PRELIMINARY EVALUATION OF THE QUALITY OF BLOOD COMPONENTS FOR TRANSFUSION USE (WHOLE BLOOD, PACKED RED BLOOD CELLS, FRESH FROZEN PLASMA) IN CANINE, FELINE AND BOVINE BLOOD PRODUCTS, AND PREPARATION OF BLOOD COMPONENT FOR NON-TRANSFUSION USE (PLATELET RICH PLASMA)IN DOG

Evaluation of hematological parameters, ammonia concentration and microbial contamination in Canine Packed Red Blood Cells stored in CPD-SAGM for 42 days. Abstract Canine transfusion medicine practices have been growing rapidly over the past few decades and the use of specific blood components (packed red cells, plasma) has permitted to optimized the canine blood donations. The study was undertaken to evaluated the changes in RBC, MCV, Ht, RDW, WBC and ammonia concentration in canine PRBC stored in CPD-SAGM for 42 days. Also the presence of bacterial contamination was evaluated with blood culture. PRBC units were stored in a routine manner and were examined every 2-3 weeks. Hematological parameters changed significantly with increase of MCV, Ht and RDW, while WBC decreased. Also ammonia concentration increased significantly during the storage. RBC and WBC deteriorated somewhat during storage and ammonia concentration increased similar to what reported in canine and human in vitro studies. No bacterial contamination was reported. The results obtained in this study agree mostly with what previously is reported in canine and human medicine. Further studies are needed to better evaluated how the reported alterations influence viability of blood cells in canine PRBC. The safety and quality of feline whole blood units collected with an open system and the effect of storage on hematological parameters and ammonia concentration Abstract The veterinary transfusion medicine is constantly in progress but still now feline blood donation, collection, and conservation of whole blood and blood products present some problems. Feline whole blood collected with open system and stored in CPDA1 for 35 days at 4\ub0C was evaluated for hematological parameters, ammonia concentration and sterility during the storage period. Statistical analysis resulted in significant increase in ammonia concentration and decrease of WBC. No other significant changes resulted in hematological parameters (RBC, Ht, MCV, RDW). No units presented bacterial contamination during storage. The use a standardized protocol during blood collection, preparation and storage of feline whole blood permit to obtain a product without microbial contamination, minimum changes in haematological parameters but with a very high ammonia concentration. Preliminary Evaluation on the Stability of Protein in Bovine Fresh Frozen Plasma Abstract The aim of this study were to evaluating preliminary stability of glucose, urea, total protein and protein fractions in bovine fresh frozen plasma (FFP). Blood was collected into human sterile double-pack blood collection system containing citrate-phosphate-dextrose-adenine (CPDA) and after centrifugation the plasma units were stored within 8 hours from blood collection at \u2013 19\ub0C obtaining FFP. The analysis of biochemical parameters were performed on fresh plasma after centrifugation of whole blood unit and on thawed FFP after 1 and 6 months of storage. Pre and post storage results were compared using a one way repeated measures ANOVA. Seven FFP were obtained from 7 different whole blood units. There was no significant changes in the concentrations of glucose, urea, total protein and protein fraction during the entire period of storage. This preliminary study showed that during 6 months of storage no significant changes were appeared in the evaluate biochemical parameters in bovine FFP. Effectiveness of manual double centrifugation method for preparation of Canine Platelet-Rich Plasma Abstract The platelet-rich plasma (PRP) is a product derived from whole blood with a platelet concentration higher than normal range in a small amount of plasma. Regenerative capacities of PRP deriving from platelet growth factors. For this reason PRP is used in human and veterinary medicine for its capacity to stimulate cell proliferation, angiogenesis, wound healing, production of fibroblasts, collagen, osteoblasts and to accelerate the healing process. In veterinary medicine the methods use to produce PRP are not standardized and extremely numerous. The aim of this study was to describe and evaluate a manual double centrifugation method to produce canine PRP. 28 blood samples (5-10 ml with 1 ml of sodium citrate) from 28 healthy dogs were analyzed. The first centrifugation (2500 rpm for 10\u2019) resulted in two components, blood cell component in the bottom and serum component (SEC) in the upper fraction of the tube. All SEC, the buffy coat and the first 2 mm of red blood cell was submitted to a second centrifugation (4000 rpm for 15\u2019) and resulted in two components, platelet poor plasma (PPP) and platelet pellet in the bottom. The amount of PPP used to resuspend platelet pellet was calculated considering that all platelets previously present in the SEC was in platelet pellet. Then 50% of the calculated PPP was used to resuspend platelet pellet with the aim to obtain the final platelet concentration of about 1 million /\ub5l. The method proposed in this study permitted in 14/28 samples of PRP (50%) to reach the human target of 1 milion platelets/\ub5l \ub1 20% and/or the target of three to six fold platelet concentration in whole blood. According to a part of human literature the concentration of platelet in PRP does not seem to be necessarily linked to its effectiveness. For this reason to complete the evaluation of the proposed method will be necessary assess the platelet viability and after apply the PRP in vivo

