Analysis of canine myeloid-derived suppressor cells (MDSCs) utilizing fluorescence-activated cell sorting, RNA protection mediums to yield quality RNA for single-cell RNA sequencing.

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy.

Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII- CD14-) and monocytic-MDSC (CD11b+ MHCII- CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

In vitro effects of Yunnan Baiyao on canine hemangiosarcoma cell lines.

Yunnan Baiyao is a Chinese herbal medicine that has been utilized for its anti-inflammatory, haemostatic, wound healing and pain relieving properties in people. It has been utilized in the veterinary profession to control bleeding in dogs with hemangiosarcoma (HSA) and has been anecdotally reported to prolong survival times in dogs with this neoplasm. This study evaluated the in vitro activity of Yunnan Baiyao against three canine HSA cell lines after treatment with increasing concentrations of Yunnan Baiyao (50, 100, 200, 400, 600 and 800 µg mL(-1) ) at 24, 48 and 72 h. Mean half maximum inhibitory concentration (IC50 ) at 72 h for DEN, Fitz, SB was 369.9, 275.9 and 325.3 µg mL(-1) , respectively. Caspase-3/7 activity increased in correlation with the IC50 in each cell line which was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, APO-BRDU Kit; BD Biosciences, San Jose, CA, USA) assay. VEGF in cell supernatant was also quantified. Overall, the study found that Yunnan Baiyao causes dose and time dependent HSA cell death through initiation of caspase-mediated apoptosis, which supports future studies involving Yunnan Baiyao.

Frameless stereotactic radiosurgery for the treatment of primary intracranial tumours in dogs.

Stereotactic radiosurgery (SRS) is a procedure that delivers a single large radiation dose to a well-defined target. Here, we describe a frameless SRS technique suitable for intracranial targets in canines. Medical records of dogs diagnosed with a primary intracranial tumour by imaging or histopathology that underwent SRS were retrospectively reviewed. Frameless SRS was used successfully to treat tumours in 51 dogs with a variety of head sizes and shapes. Tumours diagnosed included 38 meningiomas, 4 pituitary tumours, 4 trigeminal nerve tumours, 3 gliomas, 1 histiocytic sarcoma and 1 choroid plexus tumour. Median survival time was 399 days for all tumours and for dogs with meningiomas; cause-specific survival was 493 days for both cohorts. Acute grade III central nervous system toxicity (altered mentation) occurred in two dogs. Frameless SRS resulted in survival times comparable to conventional radiation therapy, but with fewer acute adverse effects and only a single anaesthetic episode required for therapy.

In vitro effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine hemangiosarcoma cell lines.

This study evaluated the in vitro activity of masitinib mesylate against canine hemangiosarcoma (HSA) cell lines after treatment with increasing concentrations of masitinib mesylate (0.01-100 µM) for 24, 48 and 72 h. Results indicated that masitinib mesylate caused a dose- and time-dependent decrease in HSA cell proliferation. The 50% inhibitory concentration (IC(50) ) at 72 h for three HSA cell lines (DEN, Fitz and SB) was found to be 8.56, 9.41 and 10.65 µM, respectively. Further investigation demonstrated that masitinib mesylate induced apoptosis in all HSA cell lines, including activation of caspase-3/7. Measurement of VEGF levels in cell supernatant found a statistically significant increased VEGF in close proximity to the IC(50) of each cell line followed by a decline back towards baseline. These findings indicate that masitinib mesylate causes dose-dependent HSA cell death in vitro and supports future clinical trials of masitinib for canine HSA.

Radiosensitivity and capacity for radiation-induced sublethal damage repair of canine transitional cell carcinoma (TCC) cell lines.

Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/ß ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/ß ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.

Effectiveness of Bacillus thuringiensis-transgenic chickpeas and the entomopathogenic fungus Metarhizium anisopliae in controlling Helicoverpa armigera (Lepidoptera: Noctuidae).

