Isolation of Canine Mammary Cells With Stem Cell Properties and Tumour-Initiating Potential

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.     

Experimental studies of the role of the little raven (Corvus mellori) in surveillance for West Nile virus in Australia

Objective To study the potential role of an Australian corvid, the little raven (Corvus mellori), in the surveillance for exotic West Nile virus (WNV) in Australia. Method In a series of trials, little ravens were infected with WNV (strain 4132 New York 1999) and Kunjin virus (strain K42886) by the intramuscular route. They were observed for 20 days during which blood and swab samples were taken for virus isolation. Tissue samples were taken from ravens humanely killed during the acute infection period, and at the termination of the trials, for virus isolation, histopathology and immunohistochemistry. Results Ravens infected with WNV became mildly ill, but all recovered and seroconverted. Blood virus titres peaked around 3 to 4 days after inoculation at levels between 10^3.0 to 10^7.5 plaque forming units/mL. Virus or viral antigen was detected in spleen, liver, lung, kidney, intestine, testis and ovary by virus isolation and/or immunohistochemistry. WNV was detected in oral and cloacal swabs from 2 to 7 days post inoculation. The molecular and pathogenic characteristics of the inocula were consistent with them being of high virulence, as expected for this isolate. Ravens infected with Kunjin virus developed viraemia and seroconverted, although they did not develop disease. Conclusions Little ravens do not develop severe disease in response to virulent WNV infection and for this reason may not be important sentinel hosts in the event of an outbreak of WNV, as in North America. However, as they have relatively high viraemias, they may be able to support virus cycles.     

EFFECTS OF ANESTHETIC PROTOCOL ON NORMAL CANINE BRAIN UPTAKE OF 18F‐FDG ASSESSED BY PET/CT

The purpose of this study was to assess the effects of four anesthetic protocols on normal canine brain uptake of 2‐deoxy‐2‐[¹⁸F]fluoro‐d ‐glucose (FDG) using positron emission tomography/computed tomography (PET/CT). Five clinically normal beagle dogs were anesthetized with (1) propofol/isoflurane, (2) medetomidine/pentobarbital, (3) xylazine/ketamine, and (4) medetomidine/tiletamine–zolazepam in a randomized cross‐over design. The standard uptake value (SUV) of FDG was obtained in the frontal, parietal, temporal and occipital lobes, cerebellum, brainstem and whole brain, and compared within and between anesthetic protocols using the Friedman test with significance set at P<0.05. Significant differences in SUVs were observed in various part of the brain associated with each anesthetic protocol. The SUV for the frontal and occipital lobes was significantly higher than in the brainstem in all dogs. Dogs receiving medetomidine/tiletamine–zolazepam also had significantly higher whole brain SUVs than the propofol/isoflurane group. We concluded that each anesthetic protocol exerted a different regional brain glucose uptake pattern. As a result, when comparing brain glucose uptake using PET/CT, one should consider the effects of anesthetic protocols on different regions of the glucose uptake in the dog's brain.     

IMAGING DIAGNOSIS—FDG‐PET/CT OF A CANINE SPLENIC PLASMA CELL TUMOR

An 8‐year‐old Shih Tzu developed abdominal pain and hyperglobulinemia. A round splenic mass was noted radiographically and sonographically. The patient was evaluated by fluorodeoxyglucose positron emission tomography coupled with computed tomography (FDG‐PET/CT). There was no evidence of metastasis or bone marrow involvement on PET/CT images. The standardized uptake value (SUV) of the splenic mass was increased over the reference range (SUV=4.83). The patient was diagnosed as splenic extramedullary plasmacytoma through immunohistopathologic study. After the splenectomy, the globulin level normalized and the patient is alive without complications.     

