Frank Künzel

Encephalitozoonosis in rabbits.

Encephalitozoon cuniculi is an obligatory intracellular microsporidian parasite that can infect a wide range of mammals, including rodents, rabbits, horses, carnivores and humans, in which the organism is known as an opportunistic pathogen of immunocompromised individuals. Nevertheless, the main host for E. cuniculi is the rabbit and infections usually have a sub-clinical course. However, severe disease is recognised in pet rabbits more frequently within the last years. As the central nervous system, the kidney and the eye are predilection organs for the organism, predominant histopathological alterations comprise granulomatous meningoencephalitis, chronical interstitial nephritis and phacoclastic uveitis. A definitive diagnosis of encephalitozoonosis in vivo is difficult, but it is important for specific treatment and the determination of possible zoonotic risks. This review article covers epidemiology, pathology, pathophysiology, immunology, clinical signs, differential diagnosis, diagnosis and treatment of encephalitozoonosis in rabbits.

Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits.

Infections with Encephalitozoon cuniculi in rabbits are observed at increasing frequency and are known as opportunistic infections in immunocompromised humans. 191 pet rabbits with suspected encephalitozoonosis, presented at the Animal Hospital of the Veterinary University of Vienna (Austria), were included in this study. Rabbits were serologically examined for antibodies against E. cuniculi (144 positive out of 184 rabbits with suspected encephalitozoonosis compared to 14 positive out of 40 clinically healthy rabbits tested as part of a standard health check) and Toxoplasma gondii (8 positive out of 157). Of the 144 seropositive rabbits with clinical signs, 75% showed neurological symptoms, 14.6% demonstrated phacoclastic uveitis and 3.5% suffered from renal failure. 6.9% of the animals had combined symptoms. Vestibular disease dominated within the rabbits that showed neurological symptoms. Polymerase chain reaction (PCR) could not detect parasite DNA in urine or cerebrospinal fluid (CSF), but did so in 4 out of 5 samples of liquefied lens material in cases with phacoclastic uveitis due to lens capsule rupture. Additionally further diagnostic procedures, such as inspection of the external ear canal (N=69), radiography of the tympanic bullae (N=65) were performed to rule out differential diagnosis. 54.2% of the patients exhibiting neurological symptoms recovered within a few days, while 87.5% of the rabbits suffering from renal failure died or had to be euthanized.

Diagnostic markers for encephalitozoonosis in pet rabbits.

Encephalitozoonosis is a common disease in pet rabbits, routinely diagnosed in vivo by serological examination or post mortem by histopathology. Recently molecular techniques have become increasingly important as diagnostic tools. The application of different diagnostic markers for in vivo and post mortem determination of E. cuniculi in naturally infected pet rabbits were compared. The examined population was divided into 33 rabbits with symptoms of encephalitozoonosis (group I) and 38 animals without symptoms (group II) which were all tested by serological analysis (Indirect Immunofluorescence Test), histological examination including special spore staining (Ziehl-Neelsen, acid-fast trichrome) and conventional and nested PCR (organs, body fluids). Additionally, in group III lens material (n=10) of animals (n=9) with phacoclastic uveitis were examined by conventional PCR. Infections with E. cuniculi could be determined post mortem in 78.8% of the rabbits of group I and in 57.9% of group II by histological examination combined with spore staining. In group I 69.7% and in group II 50.0% showed seroconversion. Conventional PCR was only sufficiently sensitive in samples of eyes with phacoclastic uveitis (n=10; 100%). Therefore nested PCR was performed in tissue samples, urine and cerebrospinal fluid (CSF) with positive results in 63.6% of group I and 42.1% of group II. Positive results in serology, pathohistology (spore detection and histological changes in the brain and/or kidneys) and nested PCR were obtained in 52.1% of the rabbits (group I and II, n=71), whereas 31.0% showed negative results in all three diagnostic techniques. 5.6% of the rabbits were positive in two methods and 11.3% in one method. In 37 rabbits (group I and II) with positive results in nested PCR, E. cuniculi DNA could be detected more frequently in the brain (91.9%) than in the kidney (54.1%). Furthermore nested PCR of urine revealed positive results in 29.7% of the rabbits (n=37) with seroconversion and/or confirmed E. cuniculi infection by spore detection. All 25 samples of CSF tested negative in nested PCR. Conventional PCR of eyes with phacoclastic uveitis was an excellent diagnostic marker in living rabbits, while nested PCR of urine or CSF was not reliable. Histological examination combined with special staining was the most sensitive method in post mortem diagnostics. Nested PCR appears to be a good post mortem method to investigate organs, especially brains, of chronically infected animals.