The use of genetically modified (Bt) crops expressing lepidopteran-specific Cry proteins derived from the soil bacterium Bacillus thuringiensis is an effective method to control the polyphagous pest Helicoverpa armigera. As H. armigera potentially develops resistance to Cry proteins, Bt crops should be regarded as one tool in integrated pest management. Therefore, they should be compatible with biological control. Bioassays were conducted to understand the interactions between a Cry2Aa-expressing chickpea line, either a susceptible or a Cry2A-resistant H. armigera strain, and the entomopathogenic fungus Metarhizium anisopliae. In a first concentration-response assay, Cry2A-resistant larvae were more tolerant of M. anisopliae than susceptible larvae, while in a second bioassay, the fungus caused similar mortalities in the two strains fed control chickpea leaves. Thus, resistance to Cry2A did not cause any fitness costs that became visible as increased susceptibility to the fungus. On Bt chickpea leaves, susceptible H. armigera larvae were more sensitive to M. anisopliae than on control leaves. It appeared that sublethal damage induced by the B. thuringiensis toxin enhanced the effectiveness of M. anisopliae. For Cry2A-resistant larvae, the mortalities caused by the fungus were similar when they were fed either food source. To examine which strain would be more likely to be exposed to the fungus, their movements on control and Bt chickpea plants were compared. Movement did not appear to differ among larvae on Bt or conventional chickpeas, as indicated by the number of leaflets damaged per leaf. The findings suggest that Bt chickpeas and M. anisopliae are compatible to control H. armigera.

The immune response to disialoganglioside GD3 vaccination in normal dogs: a melanoma surface antigen vaccine.

As a result of its metastatic potential, canine malignant melanoma like its human counterpart like its human counter part, has a poor response to conventional treatment protocols. This prompted us to investigate the possibility of enhancing the immune response against the melanoma cell surface antigen, disialoganglioside GD3. Initially a flow cytometric study was designed in which the incidence of GD3 on the cell surface, recognized by the monoclonal antibody Mel-1 (R24), was established in canine melanoma cell lines. Results from the flow cytometry found GD3 to be highly expressed (94.2%) in six out of seven canine melanoma cell lines. Since it was thus potentially a good target, a study in which normal dogs were vaccinated intradermally with a vaccine containing GD3 plus adjuvants was designed. The adjuvant included CpG oligodeoxynucleotide (CpG-ODN) sequences and RIBI-adjuvant, which are known to target toll-like receptors (TLR) of the innate immune system. From a cohort of 10 dogs, 4 were vaccinated 3 times, at 4 weekly intervals with GD3 plus adjuvant, and 4 received only RIBI-adjuvant, and 2 phosphate buffered saline. Caliper measurements were collected to assess skin reaction at the vaccination site and sera assayed for IgM and IgG antibodies against GD3 and cell-mediated cytotoxicity against a melanoma cell line. Results from the study found significant differences (P<0.05) in the vaccine site reactions, IgM/IgG levels and cell-mediated cytotoxicity in the vaccinated versus unvaccinated dogs. The addition of CpG-ODN sequences and increasing GD3 concentration in the vaccine increased the inflammation response at the injection site. GD3 IgG and IgM antibodies in vaccinated dogs showed increasing titers over time and achieved significance at weeks 9 and 12, respectively. Cell-mediated cytotoxicity was only detected in peripheral blood mononuclear cells from vaccinated dogs. In conclusion, by combining the tumor antigen GD3 (a known weak self-antigen) and an adjuvant, tolerance was overcome by an innate and adaptive immune response in this population of normal dogs.

Efficacy and toxicity of BOPP and LOPP chemotherapy for the treatment of relapsed canine lymphoma.

Mechlorethamine (Mustargen), Oncovin) (vincristine), procarbazine and prednisone (MOPP) chemotherapy is useful for relapsed canine lymphoma. This study evaluates the efficacy of MOPP after substitution of CCNU (lomustine, LOPP protocol) or BCNU (carmustine, BOPP protocol) for mechlorethamine in 60 dogs with relapsed lymphoma. Seven of 14 (50%) dogs treated with BOPP responded, for a median of 129.5 days for complete responders (range 9-354 days) and a median of 140 days for partial responders (range 4-276 days). Twenty-three of 44 (52%) dogs treated with LOPP responded for a median of 112 days for complete responders (range 48-250 days) and a median of 84.5 days for partial responders (range 69-290 days). Two dogs receiving a combination of LOPP and BOPP partially responded for 28 and 163 days, respectively. With BOPP chemotherapy, nine dogs (20.5%) and seven dogs (50%) had one or more episodes of Grade II or higher neutropenia and thrombocytopenia, respectively. Seven dogs (50%) had one or more episodes of Grade II or higher gastrointestinal toxicity. While receiving LOPP chemotherapy, 28 dogs (63.6%) and 17 dogs (38.6%) had one or more episodes of Grade II or higher neutropenia and thrombocytopenia, respectively. Seventeen dogs (38.6%) had one or more episodes of Grade II or higher gastrointestinal toxicity. Overall, there were 17 non-fatal treatment-related episodes of sepsis requiring hospitalization. Eight dogs (13%) died or were euthanized because of treatment-related sepsis and/or chemotherapy-related complications. Severe haematologic toxicity, coupled with the improved response duration observed in dogs receiving reduced doses during B/L-OPP rescue, underscores the need for protocol optimization.

