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SMALL ANIMALS 2 Australian Veterinary Journal Volume 84 Nos 1 84 2 7 F IP is a progressive debilitating ultimately fatal systemic disease of domestic and wild felidae caused by infection with mutant variants of the ubiquitous FeCV These mutant variants are commonly referred to as feline infectious peritonitis virus or FIPV The enigmatic nature of FIPV infec tion coupled with the uniqueness and gravity of the disease has generated a great deal of interest amongst researchers Despite considerable advances in our understanding much remains unknown and FIP continues to be one of the most significant infectious diseases of cats One of the most challenging practical aspects of FIP is procuring a definitive antemortem diagnosis A wide range of tests each with limitations has been developed to facilitate a definitive diagnosis of FIP Consideration of these limitations is imperative for the proper use and interpretation of these test results Since the development of the first technique to detect FCoV serum antibody titres in 1976 2 interpretation of FCoV serology has been a controversial topic in feline medicine Serum anti body tests are considered by some workers to be helpful in supporting a clinical diagnosis of FIP 3 although they must be considered together with the patient s history and other clinico pathological findings and can never be used alone 4 5 While an indirect immunofluorescent antibody titre for FCoV of 1 1600 is claimed to have a positive and negative predictive value for FIP of 0 94 and 0 88 respectively antibody titres below this end titre are unreliable and fraught with interpretative difficulties 3 Furthermore qualitative tests that simply detect the presence or absence of FCoV antibodies are not helpful in diagnosing FIP Despite this recommendations for the use of serology and the management of seropositive cats have varied The current view however is that the euthanasia of cats on the basis of serological tests alone irrespective of the magnitude of the titre can never be justified 6 7 FCoV serology can however provide valuable information about the dynamics of infection within groups of cats either as part of control programs within breeding catteries and shelters 8 or when used to conduct epidemiological studies within wider feline populations 9 While FCoV serology is unable to predict which cats will go on to develop FIP it is accepted that the magnitude of the FCoV antibody titre reflects the viral load within a given host 10 11 Although this is not a direct predictive relationship 12 13 some researchers have speculated that certain cats with persistent and or high FCoV antibody titres given the right host susceptibility may be at a greater risk of developing FIP A range of serological tests used to measure FCoV antibody titres is available and marketed for veterinary use The two most common uses for FCoV serology are i as an aid to the diagnosis of FIP in cats with history and signs consistent with the disease and ii to determine if a presently healthy cat has been exposed to FCoV The latter is most commonly requested as part of a a Faculty of Veterinary Science Building B14 The University of Sydney New South Wales 2006 b Post Graduate Foundation in Veterinary Science Veterinary Conference Centre B22 The University of Sydney New South Wales 2006 c Corresponding author jnorris mail usyd edu au BSH British Shorthair cats DSH Domestic Shorthair cats ELISA Enzyme linked immunosorbent assay FCoV Feline Coronaviruses FeCV Feline enteric coronavirus FIP Feline infectious peritonitis FIPV Feline infectious peritonitis virus OLR Ordinal logistic regression PCR Polymerase chain reaction NewJan Feb qxd 19 01 2006 3 07 PM Page 2 SMALL ANIMALS SMALL ANIMALS 3Australian Veterinary Journal Volume 84 Nos 1 and ii tests used to determine if a presently healthy cat s had been exposed to FCoV for example as part of routine cattery management programs or in a multicat household in which one cat had or was suspected of having FIP Additionally results from cats with one or more physical or biochemical finding suggestive of FIP ascites pleural effusion persistent pyrexia uveitis neurological signs hyperglobulinaemia weight loss were further divided into groups for additional assessment Data analysis All statistical analyses were performed using a commercial statis tical software package Minitab v 13 32 for Windows For all tests P values 0 05 were considered significant Results from each antibody test were related to four explanatory variables age breed gender and health status at the time of blood collec tion by means of two separate univariate OLR analyses Selected factors for both tests were further examined independ ently The median titres of cats with clinical signs suggestive of FIP group 1 and clinically healthy cats group 2 within each breed were compared by Mann Whitney U tests Median titres of domestic versus purebred and male versus female cats for both tests