of cytoplasmic chlamydial inclusions. Each sample was accompanied by data relating to cat breed, sex, age, vaccination status, habitat and contact possibility with other cats, clinical signs and their duration (acute, 2 weeks; chronic, 2 weeks), and medications given within the past 14 days. The vaccination status was divided as follows: regularly vaccinated, partially vaccinated (kittens that had received the 1st injection but not the booster and older cats for which the vaccination was not current), and unvaccinated. It was also recorded whether the vaccine was directed against feline panleucopenia virus, FCV, and feline herpesvirus only or against C felis as well. In accordance with previous studies, 12,13 the age of C felis positive cats was divided into 3 groups: 6 weeks, 6 weeks to 9 months, and 9 months. Conjunctival Scrapings Conjunctival scrapings were obtained by rolling a sterile microbrush a firmly over the ventral conjunctiva after topical anesthesia (oxibuprocaine chlorohydrate). The smears were then air dried and stained with May-Grünwald-Giemsa. To standardize the procedure, microbrushes were given to the collaborating veterinarian practitioners, along with instructions for collecting scrapings. The smears were then sent to the University of Torino and stained in the same laboratory. All the slides were examined by the same cytologist before PCR assays were performed. PCR Procedures Conjunctival swabs were obtained by rolling a sterile cotton-tipped swab firmly over the ventral conjunctiva and were placed in a 2-mL tube containing 200 L of sterile physiologic solution (0.9% NaCl). To standardize the procedure, sterile 2-mL tubes containing 200 L of physiologic solution were given to the cooperating veterinarian practitioners. After sample collection, tubes were frozen at 20 C until testing. Specimens from private clinics were stored at 20 C before being sent on ice to the University of Torino and were kept at 20 C until processing. DNA was extracted with a commercial kit. b PCR for the detection of C felis was performed with primers designed by Buxton et al, 43 which corresponded to the conserved regions in the upstream noncoding region and 5 coding region of the chlamydial major outer membrane protein gene. The primer sequences were as follows: oligo 420 (5 -CAG GAC ATC TTG TCT GGC TTT AA-3 ) and oligo 423 (5 -CGG ATG CTG ATA GCA TCA CAC CAA GT-3 ), which amplify a 277-base pair (bp) DNA fragment. c The reaction was performed in a 25- L amplification mix composed of 2.5 UofTaq DNA polymerase d ; 10 mm Tris-HCl (ph 8.3); 50 mm KCl; 0.01% gelatin; 1.5 mm MgCl 2 ; 200 M each of 2 deoxyadenosine 5 triphosphate, deoxythymidine, deoxyguanosine, and deoxycytidine; 1 M of each primer; and 5 L of the extracted DNA. The samples were subjected to 35 cycles of 30 seconds at 94 C for denaturation, 30 seconds at 47 C for annealing, and 1 minute at 72 C for extension. A final extension step of 7 minutes at 72 C was included before the PCR was completed. Both positive and negative controls were routinely included. A 20- L aliquot of each reaction was subjected to electrophoresis on 7% polyacrylamide gel. PCR products were detected by silver nitrate staining. PCR for FHV-1 detection was performed with the primers designed by Suchy et al, 44 which were derived from the sequences of the (tk) gene that produces FHV-1 thymidine kinase. The sense primer FHV-

3 Conjunctivitis in Cats 801 tkf (5 -GTT GTC GGT GGT ATC TAT GC-3 ) and the antisense primer FHV-tkr (5 -GAG GTT CTC GTG GAA GTG TT-3 ) amplify a 306-bp product. e The reaction was performed in a 25- L amplification mix as described above. After an initial denaturation period of 5 minutes at 94 C, reactions were subjected to 35 cycles of 1 minute at 94 C for denaturation, 30 seconds at 56 C for annealing, and 30 seconds at 72 C for extension. Then, the samples were subjected to a final extension step of 7 minutes at 72 C. As for C felis, both positive and negative controls were routinely included. Reaction products were subjected to electrophoresis on 7% polyacrylamide gel and detected by silver nitrate staining. Statistical Methods The median ages of the C felis positive cats, FHV-1 positive cats, and control cats were compared by nonparametric Kruskal-Wallis 1- way analysis of variance. The Wilcoxon rank sum test for independent data was used to determine which pairs of groups were significantly different. Finally, to avoid spurious significant P-values from repeated tests on the same data set, the Bonferroni correction was applied. 