Suppurative rhinitis associated with Haemophilus species infection in a cat.

A young cat with signs of chronic rhinitis was evaluated for underlying anatomical, inflammatory, or infectious disease. Initial diagnostics were significant for the isolation of an unusual pathogen, Haemophilus species. Isolation using a human RapID NH system erroneously identified the isolate as H. segnis, a human pathogen. No database of veterinary pathogens (Haemophilus) are included in the system and animal pathogens will either be erroneously identified or yield a unique biocode not listed. Because of the unique nature of the pathogen we explored the possibility of immunosuppression as a contributory factor to infection. A variety of laboratory tests were employed to evaluate immune function. The clinical indications and utility of immune function testing are discussed. No immune dysfunction was identified.

Hepatic osteodystrophy in rats results mainly from portasystemic shunting.

BACKGROUND AND AIMS: In chronic liver disease, bone disease frequently develops. The contributions of the different features of liver disease such as parenchymal inflammation, portal hypertension, and portasystemic shunting on bone metabolism have not been systematically studied. The aim of this study was to identify the features of liver disease contributing to bone disease using rat models. METHODS: Parenchymal liver disease was induced by carbon tetrachloride administration, portal hypertension by partial portal vein ligation, and portasystemic shunting by end to side anastomosis of the portal vein to the inferior vena cava. Normal and sham operated surgical animals served as controls. Serum calcium, 25-hydroxy vitamin D (25-OH vit D), and osteocalcin levels, and urinary deoxypyridinoline excretion were analysed. Testosterone and oestradiol levels were determined in male and female rats, respectively. Interleukin 1, interleukin 6, and tumour necrosis factor alpha (TNF-alpha) were determined in serum. Bone density was measured in all groups and in addition, in the surgical groups, histomorphometry was performed on undecalcified specimens of the proximal tibia. The calcium content of the femurs, removed at termination and ashed, was determined. RESULTS: Early parenchymal disease and portal hypertension did not affect bone metabolism or body mass. Portasystemic shunting increased bone resorption, decreased bone formation, bone density, and trabecular bone volume which were commensurate with a reduction in body mass. TNF-alpha levels were elevated and testosterone levels were low in male portasystemic shunted rats. CONCLUSIONS: Portasystemic shunting in the rat adversely affects bone metabolism as part of a generalised catabolic state where high TNF-alpha and low testosterone and 25-OH vit D levels may play a role.

Biodistribution and pharmacokinetics of variously sized molecular radiolabelled polyethyleneiminomethyl phosphonic acid as a selective bone seeker for therapy in the normal primate model.