and 2 years of age for the Coronase test were also compared by Mann Whitney U tests One sample sign tests were used to identify differences between the median titres of cats with certain physical findings consis tent with FIP and the overall median for the Coronase test Similarly Mann Whitney U tests were used to compare median titres of cats with and without a recorded history of certain clin ical signs Kruskal Wallis analyses were used to identify signifi cant differences in median titres within each breed for each test The correlation between Coronase and 7B FIP test results for blood samples that were tested concurrently was calculated to determine the degree of association between the two tests Results Coronase antibody test results Results from 637 Coronase antibody tests performed at VETPATH on samples sent from all states and territories of Australia between June 2001 and June 2003 were recorded Results were reported as one of the following antibody titres noted here and subsequently as a reciprocal titre 0 25 100 400 800 or 1600 The overall median Coronase antibody titre was 100 range 0 to 1600 Breed The distribution of median Coronase antibody titres for indi vidual cat breeds represented by a minimum of eight cats is shown in Figure 1 There was a wide range of median antibody titres in these 12 breeds from the lowest median antibody titre of 62 5 in the Siamese and Burmilla to the highest median anti body titre of 400 in the Burmese Cornish Rex and BSH There were significant differences in median Coronase antibody titres of these 12 breeds P 0 0005 The median antibody titres of Siamese Persians DSH and Bengal cats 100 were significantly lower than that of BSH Cornish Rex and Burmese cats 400 P 0 0001 Similarly using geometric means Siamese Persians DSH and Bengal cats mean 43 had significantly lower anti body titres than BSH Cornish Rex Birman and Burmese cats mean 138 Figure 2 Age No significant association between the age of cats and the Coronase antibody titre was identified P 0 116 although there was a trend towards an inverse relationship between these two variables The distribution of median Coronase antibody titres by age is illustrated in Figure 3 NewJan Feb qxd 19 01 2006 3 07 PM Page 3 SMALL ANIMALS 4 Australian Veterinary Journal Volume 84 Nos 1 P 0 2825 There were no significant differ ences in the median titres of group 1 and group 2 cats when divided into individual breeds with the exception of Persian cats for which the median Coronase antibody titre of group 1 cats 12 5 was significantly lower than for group 2 cats 250 Table 1 There was no significant difference between the median titres of group 2 domestic and purebred cats P 0 7573 Overall no significant association between the health status of cats and the Coronase antibody titre could be identified P 0 516 Clinical findings associated with FIP The median Coronase antibody titres of cats with one or more of the physical or biochemical abnormalities suggestive of FIP as stated in the methods were compared to the overall median Coronase antibody titre There was no significant difference between the median Coronase antibody titres of cats for any of the physical or biochemical findings examined and the overall median Table 2 7B FIP antibody test results Results from 191 7B FIP antibody tests performed at VETPATH between June 2001 and June 2003 were recorded The results of this 7B FIP test were reported by VETPATH as one of the following reciprocal antibody titres 0 40 80 160 320 or 640 The overall median 7B FIP antibody test titre was 40 range 0 to 640 Breed The range of 7B FIP antibody titres of the breeds represented by six or more cats is shown in Figure 4 There was no significant difference in the median 7B FIP antibody titres of these seven breeds P 0 124 The median 7B FIP antibody titre 0 of domestic cats n 49 was not significantly different from the median 7B FIP antibody titre 40 of purebred cats n 123 P 0 6661 Overall a significant relationship between 7B FIP antibody titre and breed was not identified P 0 797 Age No significant association between age of tested cats and 7B FIP antibody titre was identified P 0 263 Gender The 7B FIP test results were recorded for 102 female and 72 male cats while the gender of 17 tested cats was not recorded The median 7B FIP antibody titre of female cats 40 was not significantly different from the median 7B FIP antibody titre 0 200 400 600 800 1000 1200 1400 1600 1800 Coronase titre 1 n Persian n 41 DSH n 140 Siamese n 4 Burmilla n 8 Bengal n 40 Australian Mist n 18 Tonkinese 8 Birman n 12 Cornish Rex n 9 Burmese n 156 Ragdoll n 36 BSH n 14 0 50 100 150 200 250 300 350 400 P ersian n 41 DS H n 140 5 S iamese n 4 B engal n 40 B urmilla n 8 Ra gdoll n 36 r A ust alian Mist n 18 B urmes e n 156 T onkin e se n 8 B irman n 12 Co rnish Re x n 9 B SH n 14 Coronase titre 1 n Figure 1 Boxplot of serum Coronase antibody titres against Feline Coronavirus versus the breed of cat median triangle interquar tile