45 The Fisher exact test was used to compare sex, breed, habitat, contact with other cats, and vaccination status prevalence among C felis positive cats, FHV-1 positive cats, and control cats. The same test was used to compare clinical signs, disease duration, and unilateral versus bilateral initial involvement in C felis positive cats and FHV-1 positive cats. The Fisher exact test was used to evaluate the relationship between conjunctivitis and PCR results. Animals positive for both C felis and FHV-1 were excluded from the statistical analysis that compared the data of C felis positive cats with those of FHV-1 positive cats. Results were considered significant at P.05. Statistical analysis was carried out by R for Windows. f Finally, the kappa coefficient ( ) was used to evaluate agreement between C felis detection by PCR and by conjunctival scraping in acute-phase infections. 46 Results The median age of the 70 cats with conjunctivitis was 1 year (range, 3 weeks to 12 years); for the 35 control cats, the median age was 3 years (range, 2 months to 14 years) (Tables 1, 2). C felis was detected in 14 of 70 (20%) cats with conjunctivitis and in none of the control cats (P.004). FHV- 1 was found in 23 of 70 (33%) cats with conjunctivitis and in 7 of 35 (20%) control cats (P.25). In cats with positive PCR results, the median age was 2.5 months (6 weeks to 11 years) and 1.5 years (6 weeks to 12 years) for C felis and FHV-1, respectively (P.16). C felis positive cats were significantly younger than control cats (P.02). Five of 9 (67%) of the C felis positive cats were aged between 6 weeks and 9 months, with none being younger than 6 weeks. There was no significant age difference between FHV-1 positive cats and control cats (P.41). The data of cats testing positive for C felis or FHV-1 are shown in Table 3. None of the 4 cats vaccinated for C felis were PCR positive for this microorganism. The median age of the 7 control cats that tested positive for FHV-1 was 3 years (range, 2 months to 8 years). All of them were domestic shorthairs. Four were males, and 3 were females. Four of 6 were indoor/outdoor cats, 1 was an indoor cat, and 1 was a rescued stray cat. Contact with other cats had been possible for 5 of 5 cats. Two of 5 cats were partially vaccinated, and 3 were unvaccinated. Regarding their clinical condition, 6 of 7 cats were healthy, whereas 1 had lower urinary tract disease and was positive for feline immunodeficiency virus. Table 1. Variable Data of the conjunctivitis-affected cats. No. Cats with Known Variable No. Cats (%) Breed 70 DSH DLH Persian Siamese Chartreux 58 (3) 1 (1) 7 (10) 2 (3) 2 (3) Sex 66 Male Female 42 (64) 24 (36) Habitat 66 Indoor/outdoor Indoor Stray Breeding colony 29 (44) 26 (39) 10 (15) 1 (2) Contact with other cats 67 Yes No 58 (87) 9 (13) Vaccination status 66 Partially vaccinated Vaccinated Not vaccinated 11 (17) 31 (47) a 24 (37) Clinical signs in addition to conjunctivitis 70 Ocular discharge Blepharospasm Follicular hyperplasia Corneal ulcerations Keratitis Symblepharon Rhinitis and nasal discharge Sneezing Pneumonia Sialorrhea Oral ulcerations 56 (80) 25 (36) 3 (4) 6 (9) 7 (10) 6 (9) 31 (44) 31 (44) 2 (3) 3 (4) 1 (1) Disease duration 62 Acute ( 2 weeks) Chronic ( 2 weeks) 31 (50) 31 (50) Initial ocular involvement 57 Unilateral Bilateral 36 (63) 21 (37) Antimicrobial topical treatment (18) b DSH, domestic shorthair; DLH, domestic longhair. a Four of 66 had vaccination for C felis. b Six of 67, rolitetracycline and chloramphenicol; 3 of 67, tobramycin; and 3 of 67, chloramphenicol. Considering cats with chronic conjunctivitis only, 6 of 31 (19%) and 10 of 31 (32%) were PCR positive for C felis and FHV-1, respectively. Coinfection with C felis and FHV-1 was detected in 5 of 70 (7%) cats. In addition to conjunctivitis, 2 of them had rhinitis and nasal discharge, and 1 was sneezing. None of the animals had corneal involvement. Three cats were in the acute phase of infection, and 2 cats were in the chronic phase. In 9 cats with ocular lesions suggestive of chronic FHV-

