Suppurative rhinitis associated with Haemophilus species infection in a cat

INTRODUCTION Cats with persistent or recurrent nasal discharge and sneezing are familiar and frustrating visitors to the general practitioner and the specialist alike. When the history, physical examination findings, or diagnostic tests rule out other causes of nasopharyngeal disease, chronic rhinitis is usually attributed to some manifestation of viral upper respiratory tract disease. Previous infection with feline herpesvirus-1 may cause permanent damage to the nasal turbinates, or recrudescence of latent viral infection may lead to recurrence of signs associated with the original infection. In either event, secondary bacterial infection is expected. Chronic rhinitis infrequently resolves; repeated courses of antibiotics may be required to palliate clinical signs. Most often, therapy is empirical; bacterial culture and susceptibility tests are costly and, owing to the variety of normal flora present, may be difficult to interpret. The mechanism by which disease persists in these cats is incompletely understood. Albeit limited, the success of immunomodulating therapies lends support to the hypothesis that the chronic rhinitis syndrome may represent an ineffective immune response to persistent viral infection. The case reported here is typical of those seen in general practice: a young adult cat with persistent nasal discharge and sneezing whose signs are only temporarily ameliorated by courses of broadspectrum antibiotics. Unexpectedly, an unusual bacterium was isolated from the nasal passages. The case is significant for at least 2 reasons: Haemophilus is only rarely reported with upper respiratory tract disease in the cat and can easily be overlooked and we believe this to be the 1st report of immune function testing (phagocytosis, oxidative burst, lymphocyte subsets) carried out by flow cytometry which was applied to a cat in the clinical setting.


INTRODUCTION
Cats with persistent or recurrent nasal discharge and sneezing are familiar and frustrating visitors to the general practitioner and the specialist alike.When the history, physical examination findings, or diagnostic tests rule out other causes of nasopharyngeal disease, chronic rhinitis is usually attributed to some manifestation of viral upper respiratory tract disease.Previous infection with feline herpesvirus-1 may cause permanent damage to the nasal turbinates, or recrudescence of latent viral infection may lead to recurrence of signs associated with the original infection.In either event, secondary bacterial infection is expected.Chronic rhinitis infrequently resolves; repeated courses of antibiotics may be required to palliate clinical signs.Most often, therapy is empirical; bacterial culture and susceptibility tests are costly and, owing to the variety of normal flora present, may be difficult to interpret.The mechanism by which disease persists in these cats is incompletely understood.Albeit limited, the success of immunomodulating therapies lends support to the hypothesis that the chronic rhinitis syndrome may represent an ineffective immune response to persistent viral infection 26 .
The case reported here is typical of those seen in general practice: a young adult cat with persistent nasal discharge and sneezing whose signs are only temporarily ameliorated by courses of broadspectrum antibiotics.Unexpectedly, an unusual bacterium was isolated from the nasal passages.The case is significant for at least 2 reasons: Haemophilus is only rarely reported with upper respiratory tract disease in the cat and can easily be overlooked 14,19 and we believe this to be the 1st report of immune function testing (phagocytosis, oxidative burst, lymphocyte subsets) carried out by flow cytometry which was applied to a cat in the clinical setting 5,27 .

