Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood.

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

Brain magnetic resonance imaging and computed tomography findings in cats and dogs with central nervous system cryptococcosis in Australia: 23 cases (2009-2020).

OBJECTIVE: To describe the imaging findings in Australian cats and dogs with CNS cryptococcosis. ANIMALS: 23 cases (10 cats; 13 dogs) with CNS cryptococcosis and brain MRI or CT studies available to review. METHODS: Retrospective, multi-institutional case series. Brain MRI or CT studies were reviewed by a board-certified radiologist. Imaging findings were described and the differences between cats and dogs explored. RESULTS: Morphologic features were consistent with extra-axial lesions in all (n = 13) dogs and either intra-axial (5/10) or extra-axial (4/10) lesions in cats, with 1 cat having no detectable lesions in low-field brain MRI scans. Meningeal abnormalities were most common, followed by forebrain and cerebellar lesions. Intracranial MRI lesions were typically T2 hyperintense and T1 hypo- to isointense. Four cases had T2 hypointense lesions affecting the brain, sinonasal cavity, or regional lymph nodes. Intracranial CT lesions were mostly soft tissue attenuating. Contrast enhancement was present in all cases with contrast series available, with ring enhancement shown only in cats. Osteolysis was more common in dogs than cats, particularly affecting the cribriform plate. All 13 dogs and many (6/10) cats had at least 1 lesion affecting sinonasal or contiguous tissues, and locoregional lymphadenomegaly was common (7/10 cats; 11/13 dogs). CLINICAL RELEVANCE: Imaging lesions in cryptococcal meningoencephalitis were extra-axial in dogs but could be intra-axial or extra-axial in cats. Careful examination for extracranial lesions (sinonasal, retrobulbar, facial soft tissue, tympanic bullae, or locoregional lymph nodes) is important to provide alternative safe biopsy sites. T2 hypointense lesions, while rare, should prompt consideration of cryptococcosis.

Retrospective study and outcome of 307 cats with feline infectious peritonitis treated with legally sourced veterinary compounded preparations of remdesivir and GS-441524 (2020-2022).

OBJECTIVES: Feline infectious peritonitis (FIP) is a serious disease that arises due to feline coronavirus infection. The nucleoside analogues remdesivir and GS-441524 can be effective in its treatment, but most studies have used unregulated products of unknown composition. The aim of the present study was to describe the treatment of FIP using legally sourced veterinary-prescribed regulated veterinary compounded products containing known amounts of remdesivir (injectable) or GS-441524 (oral tablets). METHODS: Cats were recruited via email advice services, product sales contacts and study publicity. Cats were excluded if they were deemed unlikely to have FIP, were not treated exclusively with the veterinary compounded products, or if there was a lack of cat and/or treatment (including response) data. Extensive cat and treatment data were collected. RESULTS: Among the 307 cats recruited, the predominant type of FIP was most commonly abdominal effusive (49.5%) and then neurological (14.3%). Three treatment protocols were used; remdesivir alone (33.9%), remdesivir followed by GS-441524 (55.7%) and GS-441524 alone (10.4%). The median (range) initial treatment period duration and longest follow-up time point after starting treatment were 84 (1-330) days and 248 (1-814) days, respectively. The most common side effect was injection pain (in 47.8% of those given subcutaneous remdesivir). Of the 307 cats, 33 (10.8%) relapsed, 15 (45.5%) during and 18 (54.5%) after the initial treatment period. At the longest follow-up time point after completion of the initial treatment period, 84.4% of cats were alive. The cats achieving a complete response within 30 days of starting treatment were significantly more likely to be alive at the end of the initial treatment period than those cats that did not. CONCLUSIONS AND RELEVANCE: Legally sourced remdesivir and GS-441524 products, either alone or used sequentially, were very effective in the treatment of FIP in this group of cats. Variable protocols precluded statistical comparison of treatment regimens.