4 802 Rampazzo et al Table 2. Variable Breed DSH DLH Persian Siamese Chartreux Sex Male Female Habitat Indoor/outdoor Indoor Stray Breeding colony Contact with other cats Yes No Vaccination status Partially vaccinated Vaccinated Not vaccinated Clinical condition Healthy Diseased Feline lower urinary tract disease Periodontal abscess Indolent lip ulcer Pancreatitis Hyperthyroidism Nasal squamous cell carcinoma Data of the control cats. No. Cats with Known Variable No. Cats (%) 31 (89) 16 (50) 16 (50) 17 (53) 14 (44) 22 (76) 7 (24) 7 (24) 14 (48) 8 (28) 28 (80) 2 (6) a DSH, domestic shorthair; DLH, domestic longhair. a One of the 2 cats was feline immunodeficiency virus positive. 1 infection, the virus was not detectable by PCR. In these cats, the lesions found in addition to conjunctivitis included symblepharon (n 3), keratitis (n 6), corneal ulcers (n 3), rhinitis and nasal discharge (n 8), and sneezing (n 5). On cytologic examination, we observed large numbers of polymorphonucleates along with a few lymphocytes in the smears of conjunctival scrapings that showed chlamydial inclusions. These inclusions were seen in epithelial cells, appearing as intracytoplasmic, basophilic, and discrete masses. Mostly, the inclusions were large, dark blue staining reticulated bodies with a granular appearance, frequently located adjacent to the nucleus, which could be deformed. The results of C felis detection by conjunctival scraping and PCR were compared. Thirty-one of the 70 cats affected by conjunctivitis were in the acute phase of infection; scrapings were available from 24 of these cats for cytologic examination. Six of the 24 smears showed chlamydial inclusions, and 8 of 24 were positive for C felis by PCR. Conjunctival scrapings were positive in 6 of the 8 samples that were also positive by PCR (75%). One of the 2 negative scrapings had been collected the day after the appearance of clinical signs. The kappa coefficient of agreement obtained from comparing the 2 methods for C felis detection was.80 (95% CI, ). Thirty-one of the 70 cats with conjunctivitis were in the chronic phase of infection, and 27 scrapings from these cats were available for cytology. Chlamydial inclusions were found in only 1 sample. By PCR, 6 of 31 of chronic-phase infection samples were positive for C felis, but, of those, just 2 scrapings were available, a number that was statistically insufficient for a comparison of the results. Discussion In our study, cats infected with C felis were significantly (P.02) younger than control cats; 67% of infected animals were aged between 6 weeks and 9 months, with none being younger than 6 weeks. Similarly, an age-significant effect on prevalence has been previously reported by Sykes et al 12 and Wills et al 13 that identified chlamydial infection prevalence as being highest in cats from 9 weeks to 6 months old and from 5 weeks to 9 months old, respectively. Wills et al 31 observed the presence of maternal antibodies against C felis in kittens born to chlamydia-infected queens, and, despite the continuous shedding of C felis from the conjunctiva and vagina during lactation, most kittens did not become infected until at least 5 weeks of age, which accounted for the lowest prevalence being observed in cats younger than 5 weeks. We did not find a significant age difference between FHV-1 positive cats and control cats (P.41). Conflicting results are present in the literature regarding age effects on disposition to FHV-1 infection. In a study performed on oropharyngeal swabs, 140 of 213 (65.7%) FHV-1 shedders were younger than 1 year, 47 whereas in another report, which was performed on conjunctival swabs, no difference in prevalence was found in animals younger than 1 year when compared to animals older than 1 year. 12 In agreement with Sykes et al, 12 we did not find any sex or breed disposition to C felis infection, whereas Wills et al 13 found that males and Birman cats were more frequently infected. As assessed in previous studies by Sykes et al 12 and Harbour et al, 47 we found no breed or sex disposition to FHV-1. Statistical analysis showed that none of the clinical signs reported in addition to conjunctivitis in either C felis positive animals or FHV-1 positive animals was more represented in cats infected by one of the 2 microorganisms when compared to the other. However, note that we did not describe the different features of conjunctivitis, such as more pronounced chemosis, mostly seen with C felis infection, 11,14 or more pronounced conjunctival injection, usually associated with FHV-1 24,35 ; likewise, we did not evaluate the intensity of either rhinitis or systemic reaction to infection. This aspect, together with the small number of positive cats analyzed, may explain the results of our study, which show that the 2 microorganisms appear to cause diseases with highly similar clinical presentations. Even though 67.1% of C felis positive cats had initial unilateral ocular involvement, no marked difference was seen between unilateral and bilateral initial ocular involvement in cats affected by either C felis or FHV-1.