An ideal radiopharmaceutical for the treatment of neoplastic and inflammatory (benign) bone disease would be a radiolabelled compound that predominantly accumulates in bone lesions with limited access to normal bone and other organs. Neoplastic tissue's abnormal blood supply (increased permeability) and lack of lymphatics will selectively accumulate radiolabelled macromolecules. This enhanced permeability and retention effect forms the basis of this study, using various molecular sizes of the radiolabelled macromolecule polyethyleneiminomethyl phosphonic acid (PEI-MP) for increased selectivity of the bone-seeking radiopharmaceutical. PEI-MP was synthesized by condensation of polyethyleneimine, phosphonic acid and formaldehyde, followed by fractionation into different molecular sizes by membrane ultrafiltration. Labelling efficiency to 99mTc (as radiotracer) was approximately 99% with complexes stable for 24 h. The pharmacokinetics and biodistribution of various 99mTc-PEI-MP fractions were investigated using 4 experimental baboons (Papio ursinus) per fraction. Scintigraphy was performed on the baboons under general anaesthesia of pentobarbital i.v. After an i.v. bolus of 99mTc-PEI-MP (approximately 185 MBq) both dynamic studies (30 x 1 min frames), and static studies (2 min acquisition every hour for 4 h) were done, as well as blood samples and urine collected. From the results macromolecules with sizes ranging between 30-300 kDa were characterized by excessive liver (21%-57% retained activity) and kidney (40% retained activity) uptake and accompanying long residing times (t1/2 up to 24 h). The percentage bone uptake averaged at 8% for these particles excluding sizes 100-300 kDa where very little bone uptake was seen (< 1%). In this case the blood clearance was also slow (t1/2 approximately 2 h). The fraction size 10-30 kDa had comparatively low accumulation and short residence times in the liver and kidneys (resp. 20%, t1/2 = 22 +/- 4 min; 17.5%, t1/2 = 20 +/- 3 min) and although the bone uptake of 18% in this case was high, it is still low for a bone-seeking agent. These particles cleared the blood with t1/2 = 25 +/- 2 min and seemed suitable for labelling with a therapeutic radioisotopic agent.

Aerial treatment of the Australian plague locust, Chortoicetes terminifera (Orthoptera: Acrididae) with Metarhizium anisopliae (Deuteromycotina: Hyphomycetes).

Between October 1999 and April 2000, nearly 4000 ha of nymphal bands and adult swarms of Chortoicetes terminifera (Walker) were aerially treated using a ULV oil formulation of strain FI-985 of Metarhizium anisopliae var. acridum. During the mild weather (maxima 22-30 degrees C) of spring (October), there was little change in nymphal bands during the first week but at all doses between 25-100 g (1-4 x 10(12) conidia) ha(-1), the bands rapidly declined 9-12 days after treatment reaching > 90% mortality by 14 days. Metarhizium persisted for some time as there was 50% mortality of locusts fed vegetation collected from the treated blocks seven days after treatment. Persistence was confirmed by the high mortality of bands that invaded from untreated areas and of nymphs that hatched on the plot five to seven days after treatment, though mortality was then delayed until early in the third week. During summer (January), temperatures were high (maxima 36-42 degrees C), and at all doses between 25 and 125 g (1-5 x 10(12) conidia) ha(-1), there was a rapid decline seven to ten days after treatment. By 12-14 days, there was a > 90% decline in numbers in most blocks which was confirmed by helicopter surveys two weeks after treatment that found very few adults within or near treated areas. Mortality was delayed in the high dose where there were blockages of spray equipment during treatment. The clear demonstration that Metarhizium can suppress small local populations of C. terminifera led to the limited operational use of Metarhizium on an organic farm and in a National Park where nearly 2500 ha of bands and swarms were treated. Continued research is needed to develop a commercially viable product so that Metarhizium can form a significant part of a programme of integrated pest management of locusts in Australia.

Targeted radiotherapy with Sm-153-EDTMP in nine cases of canine primary bone tumours.

Nine dogs with primary bone tumours were treated with Samarium-153-EDTMP (Sm-153-EDTMP). Conventional treatment protocols were precluded by the size of the dogs and the owners' refusal of limb amputation. All the tumours were of the appendicular skeleton; 4 were confirmed osteosarcomas. The other 5 tumours were radiologically suspect for osteosarcoma. Bone scans were performed on all dogs using Technetium-99m-methylene diphosphonate (Tc-99m-MDP) before administration of Sm-153-EDTMP. Regions of interest were identified over the contralateral limb at the same site as the tumour and counts per pixel were recorded for the tumour and contralateral limb and expressed as a ratio. The dogs were given 1 injection of 37 MBq/kg (1 mCi/kg) of Sm-153-EDTMP intravenously. Thoracic and primary tumour site radiographs were taken at monthly or 2-monthly intervals to monitor progression of the primary tumour and search for evidence of metastasis. Two dogs showed no response to treatment, with an increase in bone pain, and were euthanased within 1 month. In 1 dog, a tumour of the scapula underwent complete involution and the dog is considered free of disease at 20 months post Sm-153-EDTMP treatment. The overall tumourcidal effect of a single dose of Sm-153-EDTMP on primary bone tumours was difficult to evaluate in this group of dogs, as, with one exception, all the primary tumours progressed over time and the dogs were euthanased. Pain control, for which Sm-155-EDTMP is used in man, was not evident, except in the dog that responded completely to treatment.