Feline precision medicine using whole-exome sequencing identifies a novel frameshift mutation for vitamin D-dependent rickets type 2.

OBJECTIVES: A 14-week-old female domestic longhair kitten presented with shifting lameness and disproportionately smaller size compared with a co-housed littermate. METHODS: Hematology and serum biochemical testing were conducted to investigate causes for delayed growth, and radiographs of the appendicular skeleton were obtained. RESULTS: The afflicted kitten had marked hypocalcemia, mild hypophosphatemia and substantial elevations in alkaline phosphatase activity, as well as pathognomonic radiographic findings consistent with rickets. Skeletal changes and hypocalcemia prompted testing of concentrations of parathyroid hormone (PTH) and vitamin D metabolites. Endocrine testing demonstrated significant increases in serum concentrations of PTH and 1,25-dihydroxycholecalciferol (calcitriol), supporting a diagnosis of vitamin D-dependent rickets type 2. Provision of analgesia, supraphysiologic doses of calcitriol and calcium carbonate supplementation achieved normalization of the serum calcium concentration and restoration of normal growth, although some skeletal abnormalities persisted. Once skeletally mature, ongoing calcitriol supplementation was not required. Whole-exome sequencing (WES) was conducted to identify the underlying DNA variant. A cytosine deletion at cat chromosome position B4:76777621 in VDR (ENSFCAT00000029466:c.106delC) was identified and predicted to cause a stop codon in exon 2 (p.Arg36Glufs*18), disrupting >90% of the receptor. The variant was unique and homozygous in this patient and absent in the sibling and approximately 400 other cats for which whole-genome and whole-exome data were available. CONCLUSIONS AND RELEVANCE: A unique, heritable form of rickets was diagnosed in a domestic longhair cat. WES identified a novel frameshift mutation affecting the gene coding for the vitamin D3 receptor, determining the likely causal genetic variant. Precision medicine techniques, including whole-exome and whole-genome sequencing, can be a standard of care in cats to identify disease etiologies, and to target therapeutics and personalize treatment.

Clinical investigation and management of Brucella suis seropositive dogs: A longitudinal case series.

BACKGROUND: Brucellosis in dogs caused by Brucella suis is an emerging zoonotic disease. OBJECTIVES: To document clinical characteristics, serology, microbiology, and clinical response to treatment in B. suis-seropositive dogs. ANIMALS: Longitudinal study of 27 privately-owned dogs. Dogs that tested positive by serology, culture, or real-time polymerase chain reaction (qPCR) were included in the study. METHODS: Clinical (physical examination and imaging) and laboratory (serology, hematology, serum biochemistry, and qPCR or culture) assessments were made at baseline and after approximately 3, 6, 12, and 18 months. RESULTS: Dogs were followed for 10 895 dog days, with 17/27 dogs completing the 18-month follow-up. Ten dogs had signs consistent with brucellosis before enrollment (n = 4), at baseline (n = 2) or during follow-up (n = 6), with 2 dogs experiencing relapse of historical signs. Antibody titers persisted for the duration of follow-up in 15/17 dogs (88%). Radiographic (n = 5) and ultrasound (n = 11) findings, of variable clinical relevance, were observed. Brucella DNA and organisms were detected in 3 dogs, all of which had clinical signs, including in the milk of a bitch around the time of whelping. Brucella DNA was not detected in blood (n = 92 samples), urine (n = 80), saliva (n = 95) or preputial swabs (n = 78) at any time during follow-up. Six dogs underwent treatment, all of which achieved clinical remission although remission was not reflected by decreasing antibody titers. CONCLUSIONS AND CLINICAL IMPORTANCE: Most dogs with B. suis infections have subclinical infections. Serology is poorly associated with clinical disease. Excretion of organisms appears rare except in whelping bitches. Clinical management using antibiotics with or without surgery is recommended.

Field Performance of a Rapid Test to Detect Progressive, Regressive, and Abortive Feline Leukemia Virus Infections in Domestic Cats in Australia and Germany.