Encephalitozoonosis in pet rabbits (Oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation

Encephalitozoon cuniculi is a common infectious agent of rabbits. The aim of this study was to determine the distribution and extent of histological lesions in the brain and in the kidney of naturally infected pet rabbits with or without clinical encephalitozoonosis. In 71 animals (33 with symptoms) which died or were euthanised, histopathological examination including staining of spores (Ziehl–Neelsen, acid-fast trichrome) was performed and changes were described quantitatively. The cerebrum was the most frequently affected brain region (97.5%), whilst the cerebellum (55%) and the vestibular cores (37.5%) were less commonly concerned. Granulomas were found in 77.5% of animals with encephalitis and in 12.5% of rabbits with interstitial nephritis. Although cerebral granulomas were found irrespective of the grade of histological changes, they were significantly correlated with changes at higher grades. There was no correlation between the severity of encephalitis and neurological symptoms. Since severe lesions were also found in clinically inconspicuous animals, histological findings of inflammatory lesions are not indicative of overt encephalitozoonosis as the causative agent for neurological signs. Other diseases causing neurological symptoms, such as suppurative encephalitis, otitis media as well as malignant lymphoma were also detected in the rabbit population that was examined in the present study.

A Retrospective Study of Neurological Disease in 118 Rabbits

A retrospective pathological study of 118 rabbits presenting with neurological disease was conducted. Diagnoses were categorized on the basis of aetiopathogenesis as inflammatory, vascular, traumatic, metabolic-toxic, neoplastic, degenerative or idiopathic. Central nervous system (CNS) lesions were present in 85 (72.0%) of the rabbits and in most of these cases (70.3%) a causative agent was identified. The majority of animals (n=78, 66.1%) had disease of an inflammatory nature and 71 of these 78 rabbits had one of two zoonotic infectious diseases: encephalitozoonosis (n=69, 58.5%) and herpes simplex virus (HSV) encephalitis (n=2). Infections with zoonotic potential are therefore a major cause of CNS disease in the rabbit.

Identification and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian companion animals and horses

The aim of this study was to investigate the antimicrobial resistance, resistance gene patterns and genetic relatedness of a collection of Austrian methicillin-resistant Staphylococcus aureus (MRSA) isolates from companion animals and horses. A total of 89 non-repetitive MRSA isolates collected during routine veterinary microbiological examinations from April 2004 to the end of 2012, and one isolate from 2013 were used for this study. The presence of mecA and other resistance genes was confirmed by PCR. Isolates were genotyped by spa typing, two multiple-locus variable-number tandem repeat analyses (MLVA) analyses, SCCmec typing and multilocus sequence typing (MLST). PCR targeting Panton-Valentine leukocidin (PVL) and detection of staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST) was performed using PCR assays. Antimicrobial susceptibility testing was performed. Five sequence types (STs-ST398, ST254, ST22, ST5 and ST1), SCCmec types II, IVa, V, and non-type-abele, 8 spa-types (t003, t011, t036, t127, t386, t1348, and t4450), and two isolates could not be assigned, 21 MLVA-14Orsay types Multiplex-PCR MLVA (mMLVA) displayed 17 different MLVA types. The present study is the most comprehensive dealing with MRSA from Austrian companion animals and horses. The results confirm that MRSA ST398 is present in a wide range of animal species and is predominant especially in horses. In other companion animals it is unclear whether the infections with the different MRSA isolates investigated in the present study truly represents a rare phenomenon or may be an emerging problem in companion animals.

Thymomas in Rabbits: Clinical Evaluation, Diagnosis, and Treatment

Thymomas are rarely recorded in rabbits, and the literature includes comparatively few cases. Medical records were reviewed to identify all pet rabbits in which a mediastinal mass was diagnosed between Feb 2007 and Jan 2010. Signalment, history, clinical signs, diagnostic work-up (including laboratory data, diagnostic imaging, and ultrasound-guided fine-needle aspiration of the mediastinal mass), treatment modalities, survival time, and histologic findings were evaluated. Cytologic and/or histopathologic examinations revealed thymomas in all rabbits with mediastinal masses (n=13). Rabbits with thymomas showed clinical signs of dyspnea (76.9%), exercise intolerance (53.9%), and bilateral exophthalmos (46.2%). In seven rabbits the thymoma was removed surgically. Two rabbits were treated conservatively, and four rabbits were euthanized because of their poor clinical condition. The two rabbits that underwent surgery were euthanized 6 mo and 34 mo later. Mediastinal masses in rabbits appear to be more common than previously believed and consist primarily of thymomas rather than thymic lymphomas. Cytology of samples collected by ultrasound-guided fine-needle aspiration is an accurate diagnostic tool for the identification of thymomas in rabbits. Due to a high rate of perioperative mortality, intensive perioperative care and the provision of a low-stress environment are recommended for a successful thoracotomy.