CASE HISTORY
A 13-month-old, 5.3 kg Sphinx, castrated male cat was referred to the Veterinary Medical Teaching Hospital, University of Florida, for evaluation of chronic sneezing and bilateral purulent nasal discharge.The present owner acquired the cat from the breeder 6 weeks previously: signs of sneezing and nasal discharge were already evident at that time.The referring veterinarian had prescribed courses of amoxicillin (Amoxi-Drops, Pfizer, New York) (22 mg/kg per os (PO) q 12 h), enrofloxacin (Baytril, Bayer, Shawnee Mission, Kansas) (4 mg/kg PO q 12 h) and amoxicillin-clavulanic acid (Clavamox, Pfizer, New York) (12 mg/kg PO q 12 h).Clinical signs resolved for the duration of treatment, but returned as soon as treatment was discontinued.The cat had last received antibiotics 3 days prior to presentation.Immunisation history was not available, but the cat was believed to have been immunised against feline herpesvirus-1 (FHV1), feline calicivirus (FCV) and feline panleukopaenia at age 9 weeks and again at age 12 weeks.The cat had tested negative for feline leukaemia virus and feline immunodeficiency virus as a kitten, and again 2 weeks prior to referral.No other previous or concurrent health problems were reported and the cat had a normal appetite and activity level.
Abnormalities found on physical examination were limited to the eyes and upper respiratory tract.The left eye had hyperaemic conjunctivae and clear watery discharge.A yellowish, clear to mucopurulent discharge was evident from the right nares.Respiratory sounds were mildly stertorous.Body temperature was 37.6 EC.Blood glucose, packed-cell volume, and total solids were within normal reference ranges.
Bilateral turbinate damage was identified on radiographic examination of the skull.Following induction of anaesthesia for rhinoscopy, samples for fungal and aerobic bacterial culture were obtained by aspirating the right nares using a sterile 22G intravenous nylon catheter.Rhinoscopy revealed bilateral hyperplastic nasal tissue, a large amount of purulent nasal discharge within the nasal cavities, and a hyperaemic oropharynx.Biopsies of nasal tissue were collected for histological evaluation.While results were pending, the cat was discharged with a 14-day course of amoxicillinclavulanic acid (Clavamox, Pfizer, New York) (12 mg/kg PO q 12 h).
Histology of the nasal mucosa was interpreted as chronic, active, and moderate to severe, suppurative rhinitis.Fungal culture was negative.Initial aerobic culture at 24 hours using regular media (Columbia Agar 5% Sheep's blood) yielded small pinpoint-sized colonies which were visible only by dissecting microscope.Gram's staining identified 0038-2809 Jl S.Afr.vet.Ass.( 2004) 75(2): 103-107 them as Gram-negative cocci-bacilli, which increased our suspicion of Haemophilus species.The colonies where then transferred to chocolate agar.Culture on chocolate agar media was positive for pure culture of a Haemophilus species (see Table 1 for a description of the bacterial isolation).Further identification was deemed necessary and the isolate was submitted to a human laboratory for species identification.Using RapID™ NH System (Remel (Apogent), Lenexa, Kansas) the isolate was identified as Haemophilus segnis, a human pathogen 4,17 (see Table 1 for details).
Growth of a single organism was thought to represent significant colonisation, as opposed to the mixed flora typically isolated in cases of chronic rhinitis.The isolate was found to be susceptible to a variety of antimicrobials, including amoxicillin-clavulanic acid (Clavamox, Pfizer, New York) and enrofloxacin (Baytril, Bayer, Shawnee Mission, Kansas) which the cat had previously received.
Upon re-evaluation 2 weeks later, the owner reported the cat was still sneezing, but that the nasal discharge had decreased somewhat and was no longer purulent.Ocular discharge was still present from the left eye.No other clinical signs were reported, and the cat continued to have a normal appetite and activity level.
Physical examination was unremarkable except for stertorous breathing sounds attributed to obstructed nasal passages and watery ocular discharge from the left eye.Schirmer tear tests were within reference ranges for each eye.Fluorescein staining of each eye was negative for corneal ulcer or erosion, but the left nasolacrimal duct was not observed to drain properly.
Because of the unusual culture results obtained from the 1st visit, repeat cultures were attempted.A conjunctival swab grew very scant growth of Corynebacterium species.Culture from a retropharyngeal swab identified predominately Mycoplasma sp. and Pasteurella sp.No Haemophilus species were isolated.Virus isolation tests utilising kidney cell cultures were negative.
Because of the persistence of clinical signs in this patient, and the previous isolation of an unusual bacterium, tests were conducted to evaluate immune function.Tests were selected on the basis of availability and included a complete blood count and differential quantitation of serum immunoglobulins, immunophenotyping of lymphocyte populations by flow cytometry, and quantitation of neutrophil phagocytosis and induced oxidative burst.The white cell count and differential were within normal range.Serum concentrations of IgA, IgM, and IgG were normal and are reported in Table 2. Flow cytometry of lymphocyte subsets are reported in Table 3.The numbers of T and B lymphocytes, and the number of CD4+ and CD8+ T lymphocytes were normal; the expansion of B cells relative to T cells was interpreted as being consistent with antigenic stimulation.
Neutrophil phagocytosis and oxidative burst responses to the Haemophilus segnis-like isolate were compared to those for Staphylococcus aureus using a modified dual-colour flow cytometry assay 6,13 .The neutrophil responses to both bacteria were within the normal range reported for cats 6,13 (Table 4).
On reviewing the literature on Haemophilus species in cats 14,19 , it became evident that the isolate was unlikely to be H. segnis