Relationship between Leishmania IFAT Titer and Clinicopathological Manifestations (Clinical Score) in Dogs

During canine leishmaniasis (CanL) due to Leishmania infantum, high levels of antibodies production are associated with the presence of various clinical signs, because of the deposition of soluble immune complexes in organs and tissues. The immunofluorescence antibody test (IFAT) is one of the most commonly used techniques for detection of anti-Leishmania antibodies. The purpose of this study was to assess whether there is a correlation between clinical signs and IFAT titers in dogs naturally infected with Leishmania. A retrospective study was performed on medical records of 49 dogs diagnosed with CanL. Information extracted from the medical records of each dog with CanL was clinical score, IFAT titer, serum total protein (TP), gamma globulin (IgG) and creatinine concentration, and protein creatinine ratio in urine sample (UP/UC) at each follow-up examination. Results show that dogs with highest IFAT titers recorded had higher mean clinical scores indicating a positive relationship (P<0.0001) between anti-Leishmania antibodies (IgG) and clinical manifestations, which becomes more evident in severe clinical forms of canine leishmaniasis. Higher TP and IgG serum concentrations were recorded in dogs with higher clinical scores. Significant association was observed between UP/UC and the IFAT titer (P=0.004)

Comparison of a Clinic-Based ELISA Test Kit with the Immunofluorescence Antibody Test for Assaying Leishmania infantum Antibodies in Dogs

This study compares a rapid Immunospecific Kalazar Canine Rapid Spot IF with the gold standard test (indirect fluorescent antibody test (IFAT)) for detection of Leishmania infantum specific IgG serum antibodies in naturally exposed dogs. Serum samples were obtained from 89 healthy dogs and dogs affected by canine leishmaniosis (CanL). IgG-IFAT titers 6580 were considered positive. Anti-L. infantum IgG antibodies were found in 54 samples with titers ranging from 1\u2009:\u200980 to 1\u2009:\u20095120. The performance of the rapid Immunospecific Kalazar was evaluated using a ROC curve. The area under the ROC curve of 0.957 was significantly different from 0.5 (P 0 (sensitivity 92.6%, specificity 97%). The test can be used for disease screening if the cutoff is >0 (highest sensitivity, 92.6%) and is recommended as confirmatory test for the presence of L. infantum IgG antibodies if the cutoff is set >2 (highest specificity, 100%)

Comparison of VIDAS and radioimmunoassay methods for measurement of cortisol concentration in bovine serum

Radioimmunoassay (RIA) is the "gold standard" method for evaluation of serum cortisol concentration. The VIDAS cortisol test is an enzyme-linked fluorescent assay designed for the MiniVidas system. The aim of this study was to compare the VIDAS method with RIA for measurement of bovine serum cortisol concentration. Cortisol concentrations were evaluated in 40 cows using both VIDAS and RIA methods, the latter as the reference method. A paired Student's t-test, Pearson's correlation analysis, Bland-Altman plot, and Deming regression analysis were used to compare the two methods. There was no statistically significant difference between mean serum cortisol concentrations measured by VIDAS or RIA methods (P = 0.6570). Both methods were able to detect significant differences in mean low and high cortisol concentrations (P < 0.00014 RIA and P < 0.0016 VIDAS). The correlation coefficient was low, but a Bland-Altman plot and Deming regression analysis show neither constant nor proportional error. The VIDAS method produced slightly higher values than RIA, but the difference was small and in no case did the mean value move the normal range. Results suggest that VIDAS method is suitable for the determination of bovine serum cortisol concentration in studies of large numbers of animals

Par&#225;metros hematol&#243;gicos, concentraci&#243;n de amon&#237;aco y morfolog&#237;a celular en unidades de concentrados de hemat&#237;es caninos almacenados en SAG M