Deleterious Effects of Progestagen Treatment in VEGF Expression in Corpora Lutea of Pregnant Ewes

The aim of the current study was to determine the possible effects of progestagen oestrous synchronization on vascular endothelial growth factor (VEGF) expression during sheep luteogenesis and the peri‐implantation period and the relationship with luteal function. At days 9, 11, 13, 15, 17 and 21 of pregnancy, the ovaries from 30 progestagen treated and 30 ewes cycling after cloprostenol injection were evaluated by ultrasonography and, thereafter, collected and processed for immunohistochemical evaluation of VEGF; blood samples were drawn for evaluating plasma progesterone. The progestagen‐treated group showed smaller corpora lutea than cloprostenol‐treated and lower progesterone secretion. The expression of VEGF in the luteal cells increased with time in the cloprostenol group, but not in the progestagen‐treated group, which even showed a decrease between days 11 and 13. In progestagen‐treated sheep, VEGF expression in granulosa‐derived parenchymal lobule capillaries was correlated with the size of the luteal tissue, larger corpora lutea had higher expression, and tended to have a higher progesterone secretion. In conclusion, the current study indicates the existence of deleterious effects from exogenous progestagen treatments on progesterone secretion from induced corpora lutea, which correlate with alterations in the expression of VEGF in the luteal tissue and, this, presumably in the processes of neoangiogenesis and luteogenesis.     

Tick paralysis in dogs and cats in Australia: treatment and prevention deliverables from 100 years of research

This review of tick paralysis caused by Ixodes holocyclus in Australia addresses the question: What are the key discoveries that have enabled effective treatment and prevention of tick paralysis in dogs and cats? Critical examination of 100 years of literature reveals that arguably only three achievements have advanced treatment and prevention of tick paralysis in animals. First, the most significant treatment advance was the commercial availability of tick antiserum in the 1930s. Hyperimmune serum currently remains the only specific anti-paralysis tick therapy available to veterinarians in Australia. Second, advances in veterinary critical care have increased survival rates of the most severely affected dogs and cats. Critical care advancements have been enabled through specialised veterinary hospitals that can provide appropriate care 24 h a day, and advanced training of veterinarians, veterinary nurses and technicians. Third, perhaps that biggest advance of all in the last 100 years of research has been the commercial availability of the isooxazoline class of acaricidal preventatives in Australia specifically for I. holocyclus. This highly effective class of preventatives offers long duration of action, low cost, spot-on or oral formulations and a low rate of adverse reactions. Animal owners and veterinarians now have the most useful tool of all – a reliable preventative. This review reveals the key events in research over the last 100 years and the tortuous pathway to delivering better treatment and preventative options for this enigmatic Australian parasite.     

The toxicity of Castanospermum australe seeds for cattle

Two calves given a mean of 16.1 g and 16.4 g ripe Castanospermum australe seeds/kg body weight daily for 13 and 16 days respectively developed haemorrhagic gastroenteritis. The first calf died. The second calf had mild myocardial degeneration and necrosis and mild nephrosis at necropsy. Two calves given a mean of 16.8 g unripe C. australe seeds/kg body weight daily for 18 days remained clinically normal and had mild gastritis at necropsy. The activity of alpha-glucosidase was reduced in the mononuclear cells of peripheral blood and in skeletal muscle. This was attributed to the presence of the indolizidine alkaloid, castanospermine, in the seeds. The toxin causing the gastroenteritis and other lesions is unknown.     

Does blood contamination of urine compromise interpretation of the urine protein to creatinine ratio in dogs?

AIMS: To determine the effect of contamination of urine with 0–5% blood, varying in haematocrit and protein concentrations, on the urine protein to creatinine ratio (UPC) in dogs, and to determine whether the colour of urine can be used to aid interpretation of UPC results.

METHODS: Urine samples were collected by free catch from 18 dogs, all of which had UPC?<0.2. Venous blood samples were also collected from each dog, and the blood from each dog was added to its own urine to produce serial concentrations of 0.125–5% blood. The colour of each urine sample was recorded by two observers scoring them as either yellow, peach, orange, orange/red or red. Protein and creatinine concentrations were determined, and dipstick analysis and sediment examination was carried out on each sample. Based on colour and dipstick analysis, samples were categorised as either having microscopic, macroscopic or gross haematuria. A linear mixed model was used to examine the effect of blood contamination on UPC.