Uterine Disorders in 50 Pet Rabbits

Although the incidence of uterine disorders in pet rabbits is high there are only a few retrospective studies and case reports on genital tract disease in female rabbits. Uterine disorders were assessed in 50 pet rabbits. In 31 pet rabbits with suspected clinical uterine disease, medical records were further reviewed regarding clinical signs, diagnostic workup, treatment as well as the outcome itself. Uterine adenocarcinoma (54%) was most frequently diagnosed, followed by endometrial hyperplasia (26%). Serosanguineous vaginal discharge was the predominant clinical sign observed by the rabbit owners. In approximately 50% of the rabbits with suspected uterine disorders, abdominal palpation revealed enlarged and/or irregular masses in the caudoventral abdomen indicating uterine lesions. Out of 23 rabbits undergoing ovariohysterectomy, four were either euthanized or died shortly after surgery because they were clinically unstable. Overall, 80% of the ovariohysterectomized animals were still alive 6 mo after surgery. In female pet rabbits that are not breeding, either ovariohysterectomy should be performed at an early age or routine checks including ultrasonography of the abdomen are recommended on a regular basis.

Comparative evaluation of specific methods for labeling of Encephalitozoon cuniculi in paraffin wax–embedded tissue samples

Detection of the microsporidian Encephalitozoon cuniculi in tissue samples is considered difficult. The aim of the current study was to determine whether immunohistochemistry (IHC) and in situ hybridization (ISH) represent reliable methods for the detection of E. cuniculi in postmortem tissue samples of rabbits. Paraffin-embedded tissue sections of brain and kidneys of 48 naturally infected pet rabbits, 10 negative controls, and the eyes of 3 further rabbits were used for all investigations. By IHC in 19 animals (37.3%), spores could be clearly detected and were all equally stained. By ISH using a digoxigenin-labeled oligonucleotide probe, only 6 animals (11.8%) proved undoubtedly positive. In these cases, many parasite-like objects revealed strong typical purple-black positive signals. However, several of the examined samples showed only partial staining of the pathogen or unclear results. Thus, in order to find an explanation for these inconsistent ISH results and to take a more detailed look at the different developmental stages of the organism, electron microscopy was applied. Empty spores, which had already discharged their polar filaments, prevailed in total number. Taken together, both techniques are rather insensitive, but under the condition that sufficient numbers of microsporidia are present, IHC can be recommended for specific identification of E. cuniculi in tissue samples. In contrast, ISH failed to detect some developmental stages of the organism, and, as such, ISH is therefore considered an inappropriate diagnostic method.

Sequence type 398 meticillin-resistant Staphylococcus aureus infection in a pet rabbit

BACKGROUND Meticillin-resistant Staphylococcus aureus (MRSA) is a frequent pathogen of humans and many animal species, and has become established as a veterinary pathogen. CASE REPORT In this case report we describe an MRSA infection in a dwarf rabbit, treatment of the infection and, subsequently, the genetic analysis of the isolated strain. The pet rabbit was presented to an animal hospital due to recurrent swellings on the head and on the neck. Bacteriological examination yielded Staphylococcus aureus, which was resistant to β-lactam antibiotics. The isolate was confirmed as an MRSA by mecA PCR, Panton-Valentine leukocidin (PVL) negative, and typed as multilocus sequence type (ST)398/staphylococcal protein A (spa) type t011/staphylococcal cassette chromosome mec (SCCmec) type SCCmecIVa. The rabbit was treated with rifampicin for 2 weeks. After 2.5 weeks the rabbit was clinically normal and control swabs were negative for MRSA. CONCLUSIONS AND CLINICAL IMPORTANCE This study is the first to report MRSA in a rabbit in Austria. This study contributes to the growing evidence that MRSA ST398 could be isolated from a variety of animals.