range box range line DSH Domestic Shorthairs BSH British Shorthairs Figure 3 Box plot of Coronase antibody titre against Feline Coronavirus versus age of cat median triangle interquartile range box range line Figure 2 Geometric mean Coronase antibody titres against Feline Coronavirus versus the breed of cat DSH Domestic Shorthairs BSH British Shorthairs 0 200 400 600 800 1000 1200 1400 1600 0123456 78 910 Age years Coronase titre 1 n NewJan Feb qxd 19 01 2006 3 07 PM Page 4 SMALL ANIMALS SMALL ANIMALS 5Australian Veterinary Journal Volume 84 Nos 1 interquarttile range box range line DSH Domestic Shorthairs 0 of male cats P 0 7932 and therefore no significant associa tion between the gender of tested cats and 7B FIP antibody titre was identified Health status at the time of blood collection Results from the 7B FIP test were divided into two separate groups on the basis of the reported health status of the cat at NewJan Feb qxd 19 01 2006 3 07 PM Page 5 SMALL ANIMALS 6 Australian Veterinary Journal Volume 84 Nos 1 and or ii the generation of a more intense humoral immune response to FCoV infection in these breeds than in other breeds The production of a humoral response to FCoV infection in the absence of an effective cell mediated immune response is ineffective in controlling FCoV infection and may enhance uptake of FCoV into macrophages The identification of parallels between breed susceptibility to FIP and high antibody titres suggests that predisposition to disease is at least partly correlated with the existence of greater viral loads in these breeds with a corresponding increased chance of FIPV arising by mutation in vivo Although the actual scenario is likely to be more complex it is possible that concerted efforts to decrease viral loads in susceptible breeds may yield a lower incidence of FIP Breed associated variation in the immune response to FCoV is likely to be an important factor determining the susceptibility or resistance of certain breeds to FIP For example differences in the function of T lymphocyte subpopulations and their cytokines may exist between individuals Further characterisa tion of the immune response in cats with FIP is required to investigate possible breed specific aspects of the immunopatho genesis of FCoV infection and FIP No significant association between the gender of tested cats and either the Coronase or 7B FIP test titres was identified in this study which is comparable to results from other studies 18 21 This observation suggests that any gender related differences do not have a significant impact on transmission of FCoV A trend towards an inverse relationship between Coronase titre and age was identified in this component of the study This is similar to reported findings of others 19 Cats are commonly infected with FCoV at a young age This may reflect a combina tion of the cat dense environments from which most kittens regardless of breed are acquired as well as the general immuno naivety of young animals which predisposes them to many infectious diseases Most infected cats shed FCoV for less than 1 year after infection 8 They are therefore able to reduce viral loads over time particularly if they are rehoused in single or two cat households where they will be much less likely to be continually re infected by other shedding cats especially young cats The high proportion of test results available from cats with suspected FIP was used to investigate the value of FCoV sero logical antibody tests for the diagnosis of FIP One of the most significant findings of this study was the absence of an identifi able association between the health status of tested cats at the time of blood collection and either Coronase or 7B FIP anti body titres The median Coronase antibody titre of cats for which FIP was considered to be a differential diagnosis was not significantly different from the median titre of presently healthy cats for which the Coronase test was used to determine previous exposure to FCoV The median 7B FIP antibody titre of cats suspected of having FIP was ironically lower than the median titre of healthy cats although this difference was not significant The most obvious limitation of this study was the lack of follow up information regarding the outcome of disease in those cats tested due to suspected FIP It is not known whether these cats recovered died of another disease that resembled FIP clini cally or whether they indeed had histologically confirmed FIP It is therefore possible that the inclusion of an indeterminate proportion of cats which did not have FIP within group 1 decreased the overall median of group 1 cats to a lower value than would have been recorded had only cats with confirmed FIP been included However when both Coronase and 7B FIP antibody titres from cats with specific clinical findings commonly associated with FIP were examined individually there was likewise no significant distinction between the median titres of cats with any of these clinical findings and the