Joural of Felie Medicie ad Surgery (2009) 11, 1023e1027 doi:10.1016/j.jfms.2009.05.023 SHORT COMMUNICATION Felie herpesvirus 1 ad felie calicivirus ifectios i a heterogeeous cat populatio of a rescue shelter Agélique Zicola DVM, MSc 1, Claude Saegerma DVM, MSc, PhD, DipECVPH 2, Domiique Quatpers DVM 1, Julie Viadier DVM 1, Etiee Thiry DVM, MSc, PhD, DipECVPH 1 * 1 Virology ad Viral Diseases, Departmet of Ifectious ad Parasitic Diseases, Faculty of Veteriary Medicie, Uiversity of Liège, B-4000 Liège, Belgium 2 Epidemiology ad Risk Aalysis Applied to Veteriary scieces, Departmet of Ifectious ad Parasitic Diseases, Faculty of Veteriary Medicie, Uiversity of Liège, B-4000 Liège, Belgium Date accepted: 29 May 2009 Felie herpesvirus 1 () ad felie calicivirus (), associated with upper respiratory tract disease, are highly prevalet i cats worldwide. With the aim to ivestigate the importace of felie respiratory viruses i a heterogeeous populatio of cats, samples were take i a rescue shelter i Liège, Belgium, betwee March 2005 ad August 2006. Reverse trascriptio polymerase chai reactio (RT-PCR) ad polymerase chai reactio (PCR) were performed to diagose ad ifectio i the sampled cats. The prevalece rate (33.1%) was higher for tha for (20.1%) whereas prevalece rate of co-ifectio with both viruses was 10%. Gigivitis was more commo i ifectios (odds ratio (OR) ¼ 2.83) whereas respiratory sigs were more ofte observed with ifectios. The average age was sigificatly higher i positive cats (38 moths) tha i positive cats (29.9 moths). The secod ad the fourth quarters of the year ad the two first quarters were sigificatly more at risk tha the others i the case of ad ifectio, respectively. Age was foud to be a cofoudig factor. High prevalece of both ifectios stregthes the importace of applyig hygieic ad prevetive measures i rescue shelters where cats with a ukow status of vacciatio are itroduced. Ó 2009 Published by Elsevier Ltd o behalf of ESFM ad AAFP. Felie herpesvirus 1 () ad felie calicivirus () are the mai agets ivolved i the felie upper respiratory tract disease (URTD). 1,2 These viruses are resposible for acute illess ad may also be the cause of recurret or chroic lesios. is a double-straded DNA virus, member of the Varicellovirus geus of the subfamily Alphaherpesviriae. 3,4 It has a tropism for asal epithelial ad cojuctival cells ad for eurocytes. 5 Trigemial gaglia are the site of latet ifectio 6 ad viral reactivatio ca occur durig a stress period. 7 is a sigle-straded positive-sese RNA virus i the family Caliciviridae, geus Vesivirus. There is cosiderable geetic diversity withi ; withi a strai, geetic variatio teds to be higher i edemically ifected cat populatios. 8,9 The most likely hypothesis is that circulatig virus is subjected to a positive selectio pressure iduced by the immue respose of ifected cats, leadig to virus evolutio ad the geeratio of ew strais. 9 Furthermore, *Correspodig author. E-mail: etiee.thiry@ulg.ac.be such evolutio may also i part explai vaccie protectio failures agaist wild-type. 9,10 The aim of this logitudial study was to gai a better kowledge of the epidemiology of felie respiratory viruses i a heterogeeous populatio of cats i a rescue shelter that experieces a cotiuous turover of aimals. Oral swabs ad polymerase chai reactio (PCR) aalyses allowed the study of ad prevalece, ad also cliical sigs caused by sigle or mixed ifectios. Viral ifectios were ivestigated irrespectively of the cliical status of the aimal. The ivestigatios aalysed the effect of several risk factors, ie, sex, age ad seaso, o ad ifectios. For each cat, age, sex, vaccie used at his arrival ad cliical sigs were recorded by the same two veteriarias: presece or absece of gigivitis, salivatio, oral ulcers, lacrymatio, cojuctivitis, asal discharge, seezig, stertor or cough. A total of 299 cats from the Société Royale Protectrice des Aimaux (SRPA), Liège, were icluded i the study. The sample size was calculated accordig to a populatio size of 1500 cats (average umber of cats eterig SRPA durig 1 year), a expected 1098-612X/09/121023+05 $36.00/0 Ó 2009 Published by Elsevier Ltd o behalf of ESFM ad AAFP.