DISCUSSION
Feline upper respiratory tract disease has been amply reviewed in the veterinary literature 1,7,10,11,18,25 .Chronic rhinitis has been defined as inflammation of the nasal passages that has been present for at least 4 weeks and typically presents clinically as sneezing and bilateral nasal discharge 1,3 .Differential diagnoses may include allergic, infectious and inflammatory causes, as well as nasal foreign bodies, neoplasms and advanced dental disease 1,3, 25 .
The most common infectious causes of rhinitis in the cat are FHV-1 or FCV 7,10,11,24,25 .Viral infections may produce both acute and chronic disease.Persistent or recurrent signs of rhinitis may result either from permanent viral-induced damage to the protective mucosal barriers that leads to impaired local immunity and repeated bacterial colonisation, or from reactivation of latent viral infection 18,24 .Diagnosis of viral infection is complicated by the ubiquity of these viruses: most cats are seropositive from prior infection or routine immunisation, and as many as 80-90% of these cats become carriers 10,18,25.However, latently infected cats, even when exhibiting signs of upper respiratory tract disease, may shed few virus particles, and may only shed intermittently 24 .
Primary bacterial infection is considered uncommon.Rhinitis may be seen, but infection with Chlamydophila felis should be considered unlikely in cats without conjunctivitis 20 .Bordetella bronchiseptica and Mycoplasma species can be isolated from healthy cats, but are occasionally implicated as primary agents in disease of the upper and lower airways 11,23 .Chronic rhinitis, regardless of underlying cause, is commonly complicated by secondary bacterial infection.Pasteurella, Streptococcus, coliforms, Staphylococcus and Pseudomonas species are considered normal flora of the feline nasopharynx and are typically isolated from cats with secondary bacterial infections 1 .Only 2 reports exist in the literature where H. felis was isolated from clinical cases.Inzana et al. 14 reported isolating H. felis from the nasopharynx in 6 of 28 normal laboratory cats and a clinical case with respiratory disease.Olsson and Falsen 19 reported isolating H. felis from 5 young cats with signs of conjunctivitis and upper respiratory tract infections.The general characteristics of the genus Haemophilus are small to medium-sized (0.5-1.0 µm), non-motile Gram-negative coccobacilli and following exposure to antibiotics threads or filaments may form 2,14 .Some species require carbon dioxide, especially during primary isolation and are capable of fermenting sugars.Importantly, owing to their fastidious nature, most species of Haemophilus will not grow on regular aerobic laboratory media (Columbia Agar 5% Sheep's blood, MacConkey Agar, and Columbia Agar CAN 5% Sheep's blood) used for most bacterial isolation but require X factor (hemin), or V factor (nicotinamide adenine dinucleotide), or both.The media of choice is chocolate agar.Haemophilus are confined to the mucosal surfaces of animals and humans and are host-specific.The host-specificity is due to the absolute requirement of iron only from iron-bound proteins of their natural host.Since Haemophilus species are speciesspecific, it seems unlikely that the specimen we isolated was the human pathogen H. segnis.Based on RapID™ NH system and morphology it was not possible to prove that the isolate was the same as H. felis.The colour of the isolate (grey versus yellow) and the oxidase-positive nature of the isolate in addition to the negative p-nitrophenyl phosphate results indicate that it may a different species.Precise identification would require sequencing of the bacteria's 16S rRNA gene which was not done.
In an informal (telephone) survey of diagnostic laboratories in southeastern USA, none reported using chocolate agar plates for routine aerobic culture of respiratory samples, and therefore it is likely that Haemophilus felis infections will be overlooked in cats.When not grown on chocolate agar, Haemophilus species can form satellite colonies close to Staphylococcus species because of growth factors supplied by the bacteria to the Haemophilus species 2. Since no veterinary pathogens are included in the RapID™ NH Systems or any other rapid culturing system database, animal pathogens will either be erroneously identified as was our case or yield a unique biocode not listed 21,22 .Many cats with chronic rhinitis, for which a specific underlying cause cannot be identified, or where chronic or recrudescent viral infection is suspected, require frequent, longterm courses of antibiotics to palliate clinical signs 9,25 .The strain isolated in our feline patient was susceptible to a variety of commonly used broadspectrum antimicrobials but resistant to ampicillin and penicillin as has been reported for strains of H. felis 14,19 .
Uncommon infections may be variously attributed to laboratory error, unusual exposure, or to compromised immune function.Growth of a solitary organism makes contamination of the culture improbable.In retrospect, obtaining samples from people or pets in the household might have contributed important information as to where the cat acquired the organism.Although neither FHV1 nor FCV were isolated, because the prevalence of virus is much lower in cats with chronic disease, a negative result did not rule out the possibility of latent infection.Thus, chronic or latent viral infection, especially with FHV1, remained likely.Given the history of chronic signs and the isolation of an unusual pathogen, immune dysfunction emerged as a possible mitigating factor.Immunosuppression in cats can be a consequence of debilitating and systemic diseases, and is associated with feline leukaemia virus and feline immunodeficiency virus infections 12 .Specific immune deficiency syndromes can be grouped in the following categories: congenital disorders, failure of passive transfer of maternal antibodies, acquired neutrophil dysfunction or neutropaenia, and disorders of lymphocytes.As in the present case, immunosuppression may be suspected when infections are recurrent or persistent, or when an unusual pathogen is isolated 12 .Immune function tests may include the following: evaluation of leukocyte number, morphology (complete blood count, differential and bone marrow cytology); evaluation of neutrophil function (chemotaxis, phagocytosis, oxidative burst and killing), evaluation of lymphocytes (quantitation of immunoglobulins, lymphocyte blast transformation, and flow cytometry of lymphocyte subsets) and evaluation for retroviral infection (feline leukaemia virus antigen test and feline immunodeficiency virus antibody test).
In the case reported here, normal total leukocyte count and normal distribution of neutrophils and lymphocytes ruled out a deficiency in the number of circulating immune cells, and examination of the cells excluded an inherited anomaly of leukocyte morphology.Quantitation of serum immunoglobulins established normal humoral immunity and circumstantial evidence for normal B lymphocyte numbers and function.B cells are most important for extracellular infections 12 .

