Studies on the aetiopathogenesis of feline chronic gingivostomatitis S.M.J. Dolieslager DVM, MRCVS Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy College of Medicine, Veterinary and Life Sciences University of Glasgow June 2012 © Sanne Maria Johanna Dolieslager, June 2012 ii Abstract Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress. No specific treatment methods are available and little is known about its aetiology. The aims of this study were:- 1) to identify the bacterial flora, including uncultivable and potentially novel species, in healthy cats and those with FCGS, using 16S rRNA gene sequencing in combination with conventional culture methods; 2) to investigate the viral status of cats with and without FCGS; 3) to assess the immune response by investigating the expression of cytokine and Toll-like receptor (TLR) genes in tissue biopsies from normal cats and those with FCGS; 4) to investigate the histopathological changes in tissue biopsies from normal cats and those with FCGS, 5) to assess putative risk factors for FCGS by the use of a questionnaire- based study. Oral swabs, mucosal biopsies and blood were collected and the location of the oral lesions was recorded. A total of 32 cats with FCGS and 16 normal cats were included in the study. Bacteria were identified from swabs by use of 16S rRNA gene sequencing and by conventional culture methods. Blood samples and swabs were used for diagnosis of infection with feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline herpes virus 1 (FHV-1), feline calicivirus (FCV) and for blood biochemistry and haematology. Gene expression levels for TLR2, TLR3, TLR4, TLR7 and TLR9, and cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α and IFN-γ mRNA were determined using quantitative PCR in biopsy samples from healthy cats and cats with FCGS. Histopathological examination of the tissue biopsies was done using hematoxylin and eosin (H&E) staining. In the healthy group, 16S rRNA gene sequencing demonstrated that the most prevalent bacteria were part of the Proteobacteria and Bacteroidetes phyla, plus a group of uncultured bacteria. The most prevalent species in the healthy group were Xanthomonadaceae bacterium (6.2 % of clones analysed), Capnocytophaga canimorsus (5.4%), Capnocytophaga cynodegmi (4.8%), Bergeyella species (4.5%) and Pasteurella multocida subspecies septica (4.4%). Uncultured bacteria accounted for 29% of the clones analysed. In the FCGS group most of the identified species were part of the phylum Proteobacteria. The most prevalent species in the FCGS group were P. multocida subsp. multocida (14.1%) P. multocida subsp. septica (11.5%), Pseudomonas sp. (7.3%), Tannerella forsythia (6.6%) and Porphyromonas circumdentaria (5.6%). A variety of uncultured bacteria represented 7.7% of all analysed FCGS clones. The culture data showed the most prevalent bacteria in the healthy group were iii P. multocida subsp. septica (9.9%), and uncultured bacteria (30.5%). In the FCGS group the most prevalent isolates were P. multocida subsp septica and P. multocida subsp. multocida (both 9.9%). Uncultured bacteria accounted for 21.7% of all isolates. FCV was detected in 71% of cats with FCGS and in 13.3% of normal cats. FeLV antigen was detected in 33.3% of normal cats but not in any cats with FCGS. FIV antibodies were detected in 3.4% of cats with FCGS and in 33.3% of normal cats. FHV-1 was detected in 6.9% of cats with FCGS, but was not detected any of the normal cats. In the FCGS group a significant increase was seen in the expression of TLR2 and TLR7 genes as well as TNF-α, IFN-γ, IL-1β and IL-6 cytokine genes. The healthy cats and cats with FCGS in the study that were found to harbour T. forsythia and P. circumdentaria showed an increase in the expression of several TLR and cytokine genes when compared to the group of cats in which these bacterial species were absent. The most severely inflamed sites in the oral cavity of cats with FCGS included the tissue lateral to the palatoglossal folds and the maxillary attached gingiva. Histopathological analysis of the tissue from the palatoglossal folds showed two types of infiltrates:- 1) a combination of lymphocytes and plasma cells, most often seen in the milder inflamed tissue samples; 2) a predominantly plasmacytic infiltration, most often seen in the severely inflamed tissue samples. Preliminary data from a questionnaire-based epidemiological study showed that the presence of potential environmental stress factors such as no ability to roam outdoors and the presence of more than one cat in the household is significantly higher in cats with FCGS when compared to normal cats. This study highlights the possibility of a multifactorial aetiology for FCGS in which FCV, specific bacteria and stress factors may play an important role. Although species from the Bacteroidetes phylum appeared to be capable of eliciting an immune response, these were not the most prevalent species in the FCGS group. A shift could be seen in the composition of the bacterial flora when healthy cats and those with FCGS were compared. iv Table of contents Abstract ...................................................................................... ii List of Figures ............................................................................. viii Acknowledgements .......................................................................... x Author’s Declaration ...................................................................... xii List of abbreviations ..................................................................... xiii Chapter 1 General introduction .......................................................... 1 1.1 Preface ............................................................................... 1 1.2 Feline chronic gingivostomatitis .................................................. 2 1.2.1 Definition of FCGS ............................................................. 2 1.2.2 Prevalence ...................................................................... 5 1.3 Treatment and management of FCGS ............................................ 6 1.3.1 Preventive measures .......................................................... 6 1.3.2 Symptomatic treatment ....................................................... 7 1.4 Bacteriology ......................................................................... 11 1.4.1 The healthy bacterial oral flora in cats .................................... 11 1.4.2 The oral flora in the inflamed feline oral cavity ......................... 13 1.4.3 Bartonella henselae .......................................................... 16 1.5 Feline viruses and FCGS ........................................................... 18 1.5.1 Feline leukaemia virus ....................................................... 18 1.5.2 Feline immunodeficiency virus .............................................. 19 1.5.3 Feline calicivirus .............................................................. 20 1.5.4 Feline herpes virus 1 ......................................................... 21 1.6 The immune response ............................................................. 23 1.6.1 Defence mechanism in the oral cavity ..................................... 23 1.6.2 Toll-like receptors ............................................................ 24 1.6.3 Cytokines ....................................................................... 28 1.7 Risk factors in FCGS ................................................................ 31 1.7.1 Age .............................................................................. 31 1.7.2 Breed ........................................................................... 31 1.7.3 Sex .............................................................................. 31 1.8 Aims .................................................................................. 32 Chapter 2 Materials and methods ....................................................... 33 2.1 Ethical approval .................................................................... 33 2.2 Sample collection .................................................................. 33 2.3 Sample processing ................................................................. 34 2.4 Statistical analysis ................................................................. 34 2.5 List of suppliers .................................................................... 37 2.6 General stock solutions and buffers ............................................. 38 Chapter 3 Clinical signs and laboratory diagnostic evaluation ...................... 39 3.1 Introduction ......................................................................... 39 3.2 Materials and methods ............................................................ 40 3.2.1 History and clinical signs ..................................................... 40 3.2.2 Oral examination .............................................................. 40 3.2.3 Blood biochemistry and haematology ...................................... 42 3.2.4 Statistics........................................................................ 42 3.3 Results ............................................................................... 44 3.3.1 Signalment ..................................................................... 44 3.3.2 Vaccination status ............................................................ 44 3.3.3 Clinical signs