1024 A Zicola et al prevalece of 40% 2 ad a accepted error of 5% with a cofidece iterval (CI) of 95%. Average age of the sampled cats was 34 moths (2e180). A average of 21 cats (from 16 to 26) i the quaratie area was sampled (oe selected radomly i order of appearace whe eterig each cage) every moth, betwee March 2005 ad August 2006. Each cat was sampled oce. The quaratie area was used for puttig i ew arrivals, i case they were icubatig somethig. Oropharygeal swabs were collected ad placed i 2 ml of culture medium (Gibco MEM supplemeted with atibiotics) ad stored i the laboratory at 4 C. Each sample was ioculated o cofluet moolayers of susceptible Cradell Rees felie kidey (CrFK) cells ad icubated at 37 C i a moist atmosphere with 5% CO 2 for 3 days ad examied for characteristic cytopathic effects (CPEs). Viral ucleic (RNA ad DNA) acid was extracted from the superatat of each ioculated moolayer by usig QIAamp viral RNA mii spi protocol (Qiage, Hilde, Germay). Two sets of primers, with a cocetratio of 400 M, for calicivirus detectio were used. A covetioal reverse trascriptio polymerase chai reactio (RT- PCR) for polymerase gee detectio of usig the set of primers p30f/p30r was performed to amplify a 126 base pair (bp) sequece of p30 gee. 11 The primer pair V2F/V2R was used to amplify a 700 bp sequece of the capsid protei VP1gee. 12 The Access RT-PCR System kit from Promega was used with 3 ml of RNA template ad accordig to maufacturer s istructios. Aealig i the RT-PCR cycle was performed at 60 C for p30 primer pairs ad at 61 C for V2 primer pairs for 30 s. A amplificatio with oe of the two primers pairs idicated that the sample was positive for. The set of primers gc-r/gc-f, with a cocetratio of 100 M, was used to diagose by the amplificatio of a 500 bp sequece located i the gee codig for glycoprotei gc. 13 PCR was carried out usig 2e5 uits of Taq DNA polymerase (Biolabs, Leusde, The Netherlads) ad 5 ml of DNA template, aealig performed at 59 C for 1 mi. Negative cotrols were performed i each test by addig water ad superatat of uifected CrFK cells i the place of the ucleic acid template. PCR positive cotrols were performed with vaccie strais. For detectio, 5 ml of the cotrol were used (75.11 g/ml), whereas for detectio, oly 3 ml of the cotrol were used (62.72 g/ml). PCR product sizes were determied by agarose gel electrophoresis ad ethidium bromide staiig. The ifectio prevalece of ad was estimated usig a biomial exact distributio. The mea age differece of ifected cats with or was assessed with the Welch s test (P 0.05). 14 Sex ad quarter of year effects, ad relatio betwee cliical sigs observed ad virus ifectio were aalysed usig a c 2 test ad by mea of odds ratios (ORs) with 95% CI; a P value < 0.05 was cosidered as sigificat. 15 The possible relatioship betwee the ifectio level with or i cats for differet quarters of the year was assessed by a CochraeMateleHaeszel (CMH) method (OR MH ). The BresloweDay test was used to evaluate the homogeeity of the ORs. The prevalece rate of aloe, 33.1% (99/299) (95% CI: 27.8e38.8%), durig the observatio period, was higher tha the, without ifectio, prevalece rate, 20.1% (60/299) (95% CI: 15.7e25.1%), ad tha the prevalece of co-ifectio with both viruses, 10% (30/299) (95% CI: 6.8e14%). Table 1 shows that the use of two primer pairs was appropriate as some samples were positive for oly with oe of the two primers sets. Furthermore, eve without obtaiig a CPE, some samples were foud positive by RT-PCR o the ifected cell culture superatat (Table 1). Amog the 67 samples that reacted egative to RT-PCR but showed a CPE, 49 were foud positive (Table 1). The remaiig 18 samples which appeared to show CPE but were egative by PCR for both viruses may have bee cotamiated with fugi or bacteria at samplig time or alteratively the apparet CPE was due to possible toxic effects of the samples o the cells: further cell passage of the ioculated cultures may have clarified this. It is also possible that there may have bee sesitivity issues with the PCRs used, particularly for. Ideed the RT-PCR performed with the coserved polymerase p30 gee 11 was more sesitive (116 positives detected) tha that performed o the hypervariable regio of the capsid protei gee 12 (79 positives detected) (Table 1). Thus usig both primer pairs for clearly helped maximise the overall sesitivity of detectio, but because of the Table 1. RT-PCR results with the two primer pairs for detectio i ifected cell culture superatat (p30: RT-PCR with p30 primer pair; V2: RT-PCR with V2 primer pair) compared with the PCR results CPE RT-PCR PCR positive samples p30 V2 N N þ þ þ 55 10 þ 35 14 þ 9 3 67 49 þ þ 11 0 þ 15 3 þ 4 0 103 11 CPE ¼ cytopathic effect; ¼ felie calicivirus; RT- PCR ¼ reverse trascriptio polymerase chai reactio; ¼ felie herpesvirus 1; PCR ¼ polymerase chai reactio; N ¼ Number of aimals.