Canine babesiosis in South Africa: more than one disease. Does this serve as a model for falciparum malaria?

South African canine babesiosis is caused by the virulent Babesia canis rossi. In recent years, this common disease has been detected in 12% of dogs presented at the outpatients' division of the University of Pretoria's (Onderstepoort) Veterinary Academic Hospital, and 31% of the affected dogs have been hospitalized as seriously ill. Of these hospitalized cases, 50% had severe anaemia at presentation, 32% had moderate anaemia and 18% were non-anaemic (often polycythaemic), frequently with central-nervous-system signs or multiple organ failure. A retrospective survey of 662 hospitalized cases revealed that the haematology, clinical biochemistry and patient profile (signalment) of the severely anaemic dogs were distinct from those of the non-anaemic, indicating that the babesiosis in these two groups of dogs should be viewed as two different disease in terms of the postulated, underlying, 'pathomechanisms'. The severely anaemic dogs exhibited hypoxic hepatic disease and an increase in serum urea (without a concomitant increase in creatinine), seldom had profound electrolyte imbalances and tended to have a much more profound leucocytosis, consisting of a left-shifted inflammatory leucogram, with higher numbers of circulating metamyelocytes, lymphocytosis and monocytosis. In contrast, the non-anaemic dogs exhibited severe azotaemia (which could be of renal or pre-renal origin) and often showed a marked electrolyte disturbance (reflecting acid-base abnormalities) and a very mild leucocyte response; such dogs often presented as leucopenic, many being lymphocytopenic. These results indicate that the severely anaemic dogs had developed haemolytic disease (possibly immune-mediated), whereas the non-anaemic dogs had developed an acute and overwhelming inflammatory response. The mean age of the non-anaemic dogs (2.66 years) was less than the dogs in the 'severe anaemia group' (0.83 years). Dogs belonging to the traditional fighting breeds (bull terriers, pit bull terriers and Staffordshire bull terriers) were noticeably over-represented in the non-survivors of the acute inflammatory response, possibly indicating an underlying genetic basis for the different presentations. It is evident that the inflammatory-response disease presentation, which is similar to complicated falciparum malaria in humans, amy serve as an animal model for the disease.

The effect of humidity on germination and infection of termites by the hyphomycete, Metarhizium anisopliae.

The effect of relative humidities (r.h.) from 90 to 100% on germination of a termite-active isolate of Metarhizium anisopliae (isolate FI25 and FI610) was studied using a liquid germinating medium to which the appropriate amount of glycerol had been added. Germination was increasingly delayed at water activities equivalent to 99, 98, and 96% r.h. and completely inhibited at 94, 92, and 90%. Twenty-one isolates were then screened for germination at 96 and 100% r.h. All isolates showed delayed germination at 96% r.h. but most isolates eventually gave a high final percentage germination at this humidity. Two isolates, FI527 and FI638, were markedly slower to germinate at both humidities. The susceptibility of two species of termites, Nasutitermes exitiosus and Coptotermes acinaciformis, to FI610 was tested at r.h. down to 86%-the lowest humidity at which the insects would survive. No consistent effect of humidity on pathogenicity was detected. Mortality occurred over the range range of humidities tested; sporulation from the disease-killed termites, however, occurred only at r.h. above 93%. It is concluded that the microclimate around living termites is usually sufficiently humid to ensure infection under most field conditions and that humidity is unlikely to limit the efficacy of the fungus in controlling termites.

The effect of diminazene aceturate on cholinesterase activity in dogs with canine babesiosis.

A clinical trial was designed to evaluate the effects of diminazene aceturate and its stabiliser antipyrine on serum pseudocholinesterase (PChE) and red blood cell acetylcholinesterase (RBC AChE) in dogs with babesiosis. The trial was conducted on naturally occurring, uncomplicated cases of babesiosis (n = 20) that were randomly allocated to groups receiving a standard therapeutic dose of diminazene aceturate with antipyrine stabiliser (n = 10) or antipyrine alone (n = 10). Blood was drawn immediately before and every 15 minutes for 1 hour after treatment. Plasma PChE showed a 4% decrease between 0 and 60 min within the treatment group (p < 0.05). No statistically significant differences were found between the treatment and control groups at any of the time intervals for PChE. There was an increase in RBC AChE activity at 15 min in the treatment group (p < 0.05). No significant differences were found between the treatment and control groups at any time interval for RBC AChE. In view of the difference in PChE, samples from additional, new cases (n = 10) of canine babesiosis were collected to identify the affect of the drug over 12 hours. No significant depression was identified over this time interval. The results suggests that the underlying mechanism in producing side-effects, when they do occur, is unlikely to be through cholinesterase depression.