overall median antibody titre for either serological test The clinical findings commonly associated with FIP are by no means pathognomonic for the disease and it is possible that they were associated with an alternative pathological process in some tested cats However when it is considered that FIP is a common cause of ascites in young cats and that most alternative causes of this are relatively easily distinguished from FIP by basic fluid analysis a significantly higher median antibody titre of cats with this clinical sign in particular would be expected to demonstrate any diagnostic value of serology None however was identified The most commonly stated reason for testing healthy cats with either test was as part of FCoV control programs in catteries As identified in other seroprevalence studies 2 13 22 FCoV antibody titres in cats living in catteries are often higher than in owned pet cats It is therefore possible that the considerable proportion of cats living in catteries within the clinically healthy group group 2 raised the overall median titre for group 2 and potentially disguised a difference in median titres of cats in groups 1 and 2 To attempt to gauge the significance of this information on the test request form was used to classify healthy cats as cattery cats or household cats Although it was only possible to classify 140 cats in this way the median Coronase and 7B FIP antibody titres of group 2 cats that were known to live in catteries were not signif icantly different from and were actually slightly lower than the median titres of those living in households Furthermore the median antibody titre of group 2 domestic cats which were very unlikely to be tested as part of a cattery breeding population was not significantly different from the median titre of group 2 purebred cats Even after the cumulative impact of these potential limitations is considered their effect on the overall median titres of group 1 and 2 cats is unlikely to be so great as to completely obscure genuine associations between the health status of tested cats at the time of blood collection and Coronase or 7B FIP antibody titres The failure to identify significant serological differences between cats for which FIP was a differential diagnosis under consideration and clinically healthy cats was a very significant NewJan Feb qxd 19 01 2006 3 07 PM Page 6 SMALL ANIMALS SMALL ANIMALS 7Australian Veterinary Journal Volume 84 Nos 1 37 1449 1453 3 Hartmann K Binder C Hirschberger J et al Comparison of different tests to diagnose feline infectious peritonitis J Vet Intern Med 2003 17 781 790 4 Addie D Jarrett O Feline coronavirus infection In Greene CE editor Infectious Diseases of the Dog and Cat 2nd edn Saunders Philadelphia 1998 58 68 5 Sparkes AH Gruffydd Jones TJ Harbour DA Hopper CD Smerdon TN Blood tests for FIP Vet Rec 1990 126 225 6 Barlough JE Jacobson RH Pepper CE Scott FW Role of recent vaccination in production of false positive coronavirus antibody titres in cats J Clin Microbiol 1984 19 442 445 7 Addie DD Interpretation of feline coronavirus serology In Pract 1989 11 232 235 8 Addie DD Jarrett O Control of feline coronavirus infections in breeding catteries by serotesting isolation and early weaning Feline Pract 1995 23 92 95 9 Horzinek MC Osterhaus ADME Feline infectious peritonitis A worldwide serosurvey Am J Vet Res 1979 40 1487 1492 10 Addie DD Jarrett JO Feline coronavirus antibodies in cats Vet Rec 1992 131 202 203 11 Hickman MA Morris JG Rogers QR Pedersen NC Elimination of feline coronavirus infection from a large experimental specific pathogen free cat breeding colony by serologic testing and isolation Feline Pract 1995 23 12 Sparkes AH Gruffydd Jones TJ Harbour DA Feline coronavirus antibodies in UK cats Vet Rec 1992 131 223 224 13 Sparkes AH Gruffydd Jones TJ Howard PE Harbour DA Coronavirus serology in healthy pedigree cats Vet Rec 1992 131 35 36 14 Addie DD The diagnosis and prevention of FIP and recent research into feline coronavirus shedding Proceedings of the 8th Annual Congress of the European Society of Veterinary Internal Medicine 1998 Vienna Austria 15 Vennema H Poland A Hawkins KF Pedersen NC A comparison of the genomes of FECVs and FIPVs and what they tell us about the relationships between feline coronaviruses and their evolution Feline Pract 1995 23 40 44 16 Herrewegh AAPM Vennema H Horzinek MC Rottier PJM deGroot RJ The molecular genetics of feline coronaviruses comparative sequence analysis of the ORF7a 7b transcription unit of different biotypes Virology New York 1995 212 622 631 17 Kennedy MA Brenneman K Millsaps RK Black J Potgieter LND Correlation of genomic detection of feline coronavirus with various diagnostic assays for feline infectious peritonitis J Vet Diagn Investig 1998 10 93 97 18 Muirden A Prevalence of feline leukaemia virus and antibodies to feline immunodeficiency virus and feline coronavirus in stray cats sent to an RSPCA hospital Vet Rec 2002 150 621 625 19 Addie DD McLachlan SA Golder M