Different feline leukemia virus (FeLV) infection outcomes are possible in cats following natural exposure, such as progressive infections (persistent viremia), regressive infections (transient or no viremia followed by proviral persistence) and abortive infections (presence of only antibodies). Laboratory-based testing is currently required for categorization of infection outcomes in cats. The aim of this study was to evaluate the field performance of a novel, rapid, combination point-of-care (PoC) test kit commercially available in Europe (v-RetroFel®Ag/Ab; 2020-2021 version) to determine different FeLV infection outcomes by concurrent detection of FeLV antigen (p27) and antibodies against FeLV transmembrane envelope protein (p15E). A secondary aim was to evaluate the performance of the same test kit (v-RetroFel®FIV) to determine positive/negative feline immunodeficiency virus (FIV) infection status by the detection of antibodies to FIV capsid protein (p24) and transmembrane glycoprotein (gp40). Two cohorts of domestic cats were recruited and tested with v-RetroFel® using plasma or serum, including cats in Australia (n = 200) and cats in Germany (n = 170). Results from p27 antigen PoC testing, proviral DNA PCR, and neutralizing antibody testing or testing for antibodies against non-glycosylated surface unit envelope protein (p45) were used to assign cats to groups according to different FeLV infection outcomes. Testing with a laboratory-based FeLV p15E antibody ELISA was also performed for comparison. In the first cohort, v-RetroFel®Ag/Ab correctly identified 89% (109/122) FeLV-unexposed cats and 91% (21/23) progressive infections, but no regressive (0/23) or abortive (0/32) infections. In the second cohort, v-RetroFel®Ag/Ab correctly identified 94% (148/158) FeLV-unexposed cats and 100% (4/4) progressive infections, but no regressive (0/2) and only 17% (1/6) abortive infections. There was test agreement between v-RetroFel®Ab and the p15E laboratory ELISA in 58.9% of samples. As a secondary outcome of this study, the sensitivity and specificity of v-RetroFel®FIV testing in cohort 1 were 94.7% (18/19) and 98.3% (178/181), and in cohort 2, 30.0% (3/10) and 100.0% (160/160), respectively. Prior history of FIV vaccination did not produce any false-positive FIV results. In conclusion, v-RetroFel®Ag/Ab (2020-2021 version) was unable to accurately determine different FeLV infection outcomes in the field. Improvements of the test prior to application to field samples are required.

Renal Crest Proliferative Lesions in Cats with Chronic Kidney Disease.

In a histopathological study of the renal crest (RC) of kidneys of cats with chronic kidney disease (CKD), 58/90 (64%) had epithelial proliferation. Of these, 33 cats had hyperplasia of the collecting duct (CD) epithelium (CDH) alone, eight had hyperplasia of the urothelium covering the RC (RCUH), of which one had concurrent abaxial renal pelvic urothelial hyperplasia (UH), and eight had both CDH and RCUH. CDH or RCUH were present in five cats with marked dysplasia of the CD epithelium (CDD) and four cats with invasive carcinomas, which also had epithelial dysplasia. All nine cats with marked dysplasia or neoplasia of the RC also had substantially altered RC contours due to focal haemorrhage, papillary necrosis or fibrosis. Three of the carcinomas had a strong desmoplastic response. In control cats, both urothelial (RC and renal pelvis) and tubular (CD and distal tubular) cells were immunopositive for cytokeratin (CK; AE1/AE3), tubular epithelial cells were positive for vimentin (Vim) and aquaporin 2 (Aq2), while urothelial cells were positive for p63. PAX8 immunolabelling was difficult to validate. CD and UH labelling was similar to control tissue. While urothelial dysplasia had the same immunolabelling pattern as UH and control tissue, CDD was generally immunonegative for Aq2. As immunolabelling of the four carcinomas did not distinguish between tubular and urothelial origin, with three positive for both Vim and p63, all were broadly designated as RC carcinomas. Overall, proliferative epithelial lesions are common in cats with CKD and form a continuum from simple hyperplasia to neoplasia of the urothelium or CD of the RC.