5 Conjunctivitis in Cats 803 Table 3. Data of the cats with conjunctivitis PCR positive for C felis or FHV-1. Breed DSH DLH Persian Siamese Chartreux Variable Sex Male Female Habitat Indoor/outdoor Indoor (2 just introduced) Stray Breeding colony Contact with other cats Yes No Vaccination status Partially vaccinated Vaccinated Not vaccinated No. Cats/No. Cats C felis positive with Variable Known (%) 9/9 (100) 4/9 (44) 5/9 (56) 5/9 (56) 8/9 (89) No. Cats/No. Cats FHV-1 Positive with Variable Known (%) 12/17 (71) 4/17 (24) 1/17 (6) 9/15 (60) 6/15 (40) 8/18 (44) 8/18 (44) 2/18 (11) 14/17 (83) 3/17 (18) 4/16 (25) 9/16 (56) 3/16 (19) Clinical signs in addition to conjunctivitis Ocular discharge Blepharospasm Follicular hyperplasia Corneal ulcerations Keratitis Symblepharon Rhinitis and nasal discharge Sneezing Pneumonia Sialorrhea Oral ulcerations 8/9 (89) 2/9 (22) 6/9 (67) 6/9 (67) 14/18 (78) 7/18 (39) 1/18 (5) 3/18 (17) 2/18 (11) 1/18 (5) 8/18 (44) 12/18 (67) 3/18 (17) Disease duration Acute ( 2 weeks) 7/9 (78) 7/17 (41) Chronic ( 2 weeks) 2/9 (22) 10/17 (59) Initial ocular involvement Unilateral 4/6 (67) 8/17 (47) Bilateral 2/6 (33) 9/17 (53) Antimicrobial topical treatment 2/18 (11) a P-value.32*.76*.69*.73.84* DSH, domestic shorthair; DHL, domestic longhair; PCR, polymerase chain reaction; FVH-1, feline herpesvirus 1. a One of 18, rolitetracycline and chloramphenicol; one of 18 chloramphenicol. * P-values were obtained comparing C felis positive cats, FHV-1, positive cats and control cats. We found that vaccination status did not affect the likelihood of developing FHV-1 conjunctivitis, as 56% of these cats were fully vaccinated. A specific vaccination for C felis is seldom used in Italy (4 of 66 cats with conjunctivitis), and none of the vaccinated cats tested positive for C felis. Prevalence of C felis The prevalence of C felis in cats with conjunctivitis has been reported to range from 13 to 30.8%, depending on the cat population, the country, and the assay used. C felis has been isolated in roughly 30% of cats with conjunctivitis in both Canada and the UK. 10,13 Antigen-detection ELISA assays showed a 23.6% prevalence in France and an 18.4% prevalence in New Zealand. 30,48 In 1989, Hanselaer et al 7 reported C felis in 23% of cats with conjunctivitis in Belgium. In cats with chronic conjunctivitis, Nasisse et al 38 in 1993 observed the microorganism by IFA in 18% of cats. In recent years, PCR has become a widely used assay because of its high sensitivity. PCR sensitivity and specificity depend on many factors, such as the primers used, the cy-

6 804 Rampazzo et al cling conditions, and the technique chosen (ie, classic, nested, multiplex, or real-time PCR). By conventional PCR, prevalences of 20 and 13% were reported in cats with conjunctivitis in the UK 26 and Australia, respectively. 34 In the latter country, Sykes et al 12 obtained a 14.3% prevalence by a duplex PCR assay (C felis and FHV-1); similar results were obtained by a triplex reverse transcriptase-pcr/pcr (C felis, FHV-1, and FCV) 33. In the current study, C felis was detected by conventional PCR in 20% of cats with conjunctivitis. A similar prevalence (19%) was observed when narrowing the focus to cats with chronic conjunctivitis only. In agreement with previous studies, 7,10,13 none of the control cats had positive PCR results. Conversely, other authors have reported the presence of C felis in cats without conjunctivitis. However, these were cats that had been in contact with affected animals, cats for which a previous mild conjunctivitis might have passed unnoticed, or cats with resolved episodes of conjunctivitis. 12,30,49 This suggests that the detection of C felis in healthy cats may be related to a recently resolved infection, rather than to the presence of the microorganism in normal conjunctival flora. A significant association between conjunctivitis and PCR detection of C felis (P.004) was found, indicating that the presence of the microorganism is related to disease. In this work, for the 1st time to the authors knowledge, PCR for C felis and conjunctival scraping results were compared in conjunctivitis-affected cats. A substantial agreement (.80, 95% CI, ) was found when comparing the results of the 2 assays in acute-phase infections. Chlamydial inclusions were detected in 75% of acute infections that were positive for C felis by PCR. One of the conjunctival scrapings negative for chlamydial inclusions, but PCR positive, was performed the day after the appearance of clinical signs. This may be because inclusions are observed 7 14 days after exposure, 11 and it suggests that the scrapings should be repeated if the initial scraping was executed at an early disease stage. Only 1 of the scrapings performed during the chronic phase of infection had inclusions, in agreement with a previous work reporting the still-detectable, but rarer presence of inclusions at 20 days postexposure. 