Objetivos Los eritrocitos sufren algunas alteraciones complejas durante el almacenamiento conocido como \u201clesiones de almacenamiento\u201d (Zehnder et al., 2008). El objetivo de este estudio era investigar la calidad de las unidades de concentrados de hemat\uedes caninos (CHC) almacenados durante 42 d\uedas en SAGM (salino-adenina- glucosa-manitol) evaluando los cambios en los par\ue1metros hematol\uf3gicos, concentraci\uf3n de amon\uedaco y morfolog\ueda celular durante el almacenamiento. Material y metodos 33 unidades de CHC fueron obtenidas de sangre entera usando un sistema de bolsas triples est\ue9riles de recolecci\uf3n humanas y una centr\uedfuga de sangre. Los perros fueron reclutados, con un informe de consentimiento, como donantes de sangre en un programa voluntario de donantes de sangre de la Unidad de Transfusi\uf3n Veterinaria de la Universidad de Mil\ue1n, Italia. Las unidades se almacenaron verticales en un refrigerador especial para almacenamiento de sangre a 4\ub0 C \ub1 2\ub0C. Para asegurar una distribuci\uf3n uniforme de la soluci\uf3n anticoagulante y aumentar la viabilidad de los CHC las bolsas fueron agitadas y mezcladas suavemente tres-cuatro veces a la semana. Una Alicuotade CHC fue tomada inmediatamente antes del almacenamiento (D0) y realizado el recuento autom\ue1tico de las c\ue9lulas sangu\uedneas y un frotis de sangre (para evaluar la morfolog\ueda celular) se realizaron y midieron concentraciones de amoniaco. Cada 2 semanas desde D0 a D42 se extrajeron al\uedcuotas de los CHC y se realizaron las mismas pruebas. Los datos fueron analizados usando MedCalc statistical software (versi\uf3n 14.10.2). Los datos fueron analizados por pruebas de nor- malidad utilizando el test de Kolmogorov-Smirnov y la media de concentraci\uf3n de amoniaco, eritrocitos, recuento de gl\uf3bulos rojos(RBC), recuento de gl\uf3bulos blancos (WBC), Volumen corpuscular medio (VCM), Hematocrito (Ht), an- cho de distribuci\uf3n eritrocitaria (RDW) en cada punto de muestreo se compararon mediante medidas de una manera repetida ANOVA. La correlaci\uf3n de VCM y Ht fue evaluada por el coeficiente de Spearman\u2019s con un valor de p<0.05 fue considerado estad\uedsticamente significativo. Resultados No hubo cambios significativos en el RBC durante el alma- cenamiento (p=1). Todos los dem\ue1s par\ue1metros evaluados mostraron diferencias estad\uedsticamente significativas entre los puntos de tiempo. En particular hubo una incremento significativo en el Ht entre D0 y D42 (p=0,0003), un sig- nificativo y progresivo aumento de VCM y RDW entre los puntos de tiempo, un significante descenso del WBC entre D= y todos los puntos de tiempo (p<0,0001) y un significativo y progresivo aumento de amon\uedaco a trav\ue9s de todos los puntos de tiempo (p < 0,.0001).Una estad\uedstica signifi- cativa y correlaci\uf3n positiva fue evidente entre HT y VCM en D0 (rho = 0,420, p = 0,0150), en D28 (rho = 0,658, p< 0,0001) y D42 (rho = 0,628, p = 0,0002). Cambios en la morfolog\ueda celular fueron observados en todos los pun- tos de tiempo con un incremento significativo de la lisis del