Radiographical Survey of Osteochondrodysplasia in Scottish Fold Cats caused by the TRPV4 gene variant.

The unique appearance of Scottish Fold cats is caused by a single gene variant in TRPV4, which impacts the development of cartilage. This results in the ears folding forward and variable effects on articular cartilage and bone. While some find this appearance desirable, early work demonstrated that homozygous cats with two copies of this variant develop severe radiographic consequences. Subsequent breeding programs have mated heterozygous cats with straight-eared cats to ensure an equal mix of heterozygous (fold) and wild-type (nonfolded) offspring, in the hope of raising healthy cats. More recent radiological surveys suggest that these heterozygous cats may also have medical problems consisting of deformed distal extremities in the worst cases and accelerated onset of osteoarthritis. However, these previous studies were undermined by selection biases, lack of controls, unblinded assessment and lack of known genotypes. Our aim was to determine if heterozygous cats exhibit radiological abnormalities when controlling for these limitations. Specifically, DNA and radiographs were acquired for 22 Scottish Fold cats. Four reviewers, blinded to the ear phenotype, assessed the lateral radiographs. Genotyping showed that all 10 folded-ear cats were heterozygous, and none of the straight-ear cats (n = 12) had the abnormal TRPV4 variant. Although each reviewer, on average, gave a numerically worse 'severity score' to folded-ear cats relative to straight-ear cats, the images in heterozygous cats showed much milder radiological signs than previously published. This study provides additional information to be considered in the complicated debate as to whether cats with the TRPV4 variant should be bred for folded ears given the potential comorbidities.

Anti-SU Antibody Responses in Client-Owned Cats Following Vaccination against Feline Leukaemia Virus with Two Inactivated Whole-Virus Vaccines (Fel-O-Vax® Lv-K and Fel-O-Vax® 5).

A field study undertaken in Australia compared the antibody responses induced in client-owned cats that had been vaccinated using two inactivated whole feline leukaemia virus (FeLV) vaccines, the monovalent vaccine Fel-O-Vax® Lv-K and the polyvalent vaccine Fel-O-Vax® 5. Serum samples from 428 FeLV-uninfected cats (118 FeLV-vaccinated and 310 FeLV-unvaccinated) were tested for anti-FeLV neutralising antibodies (NAb) using a live virus neutralisation assay to identify 378 FeLV-unexposed (NAb-negative) and 50 FeLV-exposed (NAb-positive; abortive infections) cats, following by anti-surface unit (SU) FeLV-A and FeLV-B antibody ELISA testing. An additional 42 FeLV-infected cats (28 presumptively regressively infected, 14 presumptively progressively infected) were also tested for anti-SU antibodies. NAb-positive cats displayed significantly higher anti-SU antibody ELISA responses compared to NAb-negative cats (p < 0.001). FeLV-unexposed cats (NAb-negative) that had been vaccinated less than 18 months after a previous FeLV vaccination using the monovalent vaccine (Fel-O-Vax® Lv-K) displayed higher anti-SU antibody ELISA responses than a comparable group vaccinated with the polyvalent vaccine (Fel-O-Vax® 5) (p < 0.001 for both anti-FeLV-A and FeLV-B SU antibody responses). This difference in anti-SU antibody responses between cats vaccinated with the monovalent or polyvalent vaccine, however, was not observed in cats that had been naturally exposed to FeLV (NAb-positive) (p = 0.33). It was postulated that vaccination with Fel-O-Vax® 5 primed the humoral response prior to FeLV exposure, such that antibody production increased when the animal was challenged, while vaccination with Fel-O-Vax® Lv-K induced an immediate preparatory antibody response that did not quantitatively increase after FeLV exposure. These results raise questions about the comparable vaccine efficacy of the different FeLV vaccine formulations and correlates of protection.