11 The appearance of chlamydial inclusions we observed coincides with what has been reported and widely described in the literature. 1,11,14,50,51 Note that there are a number of structures that may be confused with chlamydial inclusions, such as melanin granules, herniated chromatin, superimposed nuclei of other cells, and eosinophilic granules. C felis inclusions differ from these artifacts because they have a well-defined and particulate structure. 51 Because of the expertise required to differentiate artifacts from inclusions and because the sensitivity and specificity of the assay rely on the accuracy of smear interpretation, slides should be examined by a skilled cytologist. Given the agreement seen in this study between PCR and conjunctival scraping results in acute-phase infection and considering the rapidity and low cost of execution, cytologic examination is confirmed to be useful for the routine screening of cats suspected of having chlamydial conjunctivitis. This would allow the number of animals that need to be tested by PCR to be reduced to include only those whose scrapings are not immediately diagnostic (eg, in smears of low quality, in chronic-phase infections, and in antibiotic-treated animals). Moreover, cytology would at the same time allow the exclusion of other causes of conjunctival inflammation, such as eosinophilic conjunctivitis. Prevalence of FHV-1 FHV-1 was detected by conventional PCR in 33% of cats with conjunctivitis and in 20% of control cats. A 32% prevalence was observed when considering cats with chronic conjunctivitis only. Similar prevalences have been reported in conjunctival swabs or biopsy specimens from cats with conjunctivitis, upper respiratory tract disease, or both. In 1993, Nasisse et al 38 isolated FHV-1 from 19% of cats with chronic conjunctivitis. In 1997, Stiles et al 25 found a prevalence of 54% by nested PCR. In 1999, Burgesser et al 36 reported FHV-1 in 13.7% of cats by nested PCR but in only 8.5% of cats by direct virus isolation. In 1999 and 2001, Sykes et al 12,33 found FHV-1 in 21.2% of cats with conjunctivitis by a duplex PCR (C felis and FHV-1) and in 17.3% of cats by a triplex PCR (C felis, FHV-1, and FCV). Importantly, the presence of FHV-1 has been observed in samples taken from the conjunctiva (swabs, scrapings, or biopsy) of control cats in different studies. Stiles et al 25 observed FHV-1 by nested PCR in 12% of normal cats. In 1997, Stiles et al 37 reported no viral isolation from 15 control cats, whereas nested PCR detected the virus in 2 of 15 (13.3%) animals. Maggs et al 27 and Burgesser et al 36 isolated FHV-1 in 10.9 and 10.7% of normal cats, respectively, from conjunctival swabs. Burgesser et al 36 obtained a higher prevalence in the same control animals by nested PCR (30.9%). Finally, by a duplex PCR, Sykes et al 12 observed FHV-1 in just 1.1% of control cats. Other studies, performed on oropharyngeal swabs, obtained FHV-1 isolation rates in healthy cats that ranged from 0 to 7.7%. 37,52 57 The finding, with different assays, of FHV-1 in the conjunctiva of clinically normal cats is most certainly due to the large percentage of cats latently infected, undergoing viral reactivation and subclinical shedding, as well as to the persistence of the virus in extraneural tissues of the head, including the cornea, lacrimal glands, and conjunctiva of normal or chronically infected cats. 40 At present, one of the major concerns about PCR (particularly nested PCR) as a diagnostic test for FHV-1 is related to the high sensitivity of the technique, permitting the detection of very low numbers of genomic copies of the virus. However, note that viral isolation from conjunctival swabs showed FHV-1 in approximately 10% of normal cats in 2 studies 35,37 and in 19% of chronically affected animals in another report. 38 This suggests that problems in interpreting FHV-1 detection in conjunctival samples and its relationship with ocular lesions arise not only with very sensitive assays, such as PCR, but with other techniques, such as virus isolation, as well. Much like the authors of a previous study, 36 we found no significant difference in the prevalence of FHV-1 between conjunctivitis-affected cats and control cats (P.25) by PCR, indicating that the assay, by itself, is not suitable for the diagnosis of FHV-1 related conjunctivitis. Re-