Flow cytometry was used to quantify lymphocyte subset populations: B cells, T cells, CD4 T cells (T-helper cells) and CD8 T cells (T-cytotoxic cells). Activated
T-helper cells are key to generating both humoral and cell-mediated immune responses 8 .The major function of cytotoxic T cells is the killing of cells that express nonself antigens such as tumour cells and virus-infected cells 8 .Although not a comprehensive examination of the innate immune system, testing of phagocytosis and oxidative burst contributed to establishing normal function of this important arm of the immune response.
Results of retroviral and immune function testing were negative.With appropriate levels of serum immunoglobulins, B and T lymphocytes, and normal neutrophil function, the hypothesis of an immune abnormality appeared unlikely.It was not, however, able to measure secretory IgA to assess the strength or specificity of mucosal immunity.In addition, although neutrophilic phagocytic and oxidative burst functions were normal, actual killing of Haemophilus species was not quantified.It may be that disruption of the physical barriers of the nasal mucosa due to prior viral respiratory disease represented the most significant impairment of the immune response in this patient.Interestingly, the ability of some Haemophilus species to cause disease is related to its ability to cleave IgA1 (via IgA1-protease), rendering it non-functional 16 .While not reported for Haemophilus animal pathogens, it may be important in understanding the colonisation and pathogenesis of Haemophilus species on animal mucosal surfaces.

CONCLUSION
Cats with chronic or recurrent signs of rhinitis are often presumed to have bacterial infections secondary to previous infection with FHV-1 or FCV.Viral-induced cytopathic damage to the nasal mucosa, and host reaction to latent viral antigen, are thought to be factors involved in weakening host defenses against opportunistic pathogens.While it may be speculated that cats with latent viral infections suffer from some degree of immunosuppression, comprehensive immune profiling has not been performed.
The unusual bacterial isolate identified in this cat is important since culture technique employed in veterinary diagnostic laboratories for samples from companion animals generally do not include chocolate agar media.It is therefore likely that Haemophilus species are not going to be isolated.However, should a Haemophilus species be recovered, human rapid identification systems do not have animal pathogens listed on their database and give erroneous results.

Table 2 :
Quantification of serum immunoglobulins a (units in mg/d serum).a Cat IgG, IgA, IgM VET-RID kit, Catalog Number R40-103, Bethyl Laboratories, Montgomery, Texas.Tests performed by Clinical Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York.

Table 4 : Flow cytometric quantitation of neutrophil phagocytosis and induced oxidative burst
myristate acetate; PMA stimulates oxidative burst independent of phagocytosis: this is a measurement of the maximal oxidative burst capacity of the neutrophil.
0038-2809 Jl S.Afr.vet.Ass.(2004) 75(2): 103-107 105 a .b PMA = phorbol Infectious diseases of the dog and cat.W B Saunders, Philadelphia: 683-693 13.Hanel R M, Crawford P C, Hernandez J, Benson N A, Levy J K 2003 Neutrophil function and plasma opsonic capacity in colostrum-fed and colostrum-deprived kittens.American Journal of Veterinary Research 64: 538-543 14.Inzana T J, Johnson J L, Shell L, Moller K, Kilian M 1992 Isolation and characterization of a newly identified Haemophilus species from cats: "Haemophilus felis".Journal of Clinical Microbiology 30: 2108-2112 15.Kilian M 1976 A taxonomic study of the genus Haemophilus, with the proposal of a new species.Journal of General Microbiology Gaskell C J, Hart C A, Gaskell R M 1999 Bordetella bronchiseptica infection in the cat.