Gross, microscopic, radiologic, echocardiographic and haematological findings in rats experimentally infected with Angiostrongylus cantonensis.

Although the gross and microscopic pathology in rats infected with Angiostrongylus cantonensis has been well described, corresponding changes detected using diagnostic imaging modalities have not been reported. This work describes the cardiopulmonary changes in mature Wistar rats chronically infected with moderate burdens of A. cantonensis using radiology, computed tomography (CT), CT angiography, echocardiography, necropsy and histological examinations. Haematology and coagulation studies were also performed. Thoracic radiography, CT and CT angiography showed moderately severe alveolar pulmonary patterns mainly affecting caudal portions of the caudal lung lobes and associated dilatation of the caudal lobar pulmonary arteries. Presumptive worm profiles could be detected using echocardiography, with worms seen in the right ventricular outflow tract or straddling either the pulmonary and/or the tricuspid valves. Extensive, multifocal, coalescing dark areas and multiple pale foci affecting the caudal lung lobes were observed at necropsy. Histologically, these were composed of numerous large, confluent granulomas and fibrotic nodules. Adult worms were found predominantly in the mid- to distal pulmonary arteries. An inflammatory leukogram, hyperproteinaemia and hyperfibrinogenaemia were found in most rats. These findings provide a comparative model for A. cantonensis in its accidental hosts, such as humans and dogs. In addition, the pathological and imaging changes are comparable to those seen in dogs infected with Angiostrongylus vasorum, suggesting rats infected with A. cantonensis could be a model for dogs with A. vasorum infection.

Identification of the environmental source of infection for a domestic ferret with cryptococcosis.

Cryptococcosis, caused by the Cryptococcus gattii and C. neoformans species complexes, is an environmentally acquired mycosis affecting a broad range of host species. Among 9 communally housed ferrets, a 5-y-old castrated male ferret domiciled in an outdoor enclosure in Sydney, Australia was diagnosed with sinonasal cryptococcosis. Clinical signs resolved during 18 mo of itraconazole therapy, but the ferret was eventually euthanized because of splenic hemangiosarcoma. At postmortem, microscopic foci of persistent cryptococcosis were detected. The diagnosis raised concerns that the owners and other ferrets were exposed to a common environmental source of infection, thus prompting an investigation. Soil samples, swabs of a hollow eucalypt log (used for behavioral enrichment), and nasal swabs from 8 asymptomatic ferrets were collected. Nasal exudate (obtained at diagnosis) and tissues (collected at postmortem) were available from the clinical case. Bird seed agar culture resulted in a heavy growth of Cryptococcus spp. from one environmental site (the log), one nasal swab, and nasal exudate and tissues from the clinical case. All other samples were culture-negative. Sub-cultured isolates from the log were a mixture of C. gattii molecular type VGI and C. neoformans molecular type VNI. Ferret isolates were a similar mixture of C. gattii VGI (all disease isolates) and C. neoformans VNI (nasal-colonizing isolate). Multilocus sequence typing further revealed the ferret isolates as identical to environmental isolates collected from the log, confirming the log as the source of clinical disease and nasal colonization. The log was removed to prevent further exposure to a high environmental load of Cryptococcus spp.

The Diagnosis of Feline Leukaemia Virus (FeLV) Infection in Owned and Group-Housed Rescue Cats in Australia.