7 Conjunctivitis in Cats 805 cently developed assays such as real-time PCR and latencyassociated transcript detection may allow further definition of FHV-1 presence in healthy cats, because they would be able to determine the viral load and whether latent forms are present. Nevertheless, determining the amount and state (latent, reactivated, or persistent) of FHV-1 in the eyes of chronically affected animals may aid in the understanding of the pathogenesis of chronic ocular lesions. In humans and animals, herpes simplex virus type 1 (HSV-1) has been shown to cause ocular diseases, including chronic herpetic stromal keratitis (HSK). Recent studies investigating the pathogenesis of HSK in animals have shown that it is an immunopathologic disease, mediated mainly by CD4 T lymphocytes. 58,59 The nature of the antigens causing the immune reaction, whether they are derived from virus or host, remains to be ascertained. 60 In a mouse model, viral replication was shown to be necessary for the development of HSK, 60 and, in other studies, HSV-1 was found to persistently infect the conjunctiva, eyelid skin, and cornea of mice. 61,62 In cats with FHV-1 induced stromal keratitis, Nasisse et al 63 observed the presence of viral antigen and infiltrating mononuclear cells in the corneal stroma, compatible with an immunopathologic mechanism. In this study, we did not detect the virus in 9 cats with clinical signs strongly suggestive of chronic FHV-1 infection, whereas 20% of control animals tested positive for the virus. Four hypotheses can be formulated to explain this apparent discrepancy. 1st, there may be a lower number of viral copies in some cats with chronic lesions with respect to the normal cats that tested positive. This would be compatible with the pathogenesis of FHV-1 chronic lesions, sustained by an immune-mediated mechanism, and not related to the cytolytic damage of tissues by the virus. A 2nd possibility is that another microorganism is responsible for the ocular lesions clinically ascribed to FHV-1. A 3rd possibility is that the specimens that tested negative were collected inadequately, accounting for a lower number of cells containing the virus in the samples. Finally, a 4th explanation may be that some inhibitor was present in the PCR reactions. Studies that use real-time PCR may be useful in cats with chronic conjunctivitis, because this assay has higher sensitivity and specificity than either conventional or nested PCR, and it evaluates the number of viral copies present in the samples. 39 In the current study, 5 of 70 (7%) cats were infected with both C felis and FHV-1, whereas other authors have reported a lower prevalence of mixed infection. In cats with chronic conjunctivitis, Nasisse et al, 38 by means of an IFA assay for C felis and both virus isolation and IFA for FHV- 1, found that just 1 of 60 cats had coinfection with C felis and FHV-1. This discrepancy might be due to the lower sensitivity of the IFA assay for C felis detection, 1,29 especially in chronic-phase infections, compared to PCR. By a duplex PCR, Sykes et al 12 found a low presence of coinfection (3 of 462 cats, or 0.6%), whereas by a triplex PCR, no cats with coinfection were observed. 33 The different results we obtained may be explained by the different PCR protocol we used, which was simple PCR, or, less likely, they may be related to the different population or country from which the samples were taken. In summary, the prevalence of C felis in northern Italy among cats with conjunctivitis, as detected by PCR, was 20%, whereas none of the control cats was found to be positive. PCR positivity was significantly associated with conjunctivitis (P.004), indicating its utility for diagnosing C felis infection. In the acute disease phase, PCR and conjunctival scraping results showed substantial agreement. Although much of the sensitivity and specificity of conjunctival scrapings rely on the experience of the examiner, the results of our study show the usefulness of inclusions detection as a rapid in-practice screening that does not require the equipment necessary for PCR techniques. As determined by PCR, FHV-1 prevalence was 33% in cats with conjunctivitis and 20% in control cats. No significant difference in FHV-1 prevalence was found between cats with conjunctivitis and control animals (P.25), suggesting that PCR detection of the virus, by itself, cannot be used to definitively establish the diagnosis of FVH-1 related conjunctivitis. 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