RESULTS: The uncontaminated urine of all 18 dogs had a UPC?<0.2. Adding blood to the urine samples resulted in an increase in UPC at all contamination concentrations compared to the non-contaminated urine (p<0.001). None of the 54 samples with microscopic haematuria had UPC?>0.5. For 108 samples with macroscopic haematuria the UPC was >0.5 in 21 samples (19.4 (95% CI=13.1–27.9)%), and for 54 samples with gross haematuria 39 (72 (CI=59.1–82.4)%) had a UPC?>0.5. No samples had a UPC?>2.0 unless the blood contamination was 5% and only 3/18 (17%) samples at this blood contamination concentration had a UPC?>2.0.

CONCLUSIONS AND CLINICAL RELEVANCE: This study showed that while blood contamination of ≥0.125% does increase the UPC, if the urine remains yellow (microscopic haematuria), then there is negligible chance that a UPC?>0.5 will be solely due to the added blood. In that scenario, attributing the proteinuria present to the haematuria in the sample would be inappropriate. However blood contamination that results in discolouration of the urine sample from yellow (indicating macroscopic or gross haematuria) could increase the UPC above the abnormal range and would need to be considered as a differential for the proteinuria. Thus knowledge of urine colour, even if limited to simple colour scores (yellow, discoloured, red) could be utilised to aid interpretation of the UPC in samples with haematuria.     

The effect of different combinations of local anaesthesia,sedative and non-steroidal anti-inflammatory drugs on daily growth rates of dairy calves after disbudding

AIM: To assess the effect of sedation and local anaesthesia (LA) at disbudding, and the addition of meloxicam or ketoprofen treatment, on weight gain in dairy calves following disbudding.

METHODS: Friesian-Jersey cross calves, from four dairy farms, were enrolled when 3–6 weeks old. All calves (n=271) were disbudded by veterinary personnel and randomly assigned to six groups: 136 were disbudded without sedation or LA, of which 31 received 20 mg meloxicam S/C and 75 received 150 mg ketoprofen I/M. A further 135 were disbudded with sedation (0.25 mg/kg xylazine I/M) and LA, of which 30 also received meloxicam and 75 received ketoprofen. Calves were weighed 3 days before, and 15 and 30 days after, disbudding (Day 0). Daily weight gain was analysed using mixed models and ANOVA.

RESULTS: Complete results were obtained from 263 calves. From Day ?3 to Day 15, the growth rate of calves disbudded without pain relief (0.53 (95% CI=0.47–0.60) kg/day) was less that of calves disbudded with some form of pain relief (0.65 (95% CI=0.62–0.68) kg/d; p=0.004). There was no difference between the effect of meloxicam or ketoprofen (p=1.00). An interaction between use of sedation and LA and additional non-steroidal anti-inflammatory drugs (NSAID) meant that NSAID treatment did not increase growth rates in calves disbudded with sedation and LA but did increase growth rates for calves disbudded without pain relief (p<0.05). From Day 16 to Day 30 there was no effect of NSAID treatment on growth rate, but calves receiving LA and sedation grew faster (0.74 (95% CI=0.69–0.80) kg/day) than calves disbudded without LA and sedation (0.66 (95% CI=0.61–0.71) kg/day; p=0.018). From Day ?3 to Day 30, calves disbudded with sedation and LA grew faster (0.71 (95%CI=0.64–0.77) kg/day) than calves disbudded without sedation and LA (0.60 (95% CI=0.55–0.65) kg/day; p=0.011). However, addition of NSAID to sedation and LA made no further difference to growth rates (p=0.69).

CONCLUSIONS: Dairy calves disbudded with no pain relief had slower growth rates than calves receiving pain relief. From Day 15 to 30 calves given no pain relief, or NSAID alone, grew more slowly than those receiving sedation and LA at disbudding. The addition of NSAID treatment to sedation and LA did not further increase growth rates.

CLINICAL RELEVANCE: This study adds to the evidence that pain management when disbudding is beneficial for calf productivity as well as calf welfare.