A field study was undertaken to (i) measure the prevalence of feline leukaemia virus (FeLV) exposure and FeLV infection in a cross-section of healthy Australian pet cats; and (ii) investigate the outcomes following natural FeLV exposure in two Australian rescue facilities. Group 1 (n = 440) consisted of healthy client-owned cats with outdoor access, predominantly from eastern Australia. Groups 2 (n = 38) and 3 (n = 51) consisted of a mixture of healthy and sick cats, group-housed in two separate rescue facilities in Sydney, Australia, tested following identification of index cases of FeLV infection in cats sourced from these facilities. Diagnostic testing for FeLV exposure/infection included p27 antigen testing using three different point-of-care FeLV kits and a laboratory-based ELISA, real-time polymerase chain reaction (qPCR) testing to detect FeLV proviral DNA in leukocytes, real-time reverse-transcription PCR (qRT-PCR) testing to detect FeLV RNA in plasma, and neutralising antibody (NAb) testing. Cats were classified as FeLV-uninfected (FeLV-unexposed and presumptively FeLV-abortive infections) or FeLV-infected (presumptively regressive and presumptively progressive infections). In Group 1, 370 FeLV-unexposed cats (370/440, 84%), 47 abortive infections (47/440, 11%), nine regressive infections (9/440, 2%), and two progressive infections (2/440, 0.5%) were identified, and 12 FeLV-uninfected cats (12/440, 3%) were unclassifiable as FeLV-unexposed or abortive infections due to insufficient samples available for NAb testing. In Groups 2 and 3, 31 FeLV-unexposed cats (31/89, 35%), eight abortive infections (8/89, 9%), 22 regressive infections (22/89; 25%), and 19 progressive infections (19/89; 21%) were discovered, and nine FeLV-uninfected cats (9/89; 10%) were unclassifiable due to insufficient samples available for NAb testing. One of the presumptively progressively-infected cats in Group 3 was likely a focal FeLV infection. Two other presumptively progressively-infected cats in Group 3 may have been classified as regressive infections with repeated testing, highlighting the difficulties associated with FeLV diagnosis when sampling cats at a single time point, even with results from a panel of FeLV tests. These results serve as a reminder to Australian veterinarians that the threat of FeLV to the general pet cat population remains high, thus vigilant FeLV testing, separate housing for FeLV-infected cats, and FeLV vaccination of at-risk cats is important, particularly in group-housed cats in shelters and rescue facilities, where outbreaks of FeLV infection can occur.

Molecular detection of Pneumocystis in the lungs of cats.

The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats' lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.

TREATMENT OF MYCOTIC RHINOSINUSITIS IN A BENNETT'S WALLABY ( MACROPUS RUFOGRISEUS) USING TOPICAL VORICONAZOLE SUSPENDED IN A REVERSE THERMODYNAMIC PLURONIC GEL.

An approximately 4-yr-old female Bennett's wallaby ( Macropus rufogriseus) was evaluated for chronic left-sided facial swelling and nasal discharge. Computed tomography, endoscopy, biopsy, mycologic culture, and panfungal polymerase chain reaction were consistent with destructive mycotic rhinosinusitis. The patient's infection was treated with a long-term injectable antibiotic, oral antifungal therapy, and multiple intranasal infusions of voriconazole suspended in a reverse thermodynamic pluronic gel. This case represents the first documented case of mycotic rhinosinusitis in a macropod and underlines the importance of advanced cross-sectional imaging in the diagnosis, monitoring, and management of nasal cavity disease in zoo animals.

Progressive cutaneous viral pigmented plaques in three Hungarian Vizslas and the response of lesions to topical tigilanol tiglate gel.

