Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010-2020) and results for a novel, highly sensitive qPCR assay.

The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells µL-1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, CT values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.

Comparing immunochromatography with latex antigen agglutination testing for the diagnosis of cryptococcosis in cats, dogs and koalas.

Although the point-of-care cryptococcal antigen lateral flow assay (LFA) has revolutionized the diagnosis of cryptococcosis in human patients, to date there has been no large-scale examination of this test in animals. We therefore assessed the LFA in cats, dogs and koalas suspected of having cryptococcosis. In sum, 528 serum specimens (129 from cats, 108 from dogs, 291 from koalas) were tested using the LFA and one of two commercially available latex cryptococcal antigen agglutination test (LCAT) kits. The LCAT is a proven and well-accepted method in veterinary patients and therefore taken as the "gold standard" against which the LFA was compared. The LFA achieved a sensitivity of 92%, 100%, and 98% in cats, dogs, and koalas, respectively, with corresponding negative predictive values of 94%, 100%, and 98%. The specificity of the LFA was 81%, 84%, and 62% for cats, dogs, and koalas, respectively, with corresponding positive predictive values of 76%, 48%, and 69%. These findings suggest the most appropriate role for the LFA is as a screening test to rule out a diagnosis of cryptococcosis in cats, dogs, and koalas. Point-of-care accessibility makes it equally suited for use in the field and as a cage-side test in veterinary hospitals. The suboptimal specificity of the LFA makes it less suited to definitive confirmation of cryptococcosis in animals; therefore, all LFA-positive test results should be confirmed by LCAT testing. The discrepancy between these observations and the high specificity of the LFA in humans may reflect differences in the host-pathogen interactions amongst the species.

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.

Feline leprosy due to Candidatus 'Mycobacterium tarwinense':Further clinical and molecular characterisation of 15 previously reported cases and an additional 27 cases

OBJECTIVES: This paper, the first in a series of three on 'feline leprosy', provides a detailed description of disease referable to Candidatus 'Mycobacterium tarwinense', the most common cause of feline leprosy in Victoria, Australia. 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 tarwinense' 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. Forty-two cats were definitively diagnosed with Candidatus 'M tarwinense' infection. Typically, cats were between 3 and 11 years of age, with no gender predilection, and were generally systemically well. All had outdoor access. Most cats underwent surgical resection of lesions with adjunctive medical therapy, often utilising a combination of oral clarithromycin and rifampicin for at least 3 months. Prognosis for recovery was generally good. Resolution of lesions was not observed in the absence of treatment, but a number of untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but did not appear to disseminate to internal organs. Preliminary results of draft genome sequencing confirmed that the species is a member of the Mycobacterium simiae complex. CONCLUSIONS AND RELEVANCE: Candidatus 'M tarwinense', a fastidious member of the M simiae complex, is capable of causing feline leprosy with a tendency to produce lesions on the head, particularly involving the eyes and periocular skin. The disease has an indolent clinical course and generally responds favourably to therapy despite lesions often containing large numbers of organisms. Detailed genomic analysis may yield clues as to the environmental niche and culture requirement of this elusive organism. Prospective treatment trials and/or drug susceptibility testing in specialised systems would further inform treatment recommendations.

Practical urinalysis in the cat: 2: Urine microscopic examination 'tips and traps'.

SERIES OUTLINE: This is the second article in a two-part series on urinalysis in the cat. The specific focus is urine microscopic examination. Part 1, which appeared in the March 2016 issue, discussed urine macroscopic examination. PRACTICAL RELEVANCE: Urinalysis is an essential procedure in feline medicine but often little attention is paid to optimising the data yielded or minimising factors that can affect the results. CLINICAL CHALLENGES: For the best results, appropriately collected urine should be prepared promptly by specialist laboratory personnel for the relevant tests and assessed by a clinical pathologist. This is invariably impractical in clinical settings but careful attention can minimise artefacts and allow maximum useful information to be obtained from this seemingly simple process. AUDIENCE: Clinical pathologists would be familiar with the information provided in this article, but it is rarely available to general or specialist practitioners, and both groups can potentially benefit. EQUIPMENT: Most of the required equipment is routinely available to veterinarians. However, instructions have been provided to give practical alternatives for specialist procedures in some instances. EVIDENCE BASE: The evidence base for feline microscopic urinalysis is quite poor and information has largely been extrapolated from the human literature. Information from feline studies has been included where available. In addition, practical clinicopathological and clinical observations are provided.

Practical urinalysis in the cat: 1: Urine macroscopic examination 'tips and traps'.

SERIES OUTLINE: This is the first article in a two-part series on urinalysis in the cat. The focus of Part 1 is urine macroscopic examination. Part 2, to appear in the May 2016 issue, discusses urine microscopic examination. PRACTICAL RELEVANCE: Urinalysis is an essential procedure in feline medicine but often little attention is paid to optimising the data yielded or minimising factors that can affect the results. CLINICAL CHALLENGES: For the best results, appropriately collected urine should be prepared promptly by specialist laboratory personnel for the relevant tests and assessed by a clinical pathologist. This is invariably impractical in clinical settings but careful attention can minimise artefacts and allow maximum useful information to be obtained from this seemingly simple process. AUDIENCE: Clinical pathologists would be familiar with the information provided in this article, but it is rarely available to general or specialist practitioners, and both can potentially benefit. EQUIPMENT: Most of the required equipment is routinely available to veterinarians. However, instructions have been provided to give practical alternatives for specialist procedures in some instances. EVIDENCE BASE: Evidence for much of the data on urinalysis in cats is lacking. Validation of the human equipment used routinely, such as dipsticks, is also lacking. As such, the evidence base for feline urinalysis is quite poor and information has largely been extrapolated from the human literature. Information from feline studies has been included where available. In addition, practical clinicopathological and clinical observations are provided.