RBC y aumento del n\ufamero de macrocitos, esferocitos y esquistocitos en D42. Hab\ueda una lisis casi total del WBC desde D14 y equinocitos, que ya eran abundantes en D0, aument\uf3 ligeramente en n\ufamero hasta D42. Discusi\uf3n Muchos estudios veterinarios han evaluado el almacenamiento de unidades de CHC (Wardrop et al., 1997, Ekiz et al., 2012). En sus estudios no hubo cambios significativos en el contaje del RBC durante el almacenamiento, mientras que Ht y VCM aumentaban, seg\ufan se informa en otro estudio reciente sobre CHC almacenado en SAGM (Ekiz et al., 2012). El aumento del RDW en las unidades de CHC podr\ueda ser una medida de la anisocitosis celular, como se inform\uf3 anteriormente en estudios humanos. El contaje de WBC redujo significativamente con el almacenamiento, y esto fue confirmado por la evalua- ci\uf3n morfol\uf3gica. De hecho, la muy corta vida \ufatil de granulocitos significa que no pueden ser almacenados (Kohn et al., 2012). Finalmente, como informaron Waddell et al. (2001), las concentraciones de amoniaco aumentaron a niveles extremadamente altos en nuestro estudio. En D14 la mayor\ueda de las unidades ten\uedan concentraciones de amoniaco muy por encima del valor normal canino (valor media en D14 260.8 \u3bcg/dl). Conclusiones Nuestros datos preliminares sugieren una lisis celular progresiva de CHC con la liberaci\uf3n de hemoglobina y en consecuencia estas unidades almacenadas suponen un aumento hipot\ue9tico del riesgo de transfusi\uf3n. Puede ser conveniente revisar el tiempo de almacenamiento bolsa en los perros. La ausencia de estudios sobre los efectos del aumento de la concentraci\uf3n de amon\uedaco en CHC caninos en perros con enfermedad hep\ue1tica nos lleva a aconsejar evitar la transfusi\uf3n de unidades de m\ue1s de 14 d\uedas en estos pacientes. Ser\ueda tambi\ue9n interesante, en futuros estudios, evaluar la permanencia de las c\ue9lulas rojas de la sangre transfundidas en los estudios in vivo para evaluar otros indicadores de la calidad del producto. Bibliograf\ueda \u2022 Ekiz E, Arslan M, Akyazi I, Eraslan Uygur E, Ihal Gultekin G, Ozcan M. The effects of prestorage leukoreduction and storage duration on the vitro quality of canine packed red blood cells. Turkish Journal of Veteri- nary Animal Science 2012; 36(6): 711-717. \u2022 Kohn B, Weingart C: Feline transfusion medicine, in: Day MJ, Kohn B, eds: BSAVA Manual of Canine and Feline Haematology and Transfusion Medicine, (2nd ed). John Wiley and Sons Ltd, Gloucester, 2012. \u2022 Waddel LS, Holt DE, Hughes D, Giger U. The effect of storage on ammo- nia concentration in canine packed red blood cells. Journal of Veterinary Emergency and Critical Care 2001; 11(1):23-26. \u2022 Wardrop KJ, Tucker RL, Mugnai K. Evaluation of canine red blood cells stored in a saline, adenine, and glucose solution for 35 days. Journal of Veterinary Internal Medicine1997; 11:5-8. \u2022 Zehnder L, Schulzki T, Goede JS, Hayes J, Reinhart WH. Erythrocyte storage in hypertonic (SAGM) or isotonic (PAGGSM) conservation medium: influence on cell properties. Vox Sangunis 2008; 95(4):280-287