Chronic episodic diarrhoea associated with apparent intestinal colonisation by the yeasts Saccharomyces cerevisiae and Candida famata in a German shepherd dog.

A 3-year-old German shepherd dog was presented with a history of lifelong episodic diarrhoea. An adverse reaction to food was considered the most likely cause of the diarrhoea. The dog had received prolonged antibiotic therapy for most of its life as well as receiving probiotics containing the yeast Saccharomyces cerevisiae (syn. S. boulardi) for a year before referral. The probiotic was discontinued 2 months before to referral. Examination and culture of faecal samples identified yeast-like organisms, S. cerevisiae and Candida famata. S. cerevisiae has been isolated from humans in association with predisposing conditions such as prolonged sojourns in hospital, immunosuppression, broad-spectrum antibiotic therapy and prosthetic devices, but is regarded as non-pathogenic in humans and is rarely associated with disease in animals. C. famata has been isolated from animals, humans and the environment, but is regarded as a very rare pathogen. No evidence of immunosuppression was found in the dog. The presence of yeasts in the faecal isolates and the history of prolonged use of antibiotics and probiotics with a concurrent adverse reaction to food, suggest that conditions may have occurred within the bowel that made it possible for the yeasts to colonise parts of it. This has apparently not been reported before.

Immunophenotypic classification of canine malignant lymphoma on formalin-mixed paraffin wax-embedded tissue by means of CD3 and CD79a cell markers.

Canine malignant lymphoma (CML) is a common lymphoid tumour. Identification of the immunophenotype is of prognostic importance: T-cell lymphomas have a worse prognosis than B-cell lymphomas. Until recently, identification of T- or B-cell lymphomas was undertaken by means of flow cytometry or fluorescent immunocytochemistry on frozen sections. Whilst valid in the research field, these methods are impractical for routine diagnostic histopathology in CML. Commercially available CD3 antibody has been successfully employed in T-cell identification in dogs in formalin-fixed paraffin wax-embedded tissue sections, but the lack of a B-cell marker has been a hindrance until the recent introduction of a commercially available pan-B cell marker, CD79a (DAKO M7051), suitable for diagnostic application upon formalin-fixed paraffin wax-embedded material. Antibody markers to CD3 and CD79a show cross-reactivity across species lines for B cells and T cells respectively. In this group of five selected canine cases, two were identified as B-cell and the other three as T-cell lymphoma, by means of CD3 and CD79a. To the best of our knowledge application of CD79a in cases of CML has not been reported.

Differential expression of the presynaptic protein SNAP-25 in mammalian retina.

We have studied the expression of the nerve terminal protein synaptosomal associated protein 25 (SNAP-25) in the retina of adult rat, mouse, and monkey, as well as in the developing mouse retina. To evaluate SNAP-25 expression, its distribution was compared to those of the synaptic vesicle-associated proteins synapsin I and synaptophysin. In situ hybridization in adult rat retinas suggested that SNAP-25 mRNA is mainly expressed by ganglion, amacrine, and horizontal cells, but not by photoreceptors and bipolar cells. In all species, the SNAP-25 polypeptide was most abundant in the inner part of the inner and outer plexiform layers and was also found in the ganglion cell axons. In adult retina, synapsin I and synaptophysin were also mainly localized in synaptic fields and processes but all three proteins showed a distinct pattern of distribution. Finally, in mouse retina, the three proteins were first detectable at embryonic day 16 and subsequently showed developmentally regulated changes in their cellular localization. These results suggest that SNAP-25 is predominantly expressed in specific subtypes of conventional synapses, but not ribbon synapses, and that it may also be involved in the physiology of nonvesicular terminals of horizontal cells. Our study also suggests that combinatorial expression of different components of the presynaptic specialization may contribute to synaptic functional diversity.