Canine disorder mirrors human disease: exonic deletion in HES7 causes autosomal recessive spondylocostal dysostosis in miniature Schnauzer dogs.

Spondylocostal dysostosis is a congenital disorder of the axial skeleton documented in human families from diverse racial backgrounds. The condition is characterised by truncal shortening, extensive hemivertebrae and rib anomalies including malalignment, fusion and reduction in number. Mutations in the Notch signalling pathway genes DLL3, MESP2, LFNG, HES7 and TBX6 have been associated with this defect. In this study, spondylocostal dysostosis in an outbred family of miniature schnauzer dogs is described. Computed tomography demonstrated that the condition mirrors the skeletal defects observed in human cases, but unlike most human cases, the affected dogs were stillborn or died shortly after birth. Through gene mapping and whole genome sequencing, we identified a single-base deletion in the coding region of HES7. The frameshift mutation causes loss of functional domains essential for the oscillatory transcriptional autorepression of HES7 during somitogenesis. A restriction fragment length polymorphism test was applied within the immediate family and supported a highly penetrant autosomal recessive mode of inheritance. The mutation was not observed in wider testing of 117 randomly sampled adult miniature schnauzer and six adult standard schnauzer dogs; providing a significance of association of Praw = 4.759e-36 (genome-wide significant). Despite this apparently low frequency in the Australian population, the allele may be globally distributed based on its presence in two unrelated sires from geographically distant locations. While isolated hemivertebrae have been observed in a small number of other dog breeds, this is the first clinical and genetic diagnosis of spontaneously occurring spondylocostal dysostosis in a non-human mammal and offers an excellent model in which to study this devastating human disorder. The genetic test can be utilized by dog breeders to select away from the disease and avoid unnecessary neonatal losses.

Ulcerated and nonulcerated nontuberculous cutaneous mycobacterial granulomas in cats and dogs.

BACKGROUND: Mycobacterial granulomas of the skin and subcutis can be caused by one of a number of pathogens. This review concentrates on noncultivable species that cause diseases characterized by focal granuloma(s), namely leproid granuloma (in dogs) and feline leprosy (in cats). Clinically indistinguishable lesions can be caused by tuberculous organisms (Mycobacterium bovis and Mycobacterium microti) and members of the Mycobacterium avium complex. Rapidly growing mycobacterial species that cause infection of the subcutaneous panniculus associated with draining tracts are not discussed. Disease caused by Mycobacterium ulcerans is an important emerging differential diagnosis for ulcerated cutaneous nodules in certain localized regions. CLINICAL LESIONS: Lesions comprise one or multiple nodules in the skin/subcutis. These are generally firm and well circumscribed, and typically become denuded of hair. They may or may not ulcerate, depending on the virulence of the causal organisms and the immune response of the host. DIAGNOSIS: The most inexpensive, noninvasive means of diagnosis is by submission of methanol-fixed, Romanowsky-stained smears to a Mycobacterium Reference Laboratory after detecting negatively stained or acid-fast bacilli on cytological smears. Scrapings of material from slides usually provide sufficient mycobacterial DNA to enable identification of the causal organism using sequence analysis of amplicons after PCR using specific mycobacterial primers. THERAPY: Therapy relies upon a combination of marginal resection of easily accessible lesions and treatment using two or three drugs effective against slowly growing mycobacteria, choosing amongst rifampicin, clarithromycin, clofazimine and pradofloxacin/moxifloxacin.

Looks can deceive: molecular identity of an intraerythrocytic apicomplexan parasite in Australian gliders.

Two yellow-bellied gliders (Petaurus australis) had an intraerythrocytic parasite closely related to the cyst-forming coccidia (Apicomplexa: Sarcocystidae). The parasitaemia persisted for 3 months or more but was observed to clear within 3 years in captivity. The parasite appears not to significantly debilitate its infected host. Traditionally, using morphological identification, the intraerythrocytic parasite would have been classified within the Hepatozoon species typically found in red blood cells. However, molecular diagnostic techniques targeting the parasite's SSU rDNA and LSU rDNA demonstrated the unusual identity of this blood parasite and disputed its identity as a haemogregarine parasite of the genus Hepatozoon. The sequence was compared with available sequences from diverse mammalian and non-mammalian blood parasites (malaria, piroplasms, hemosporidia and sarcosporidia). The intraerythrocytic blood parasite was found to be most closely related to the cyst-forming coccidia including Besnoitia spp., Cystoisospora spp., Hammondia spp., Hyaloklossia lieberkuehni, Neospora caninum, Sarcocystis spp. and Toxoplasma gondii. The life cycle of this intraerythrocytic parasite remains unknown. The presented DNA identification demonstrates its suitability for an improved identification of blood parasites.