Survey of Dermatophytes in Stray Cats with and without Skin Lesions in Northern Italy

The aim of this study was to determine the prevalence of dermatophytes in stray cats with and without clinical lesions from different colonies in rural and urban areas of Milan and surroundings in northern Italy. Stray cats (273) were caught during a trap-neuter-release (TNR) program conducted in different colonies of northern Italy in both rural and urban areas. Each cat was examined in dark environment with a Wood's lamp prior to sample collection. Hair or scales exhibiting typical fluorescence were removed with a pair of sterile hemostats and cultured. The hair of all cats was then sampled by Mackenzie modified brush technique regardless of the presence or absence of skin lesions attributable to dermatophytosis. All the hair samples were subjected to fungal culture. 15 cats were positive (5.5%). Microsporum canis was the most common dermatophyte isolated (13/15). The only other isolated dermatophyte was Trichophyton mentagrophytes (2/15). Our estimated prevalence of dermatophytes in stray cats was much lower than other Italian studies on the same population

Serological and molecular evaluation of Leishmania infantum infection in stray cats in a nonendemic area in Northern Italy

Infection by Leishmania species is increasing worldwide. It was hypothesized recently that cats act as a secondary reservoir for Leishmania infection. The aim of the present study was to assess the prevalence of Leishmania infantum antibodies and DNA in blood samples collected in a sample of stray cats in metropolitan area of Milan in northern Italy, which is a nonendemic area for leishmaniasis. An indirect immunofluorescence antibody test for L. infantum showed that 59 of 233 cats (25.3%) were seroreactive, 38 samples (16.3%) had antibody titers of 1 : 40, 15 (6.4%) had antibody titers of 1 : 80, and 6 (2.6%) had antibody titers of 1 : 160. Feline immunodeficiency virus (FIV) seropositive status was statistically associated with seroreactivity to L. infantum (P = 0.01) as shown by univariate and multivariate logistic regression (P = 0.0098; OR = 7.34). All blood samples that were tested using real-time PCR were negative for parasite DNA. These results were surprising, since no autochthonous human or canine cases of leishmaniasis have ever been reported in this region of northern Italy. It is possible that this high seroreactivity to L. infantum could be due to cross-reaction with antigens from other parasites. Additional studies that include parasite isolation are needed to clarify our findings on feline leishmaniasis in this region

Frequency of Piroplasms Babesia microti and Cytauxzoon felis in Stray Cats from Northern Italy

Emerging diseases caused by piroplasms pose a health risk for man and other animals, and domestic cats have been proposed as potential reservoirs for some piroplasm infections. The aim of this study was to identify the frequency of the piroplasms Babesia microti and Cytauxzoon felis in stray cats from northern Italy and to identify possible risk factors associated with these infections. Blood samples from 260 stray cats enrolled in a trap-neuter-release (TNR) program in northern Italy were examined with conventional PCR for the presence of Babesia microti and Cytauxzoon felis DNA. No sample (0.0%) tested positive for C. felis, whilst B. microti DNA was detected in two samples (0.8%). Both infected cats were in good clinical condition and recovered well from the neutering surgery. One of these two cats had a triple coinfection with Babesia microti, Candidatus Mycoplasma haemominutum, and Anaplasma phagocytophilum. Evidence presented in this study indicates that the blood borne protozoans Babesia microti and Cytauxzoon felis are not widely distributed in stray cat populations in Milan, northern Italy, and that the significance of cats as a reservoir host for B. microti in this area is limited

Prevalence of Haemoplasma infections in stray cats in Northern Italy

This study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in Milan, Northern Italy. Blood samples from 260 stray cats were evaluated, with conventional PCR, for the presence of DNA associated with Mycoplasma haemofelis (Mhf) and "Candidatus Mycoplasma haemominutum" (CMhm). Odd ratios (OR) were calculated to identify risk factors for haemoplasma infections. PCR was positive in 86 out of 260 subjects (33.1%), with a prevalence of 10.8% (28/260 cats) for Mhf and 22.3% (58/260 cats) for CMhm. No coinfections were registered. There were significant associations between infections and season of sampling, that is, a negative association between winter sampling and a haemoplasma positive status (OR = 0.29, P = 0.001), or CMhm positive status (OR = 0.29, P = 0.01). Haemoplasma infections are common in stray cats in Milan. Thus, domestic cats with outdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition. Moreover, as these infections are transmitted via blood, feline blood donors from this area should be screened by PCR and preferably be drawn from a population of indoor cats regularly treated for fleas

Neutrophil Gelatinase-Associated Lipocalin and Noninfectious Pyuria in Dogs

Neutrophil gelatinase-associated lipocalin (NGAL) is a neutrophil-derived protein whose concentration increases in plasma and urine with ongoing renal damage. Urinary leucocytes can be a potential source of urinary NGAL. The aim of this study is to investigate the effects of urinary neutrophil count and other urinary parameters on urinary NGAL values in urine with negative culture. Urinalysis, urine culture, and determination of urinary NGAL were performed on 33 clinically healthy nonproteinuric dogs with negative urinoculture. The median uNGAL concentration in dogs in this study population was 9.74 ng/mL (IQR 1.93-25.43 ng/mL). In samples with WBCs &gt; 5 hpf (mean 15.9, 6-50 leucocytes/hpf), median uNGAL value was significantly higher than that in samples with WBCs &lt; 5 hpf (mean 0.9, 0-3 leucocytes/hpf), (4.96 pg/mL (0.29-11.34) and 23.65 pg/mL (20.04-29.80), resp.; = 0.0053). The severity of urinary pyuria and the UPC value were correlated with uNGAL concentration. The results of our study show that urinary NGAL concentration is correlated with WBCs number in urinary sediment of dogs with negative urinoculture. The present study suggests that noninfectious pyuria is significantly correlated with urinary NGAL values and might influence uNGAL values