Table 2. Adjusted ORs calculated for selected cliical sigs betwee four groups of aimals ( group ( ¼ 99), group ( ¼ 60), ad group ( ¼ 30) ad group without viral ifectio ( ¼ 110)) Cliical sigs ad lesios Number of aimals by group, FeHV- 1, &,, & OR (95% CI)* & & Gigivitis 41 19 10 22 2.83(1.53e5.23) þ0.66(0.33e1.29) 0.71(0.30e1.67) 1.85(0.90e3.80) 0.93(0.36e2.36) 2.00(0.82e4.88) Salivatio 1 4 2 0 3.37(0.14e83.57) 7.00(0.76e64.18) 7.00(0.61e80.07) 17.6 (0.93e332.73) 1.00(0.17e5.79) 19.39(0.91e415.20) Oral ulcers 5 3 1 2 2.87(0.54e15.15) 0.99(0.23e4.30) 0.65(0.07e5.78) 2.84(0.46e17.50) 1.53(0.15e15.33) 1.86(0.16e21.26) Other oral lesios 0 0 0 3 0.15(0.01e3.03) 1.64(0.03e83.97) 3.26(0.06e167.90) 0.25 (0.01e5.00) 0.50(0.01e26.03) 0.50(0.03e10.02) Lacrymatio 7 8 6 4 2.02(0.57e7.11) 2.02(0.69e5.89) 3.29(1.01e10.69) þ 4.08(1.17e14.16) þ 0.62(0.19e1.97) 6.63(1.73e25.31) þ Cojuctivitis 3 3 0 5 0.66(0.15e2.82) 1.68(0.33e8.63) 0.45(0.02e8.90) 1.11(0.25e4.79) 3.71(0.19e74.25) 0.31(0.02e5.85) Nasal discharge 11 19 10 8 1.59(0.61e4.14) 3.71(1.62e8.50) þ 4.00(1.49e10.71) þ 5.91(2.40e14.56) þ 0.93(0.36e2.36) 6.38(2.24e18.14) þ Seezig 12 30 17 11 1.24(0.52e2.96) 7.25(3.30e15.94)þ 9.48(3.70e24.30) þ 9.00(4.03e20.08) þ 0.76(0.32e1.85) 11.77(4.53e30.54) þ Stertor 4 8 3 4 1.12(0.27e4.59) 3.65(1.05e12.71)þ 2.64(0.55e12.52) 4.08(1.17e14.16) þ 1.38(0.34e5.65) 2.94(0.62e13.95) Cough 0 0 0 1 0.37(0.01e9.11) 1.64(0.03e83.97) 3.26(0.06e167.9) 0.60(0.02e15.04) 0.50(0.01e26.03) 1.20(0.05e30.12) ¼ without viral ifectio; ¼ umber of aimals with cliical sigs; þ: Risk factor statistically sigificat at P 0.05. *OR was adjusted whe values of zero caused problems for OR calculatios (adjustmet of Greier). ad ifectios i a heterogeeous cat populatio 1025
1026 A Zicola et al high sequece variability see i there may still have bee other strais which were ot detectable with the primer pairs used. c 2 tests showed o sigificat differece (P > 0.05) betwee sex ad origi of ifectio. O the other had, Welch s test demostrated that the average age of positive cats was 38 moths, which was sigificatly higher tha the average age of positive cats (29.9 moths; P 0.05). It was also higher tha the average age of uifected cats (32 moths). I breedig catteries, ad isolatio i cats of 4e11 moths old were positively associated with respiratory tract disease. 16 This differece betwee the ivestigated rescue shelter ad breedig catteries ca be explaied by stressful coditios ad high turover i the cat populatio. I the secod ad the fourth quarters of the year, cats were sigificatly more frequetly ifected by tha the two other quarters (c 2 ¼ 7.96, P ¼ 0.005) (OR ¼ 2.07 (95% CI: 1.24e3.43)). The possible relatioship betwee age ad seaso was assessed for, by a CMH test (OR MH ¼ 2.01 (95% CI: 1.23e3.35)). The age of aimals was foud to be a cofoudig factor for the seaso because the crude OR was higher tha the OR calculated for each age stratum: less tha 1 year old(or:2.60(95%ci:0.90e7.47)), 1 year old (OR: 1.00 (95% CI: 0.26e3.93)), 2 years old (OR: 2.50 (95% CI: 0.71e8.80)), 3 years old (OR: 5.27 (95% CI: 1.59e17.5)), ad more tha 3 years (OR: 1.04 (95% CI: 0.37e2.88)). The Breslow test showed that the coditioal ORs were homogeeously distributed (P ¼ 0.30). I the two first quarters, cats were sigificatly more frequetly ifected by the tha i the two later quarters (c 2 ¼ 11.4, P ¼ 0.001) (OR ¼ 2.28 (95% CI: 1.41e3.69)). The possible relatioship betwee age ad seaso was assessed for, by a CMH test (OR MH ¼ 2.33 (95% CI: 1.44e3.80)). The age of aimals was foud to be a cofoudig factor for the seaso because the crude OR was higher tha the OR calculated for each age stratum: