Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan. The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated. The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified. A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

Introduction: In the present study apoptosis was investigated in the cornu ammonis and cerebellum of 10 dogs naturally infected with rabies virus. Diagnosis of rabies was based on the results of fluorescent antibody staining and experimental inoculation.Material and Methods: The paraffin tissue sections were stained with haematoxylin and eosin, avidin-biotin complex peroxidase (ABC-P), and terminal deoxynucleotidyl transferase biotin-dUTP nick end-labelling (TUNEL) methods.Results: Histopathological examination revealed encephalomyelitis of varying severity and the presence of Negri bodies. Dense rabies antigens were determined in the motor neurons with ABC-P method. On the other hand, Bcl-2 protein and Bax protein gave positive reaction in seven and five cases, respectively. TUNEL staining demonstrated very marked apoptotic changes in the nuclei of neurons localised deep in the substantia alba of the cerebellum. Similar changes were also determined in perivascular mononuclear cells and glia cells within the substantia alba. No apoptopic changes were found in the motor neurons of the cornu ammonis.Conclusion: The absence of apoptotic changes in the neurons was considered to be the consequence of the necrotic changes that developed in these neurons.

Cyprinid herpesvirus 3 (CyHV-3) is a virus infecting carp with disease symptoms of gill necrosis, fish discoloration, sunken eyes, and mortality reaching 90%. Several research groups have examined how to potentially abate the consequences of viral activity. Recently we showed that acyclovir inhibits CyHV-3 replication in vitro and in the present study we examined the anti-CyHV-3 activity of the tricyclic derivative of acyclovir 6-(4-MeOPh)-TACV (T-ACV), a fluorescent molecule known for higher lipophilicity than acyclovir, and therefore potentially better candidate for application in vivo. CCB and KF1 cell lines were incubated with T-ACV at concentrations of 0, 66.67, and 133.33 μM for three days and toxicity examined with MTT and CV assays. To investigate the antiviral activity of T-ACV, the lines were infected with CyHV-3 or mock infected and incubated for three days with the drug at concentrations of 0 or 66.67 μM. The activity of T-ACV was evaluated by plaque assay and TaqMan qPCR. T-ACV at a concentration of 66.67 μM displayed low toxicity and inhibited CyHV-3 activity by 13–29%, varying by cell line and method. The low anti-CyHV-3 activity of T-ACV indicates that it would be reasonable to screen several tricyclic derivatives of acyclovir for such activity.

Introduction: The aim of the study was the application and evaluation of real-time PCRs based on the fluorescence of SYBR Green I intercalating dye for the detection of three Bacillus anthracis genes in contaminated liver and blood samples. The goals for detection were rpoB gene as a chromosomal marker, pag gene located on plasmid pXO1, and capC gene located on plasmid pXO2. Material and Methods: Five B. anthracis strains were used for the experiments. Additionally, single strains of other species of the genus Bacillus, i.e. B. cereus, B. brevis, B. subtilis, and B. megaterium, and strains of six other species were used for evaluation of the specificity of the tests. Three SYBR Green I real-time PCRs were conducted allowing confirmation of B. anthracis in the biological samples. Results: The observation of amplification curves in real-time PCRs enabled the detection of the chromosomally encoded rpoB gene, pag gene, and capC gene of B. anthracis. The specificity of the tests was confirmed by estimation of the melting temperature of the PCR products. The sensitivity and linearity of the reactions were determined using regression coefficients. Strains of other microbial species did not reveal real-time PCR products. Conclusion: All real-time PCRs for the detection of B. anthracis in biological samples demonstrated a significant sensitivity and high specificity.

Introduction: Exposure to zoonotic factors in veterinary practice is closely related to the nature of the work. The main aim of the study was to determine the risk of selected zoonotic infections among the occupational group of veterinarians in Poland. Material and Methods: Blood samples of 373 veterinarians (162 males and 211 females) from 12 provinces of Poland were collected by the venipuncture of a forearm for serological tests. Commercial immunoenzymatic tests (ELISA) were used for detection of specific IgG antibodies to Echinococcus granulosus, IgM and IgG to Leptospira spp., and IgM, IgA, and I and II phase IgG to Coxiella burnetii. Enzyme-linked fluorescence assays (ELFA) were used to detect IgM and IgG antibodies to Toxoplasma gondii. Results: Positive results were found in 209 (56.0%) veterinarians for at least one of the examined diseases. The overall proportion of participants found to have specific Toxoplasma gondii antibodies in the IgM and/or IgG assays amounted to 44.5%. The presence of Coxiella burnetii antibodies was found in 16 (4.3%) subjects, while Leptospira spp. antibodies were detected in 63 (16.9%) veterinarians. Among the 373 veterinarians examined, no Echinococcus granulosus antibodies were found. Conclusion: Results of the study seem to indicate a slightly elevated risk of Toxoplasma gondii infection and a moderate risk of infection with Leptospira spp. and Coxiella burnetii in veterinarians.

OBJECTIVE To determine the median time to maximum concentration (tmax) of amikacin in the synovial fluid of the tarsocrural joint following IV regional limb perfusion (IVRLP) of the drug in a saphenous vein of horses. ANIMALS 7 healthy adult horses. PROCEDURES With each horse sedated and restrained in a standing position, a 10-cm-wide Esmarch tourniquet was applied to a randomly selected hind limb 10 cm proximal to the point of the tarsus. Amikacin sulfate (2 g diluted with saline [0.9% NaCl] solution to a volume of 60 mL) was instilled in the saphenous vein over 3 minutes with a peristaltic pump. Tarsocrural synovial fluid samples were collected at 5, 10, 15, 20, 25, and 30 minutes after completion of IVRLP. The tourniquet was removed after collection of the last sample. Amikacin concentration was quantified by a fluorescence polarization immunoassay. Median maximum amikacin concentration and tmax were determined. RESULTS 1 horse was excluded from analysis because an insufficient volume of synovial fluid for evaluation was obtained at multiple times. The median maximum synovial fluid amikacin concentration was 450.5 μg/mL (range, 304.7 to 930.7 μg/mL), and median tmax was 25 minutes (range, 20 to 30 minutes). All horses had synovial fluid amikacin concentrations ≥ 160 μg/mL (therapeutic concentration for common equine pathogens) at 20 minutes after IVRLP. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that, in healthy horses, maintaining the tourniquet for 20 minutes after IVRLP of amikacin in a saphenous vein was sufficient to achieve therapeutic concentrations of amikacin in the tarsocrural joint.