Cutaneous pigmented viral plaques is a disorder of epidermal growth caused by canine papillomavirus type 4 (CPV-4). There is currently no standard of care for managing this condition and it has not been reported in the Hungarian Vizsla. This case series documents the clinical features of canine pigmented viral plaques in Hungarian Vizsla dogs and the treatment of a severe case using a novel topical agent tigilanol tiglate (EBC-46). A 4-year-old spayed Hungarian Vizsla in Australia was presented for multiple cutaneous pigmented plaques extending from the ventral cervical region. Lesions were neither painful nor pruritic. The number and size of these sessile plaques increased over time, with the largest lesions eventually taking on an exophytic (wart-like) appearance. These lesions did not affect the dog's wellbeing. Two much less severe cases in a 5-year-old Vizsla from the UK and a 7-year-old Vizsla from New Zealand were also diagnosed. Histology was consistent with papillomavirus-induced pigmented plaques and CPV-4 DNA sequences were amplified from paraffin-embedded formalin-fixed tissue using the polymerase chain reaction from the most severely affected patient. Topical imiquimod was ineffective although used for only a short time. Two topical applications of novel anti-neoplastic diterpene ester tigilanol tiglate as a gel, 9 days apart, greatly reduced the size and number of lesions in a limited portion of skin treated, over the lateral hock. While CPV-4 has been previously reported to cause pigmented plaques, most commonly on pug dogs, but sporadically on other breeds, this is the first report of this virus causing plaques in Hungarian Vizslas. The cases illustrate some of the difficulties in diagnosing papillomavirus-induced disease in dogs, especially in its early stages. Topical tigilanol tiglate is a potentially useful topical therapy for this viral-induced disorder of cell growth and represents a treatment deserving of further investigation.

Presumptive vertical transmission of Neospora caninum in related Bernese Mountain dogs.

Neospora caninum is a tissue cyst-forming coccidium capable of causing spinal cord or skeletal muscle disease in dogs. Infected bitches can transmit the parasite to their pups in utero. Seroprevalence of N. caninum was studied in naturally-infected, privately owned Bernese Mountain dogs, using antibody detection via an indirect fluorescent antibody test (IFAT) to identify infected individuals. A retrospective study was undertaken on available dogs from 14 litters. Five of eight dams tested seropositive. The index case was a bitch with a titre of 1:3200 by IFAT. Only one offspring from her first litter was seropositive. The frequency of putative congenital transmission in the breeding kennel was variable. Our results reiterate the importance of serological testing of all dams and littermates in a breeding kennel when clinical neosporosis is suspected in neonatal puppies.

Feline leprosy due to Candidatus 'Mycobacterium lepraefelis': Further clinical and molecular characterisation of eight previously reported cases and an additional 30 cases.

OBJECTIVES: This paper, the last in a series of three on 'feline leprosy', provides a detailed description of disease referable to the previously unnamed species, Candidatus 'Mycobacterium lepraefelis', a close relative of the human pathogens Mycobacterium leprae and Mycobacterium lepromatosis. METHODS: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M lepraefelis' infection. RESULTS: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory (VIDRL) records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Thirty-eight cats were definitively diagnosed with Candidatus 'M lepraefelis' infection. Typically, cats tended to be middle-aged or older when first infected, with a male predilection. Affected cats typically had widespread cutaneous lesions, in some cases after initially localised disease. Advanced cases were often systemically unwell. All cats had outdoor access. The histological picture was lepromatous in the majority of patients, although two cases had tuberculoid disease. In one case that underwent necropsy, lesions were evident in the liver, spleen and lungs. Treatment was varied, although most cats received a combination of oral clarithromycin and rifampicin. Prognosis for recovery was variable, but typically poor. CONCLUSIONS AND RELEVANCE: Candidatus 'M lepraefelis' typically causes high bacterial index (lepromatous) feline leprosy that in some cases progresses to systemic mycobacteriosis. The disease has a variable clinical course and prognosis. Many cases either died or were euthanased due to the infection. Multilocus sequence analysis reveals a heterogeneous picture and further analysis of draft genome sequencing may give clues to the taxonomy and epidemiology of this organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations. Comparative aspects: This paper finishes with a discussion of comparative aspects of infection caused by the three feline leproid disease agents that have been the subject of this series: Candidatus 'Mycobacterium tarwinense', Mycobacterium lepraemurium and Candidatus 'M lepraefelis'.

Feline leprosy due to Mycobacterium lepraemurium.