less tha 1 year old (OR: 1.18 (95% CI: 0.41e3.37)), 1 year old (OR: 6.13 (95% CI: 1.95e19.26)), 2yearsold(OR:8.00(95%CI:1.45e44.30)), 3 years old (OR: 1.68 (95% CI: 0.48e5.85)), ad more tha 3 years (OR: 1.59 (95% CI: 0.65e3.88)). The Breslow test showed that the coditioal ORs were homogeeously distributed (P ¼ 0.12). Adjusted OR revealed a relatioship betwee virus ifectio ad selected cliical sigs (Table 2). However, 31.2% of sampled cats did ot show ay cliical sig i spite of positive PCR diagosis for or (59/ 189). I cases of positive cats exhibitig cliical sigs, gigivitis was domiat (OR ¼ 2.83, CI: 1.53e5.23). Respiratory sigs like asal discharge (OR ¼ 3.71, CI: 1.62e8.50; OR ¼ 5.91, CI: 2.40e14.56), seezig (OR ¼ 7.25, CI: 3.30e15.94; OR ¼ 9.00, CI: 4.03e20.08), stertor (OR ¼ 3.65, CI: 1.05e12.71; OR ¼ 4.08, CI: 1.17e14.16) ad lacrymatio (OR ¼ 4.08, CI: 1.17e14.16) were maily reported i ifected cats (Table 2). A age effect was demostrated i cats ifected by ad, especially for that ifects youger cats. 16e18 These results could be explaied by the protectio of kittes by materally derived atibodies. Furthermore, a immue-mediated mechaism for the acquisitio of resistace to ifectio has bee postulated i older cats. 19 is rarely isolated from cliically healthy aimals 20 because latet carriers are ot expected to shed the virus at the precise time that they are sampled, 21 whereas is cotiuously shed by ifected cats. 22 I the ivestigated shelter, vacciatio coicides with the arrival of ew cats. However, vacciatio is kow to reduce but ot prevet viral excretio 4,23 ad thus cats already ifected o arrival or subsequetly ifected might still shed either or. The icrease i prevalece observed i sprig could be liked to the parturitio period i the orther hemisphere. Some sampled cats are probably latet carriers of, but virus detectio i swabs is oly possible durig reactivatioesheddig period. 2 The true prevalece rate of ifectio i the cat populatio from the SRPA i Liège is likely to be higher. The age of aimals was foud to be a cofoudig factor for the seaso. However, iteractios with other factors caot be defiitively excluded. Therefore further studies should iclude multivariable aalysis to take ito accout the possible effects of other parameters. I the case of ifectio with both viruses, the absece of lesios was rarely observed. This study shows that, i ifectio, the most commo lesios are oral lesios (47%) ad this observatio correlates with previous studies. 2 O the other had, i ifectio, respiratory lesios are ofte observed (38%). Thirtyoe per cet of sampled cats did ot show ay cliical sig i spite of positive PCR diagosis for viral ifectio. I most rescue shelters, cats with cliical sigs go ito quaratie the arrive, whereas i the absece of cliical sigs, they are directly put ito cotact with other cats. This situatio allows a quick propagatio of the ifectios from subcliical shedders. 24 As the high prevalece of ad ifectios associated with oral ad respiratory sigs are observed i rescue shelters, hygieic measures ad prevetio play a pivotal role to cotrol diseases i such facilities, 25 where the cat populatio is very heterogeeous ad where the vacciatio ad ifectio status of itroduced aimals are ukow. Ackowledgmets The authors wish to thak the Société Royale Protectrice des Aimaux i Liège ad, especially, Domiique Ziat ad Lorèe Dams for their ivaluable techical support, ad Sarah Porter for her help revisig the mauscript. Refereces 1. Baasch MJ, Foley JE. Epidemiologic evaluatio of multiple respiratory pathoges i cats i aimal shelters. J Felie Med Surg 2005; 7: 109e19.
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