OBJECTIVE To evaluate the effectiveness of a novel fluorescence tracer agent, MB-102, for conducting ocular angiography in dogs. ANIMALS 10 ophthalmologically normal dogs (2 to 4 years old) and 10 dogs with retinal degeneration or primary open-angle glaucoma (< 6 years old). PROCEDURES While anesthetized, all dogs received sodium fluorescein (20 mg/kg, IV) or MB-102 (20 or 40 mg/kg, IV) first and then the other dye in a second treatment session 2 days later in a randomized crossover design. Anterior fluorescence angiography was performed on one eye and posterior fluorescence angiography on the other. Imaging was performed with a full-spectrum camera and camera adaptor system. Filter sets that were tailored to match the excitation and emission characteristics of each angiographic fluorescent agent were used. RESULTS All phases and phase intervals during anterior and posterior segment angiography were identified, regardless of the dye used. However, agent fluorescence and visualization of the iridal blood vessels were hindered in some dogs, irrespective of agent, owing to the degree of iridal pigmentation present. No significant difference was noted between the 2 dyes in any phase or phase interval, and slight improvement in image contrast was observed with MB-102 during the venous phases owing to a reduction of vessel wall staining in both normal and diseased eyes. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that MB-102 would be useful for conducting ocular angiography in dogs.

OBJECTIVE To evaluate the usefulness of injection of indocyanine green (ICG) solution with near-infrared (NIR) fluorescence imaging for transcutaneous detection of sentinel lymph nodes (SLNs) and their associated lymphatic vessels in the oral mucosa of healthy dogs. ANIMALS 6 adult purpose-bred research hounds. PROCEDURES Each dog was sedated, and 1 mL of ICG solution was injected into the gingival mucosa dorsal to the right maxillary canine tooth. Subsequently, NIR fluorescence imaging was used to transcutaneously detect the lymphatic vessels and SLNs. The distance between the injection site and each SLN was measured. Time to first evidence of node fluorescence was recorded, and velocity of ICG movement was calculated. A slide preparation of a fine-needle aspiration sample of the fluorescing structure underwent cytologic examination (to confirm presence of lymphatic tissue) and NIR fluorescence imaging (to confirm presence of ICG). RESULTS The ipsilateral mandibular lymphocentrum was the SLN in all dogs. The time to visually detectable fluorescence ranged from 4 to 15 minutes (mean ± SD, 8.8 ± 3.76 minutes). The mean velocity was 1.94 ± 0.93 cm/min. Fluorescence was not observed in the contralateral lymph nodes. Each fluorescing structure was confirmed to be lymphatic tissue, and NIR fluorescence imaging revealed that ICG was present in the sampled SLN. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that injection of ICG solution with NIR fluorescence imaging can be used to transcutaneously identify SLNs along with associated lymphatic vessels in the oral mucosa of healthy dogs. Time from injection to identification of fluorescence was rapid with prolonged retention of material within the SLN, indicating that this procedure could be performed during surgery.

Objective—To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Sample—Citrate-anticoagulated whole blood from 30 healthy adult horses. Procedures—Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead–calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples. Results—Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP. Conclusions and Clinical Relevance—Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.

Objective: To determine the effect of various UVB radiation sources on plasma 25-hydroxyvitamin D3 concentrations in Hermann's tortoises (Testudo hermanni). Animals: 18 healthy Hermann's tortoises. Procedures: Tortoises were exposed to sunlight in an outdoor enclosure located in the natural geographic range of Hermann's tortoises (n = 6 tortoises) or a self-ballasted mercury-vapor lamp (6) or fluorescent UVB-emitting lamp (6) in an indoor enclosure for 35 days. Plasma samples were obtained from each tortoise on the first (day 0) and last (day 35) days of the study, and concentrations of 25-hydroxyvitamin D3 were determined. Amount of UVB radiation in enclosures was measured. Results: Mean ± SD plasma 25-hydroxyvitamin D3 concentrations for tortoises exposed to the mercury-vapor and fluorescent lamps were significantly lower on day 35 (155.69 ± 80.71 nmol/L and 134.42 ± 51.42 nmol/L, respectively) than they were on day 0 (368.02 ± 119.34 nmol/L and 313.69 ± 109.54 nmol/L, respectively). Mean ± SD plasma 25-hydroxyvitamin D3 concentration for tortoises exposed to sunlight did not differ significantly between days 0 (387.74 ± 114.56 nmol/L) and 35 (411.51 ± 189.75 nmol/L). Mean day 35 plasma 25-hydroxyvitamin D3 concentration was significantly higher for tortoises exposed to sunlight versus those exposed to mercury-vapor or fluorescent lamps. Sunlight provided significantly more UVB radiation than did the mercury-vapor or fluorescent lamps. Conclusions and Clinical Relevance: Plasma 25-hydroxyvitamin D3 concentrations differed between tortoises exposed to sunlight and those exposed to artificial UVB sources. Exposure to sunlight at a latitude similar to that of the natural geographic range is recommended for healthy and calcium-deficient tortoises.