In vitro comparison of the effects of two forms of hydroxyethyl starch solutions on platelet function in dogs

OBJECTIVE: To evaluate the effect of 2 hydroxyethyl starch (HES) preparations (ie, HES solution with a molecular weight of 600 kd and a degree of substitution of 0.7 [HES 600/0.7] and a calcium-containing polyionic HES solution with a molecular weight of 670 kd and a degree of substitution of 0.75 [HES 670/0.75]) on canine platelet function. SAMPLE POPULATION: Blood samples from 10 healthy adult dogs. PROCEDURES: Dilution of citrated whole blood was performed with saline (0.9% NaCl) solution, HES 600/0.7, and HES 670/0.75 at ratios of 1:9 (ie, 1 part saline solution or colloid to 9 parts whole blood) and 1:3. Measurements of time to platelet plug formation in a capillary tube (ie, closure time) were made by use of a bench-top platelet function analyzer with collagen and ADP platelet agonists. RESULTS: Mean baseline closure time was 68.0 +/- 15.3 seconds. A 1:3 dilution of whole blood with saline solution, HES 600/0.7, and HES 670/0.75 resulted in mean closure times of 85.8 +/- 15.7 seconds, 100.6 +/- 18.6 seconds, and 101.6 +/- 16.2 seconds, respectively. Closure time following 1:3 dilution of whole blood with saline solution was significantly different from baseline and from 1:9 dilution with saline solution. Closure time following 1:3 dilution of whole blood with HES 670/0.75 was significantly different from baseline, 1:3 and 1:9 dilutions with saline solution, and 1:9 dilutions with HES 600/0.7 or HES 670/0.75. CONCLUSIONS AND CLINICAL RELEVANCE: Saline solution, HES 600/0.7, and HES 670/0.75 affect canine platelet function by prolonging closure times; HES solutions prolonged closure time to a greater extent than saline solution.     

Effect of freezing goat milk samples on recovery of intramammary bacterial pathogens

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

Prevalence of antibodies against Ornithobacterium rhinotracheale in broilers and breeders in Southern Brazil

In this investigation, we determined the prevalence of the Ornithobacerium rhinotracheale (ORT) infection in broilers and broiler breeders in southern Brazil. We also correlated the presence of antibodies in broilers with performance. Sera from 1550 broilers from 50 flocks were collected during the slaughter time in nine companies with federal veterinary inspection of the state of Rio Grande do Sul. Sera from 480 meat-type breeders of 40 flocks from 14 companies in southern Brazil were also analyzed by enzyme-linked immunosorbent assay, and the prevalence of antibodies was determined. The prevalence of ORT antibodies in broiler flocks was 63.83%, but in each individual flock only 6.52% of the birds were positive. The prevalence in broiler breeder flocks was 100.00%, and in each individual flock 94.62% of the birds were positive. There was a positive correlation between the presence of antibodies to ORT and decreased body weight in broilers. There was no significant correlation between presence of antibodies to ORT and age, lineage, efficiency index, feed conversion, and mortality. There was a positive correlation between the presence of respiratory signs and antibodies to ORT, although the reverse correlation was not significant. These results confirm that ORT is present and widespread in broilers and broiler breeders in southern Brazil.     

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.     

Protostrongylid infection in meat sheep from Northwestern Spain: prevalence and risk factors

2093 Faecal samples from 74 commercial meat ovine flocks were collected and examined by the Baermann-Wetzel method for protostrongylid infection. The risk of being infected by lungworms was evaluated with a data mining classification tree (CHAID), and the intensity of infection with a general linear model (GLM). 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2-12.9). Only two species were found, Muellerius capillaris (97.9%) and Neostrongylus linearis (5.4%). 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces. All of them held animals infected by M. capillaris and seven presented mixed infections with N. linearis. Average larval output in infected sheep was 11.9 (SD 30.91). This study showed that protostrongylid prevalence in sheep for meat production was determined mainly by a positive interaction with Dictyocaulus filaria infection; other factors that have influenced over protostrongylid infection were age, introducing external animals in the flocks, mixed management with goats and animal density in pastures. Treatment effects on prevalence were only observed in flocks that did not introduce ewes. The lowest protostrongylid prevalence has been reported in flocks without D. filaria infection and without contact with goats.     

Y chromosome genetic diversity in the Lidia bovine breed: a highly fragmented population

To assess the paternal gene pool in the Lidia bovine breed (or fighting bull), a total of 603 animals belonging to 81 herds classified in 33 lineages were genotyped for six Y chromosome microsatellites, one single nucleotide polymorphism and one indel. A total of 10 haplotypes were determined with a high level of frequency variation between them, ranging from 0.2 to 74%. All the haplotypes identified belong to two previously defined major haplogroups (Y1 and Y2). Two major paternal influences were identified, corresponding to the two most common haplotypes (H1Y1 and H3Y2) with frequencies of 74 and 18%, respectively. The detection of the INRA189-104 allele evidenced an African influence in the Lidia bovine breed. Low levels of haplotype diversity have been achieved and only eight lineages showed more than one haplotype. Analysis of molecular variance showed a high level of interlineage variance (F(ST) = 86%). Network results evidenced two main clusters made for those haplotypes belonging to Y1 and Y2 haplogroups, respectively. The findings support a high level of genetic structure together with a low level of genetic diversity in the Lidia bovine breed.     

Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting.

We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases.     

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T_h1 and T_h2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.