OBJECTIVES: This paper, the second in a series of three on 'feline leprosy', provides a detailed description of disease referable to Mycobacterium lepraemurium, the most common cause of feline leprosy worldwide. METHODS: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with M lepraemurium infection. RESULTS: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Sixty-five cats were definitively diagnosed with M lepraemurium infection. Typically, cats were 1-3 years of age when first infected, with a male gender predilection. Affected cats were generally systemically well. All had outdoor access. Lesions tended to consist of one or more cutaneous/subcutaneous nodules, typically located on the head and/or forelimbs, possibly reflecting the most likely locations for a rodent bite as the site of inoculation for organisms. Nodules had the propensity to ulcerate at some stage in the clinical course. The cytological and histological picture varied from tuberculoid, with relatively low bacterial numbers, to lepromatous with moderate to high bacterial numbers. Treatment was varied, although most cats underwent surgical resection of lesions with adjunctive medical therapy, most often using a combination of oral clarithromycin and rifampicin. Prognosis for recovery was generally good, and in two cases there was spontaneous remission without the requirement for medical intervention. Untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but had no apparent tendency to disseminate to internal organs. CONCLUSIONS AND RELEVANCE: M lepraemurium causes high bacterial index (lepromatous) or low bacterial index (tuberculoid) feline leprosy. The infection typically causes nodules of the skin and/or subcutis (which tend towards ulceration) on the head and/or forelimbs. The disease usually has an indolent clinical course and infected cats have a generally favourable response to therapeutic interventions, with rare cases undergoing spontaneous resolution. Genomic analysis may yield clues as to the environmental niche and culture requirements of this elusive organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations.

Successful treatment of multifocal pedal Prototheca wickerhamii infection in a feline immunodeficiency virus-positive cat with multiple Bowenoid in situ carcinomas containing papillomaviral DNA sequences.

CASE SUMMARY: A 16-year-old, castrated male, feline immunodeficiency virus (FIV)-positive, domestic shorthair cat developed multiple skin lesions. Most of these were Bowenoid carcinoma in situ and contained DNA sequences consistent with Felis catus papillomavirus type 2. Two additional lesions that developed in the skin and subcutaneous tissues between the digital and carpal pads on the left forelimb and right hindlimb were shown by cytology, histology and culture to be caused by Prototheca wickerhamii. These lesions failed to improve in response to systemic therapy treatment with itraconazole, but excision by sharp en bloc resection with follow-up oral itraconazole therapy proved curative for one lesion, although the other lesion recurred, necessitating a second surgery. RELEVANCE AND NOVEL INFORMATION: This is only the second reported case of feline protothecosis from Australia and the first case that has been cultured and identified to the species level. Also of great interest was the presence of multiple papillomavirus-associated neoplastic lesions, which may have afforded a portal of entry for the algal pathogen and the cat's positive FIV status; the latter might have impacted on both viral and algal pathogenesis by effects on immunocompetence.

Metastatic pulmonary carcinomas in cats ('feline lung-digit syndrome'): further variations on a theme.

CASE SERIES SUMMARY: The aim of this retrospective case series was to describe seven cases where cats with primary lung tumours were presented because of metastases to extra-pulmonary sites ('feline lung-digit syndrome'). Specifically, we wanted to emphasise less typical case descriptions where tumour emboli resulted in lesions developing in the musculature (including the muscles of the head), skin or distal aorta. RELEVANCE AND NOVEL INFORMATION: The cases are presented to increase clinical awareness of this entity in feline practice, especially when characteristic combinations of clinical signs are present. When clinicians have a high index of suspicion for these presentations, radiology (thorax and digits) or ultrasound (distal aorta), combined with collection of fine needle aspirate specimens for cytology, are usually sufficient to secure a definitive diagnosis. Novel information in this series includes CT and MRI findings from some cases. Typically, needle aspiration or biopsy targeting the skin, digits and lesions in musculature is far easier compared with sampling the primary tumour site in the lungs. The differential diagnosis and investigation of multiple digital lesions is also considered.