The effect of different isolation procedures on canine leucocyte populations and on lectin-induced lymphocyte proliferation

Proliferation assays performed on peripheral blood mononuclear cells (PBMC) are commonly used in experimental and clinical immunology. A prerequisite for an in vitro assay is the ability to obtain relatively pure populations of mononuclear cells from whole blood, as contaminating polymorphonuclear cells may affect the proliferation of lymphocytes. Purification of canine leucocytes from whole blood is associated with difficulties in obtaining pure lymphocytes in high yields. The aim of this study was to optimize the lymphocyte purification from canine whole blood in terms of total cell recovery and purity, while not influencing the proliferation capacity of the isolated cells. To acquire optimal isolation of canine lymphocytes several density gradient media of different densities and osmolalities were examined. For optimal phagocyte removal, pre-treatment of whole blood with carbonyl iron/arabic gum and/or adherence to fibrinogen pre-coated polystyrene tissue flasks were examined. Lectin-induced proliferation was used as measurement of cell activity of the obtained cell fractions after the different separation procedures. Canine blood pre-treated with carbonyl iron/arabic gum followed by density gradient centrifugation with medium 'G' (density: 1.079 g/cm(3), osmolality: 256 mOsm) and adherence to pre-coated polystyrene tissue flask obtained the best PBMC cultures with a median lymphocyte purity of 88% and a median yield of recovered lymphocytes of 54%. This culture also resulted in the highest proliferation and subsequently the highest stimulation index upon lectin stimulation.     

Acral mutilation and analgesia in 13 French spaniels

Acral mutilation and analgesia (AMA) is reported in 13 French spaniels in Canada. This newly recognized disorder shares striking similarities in clinical features and biopsy findings to the other acral mutilation syndromes or hereditary sensory neuropathies reported in German short-haired pointer dogs, English pointer dogs and English springer spaniels. Clinical signs are first noted between 3.5 and 12 months of age. Affected dogs lick, bite and severely self-mutilate their distal extremities resulting in ulcers with secondary bacterial infection. Auto-amputation of claws, digits and footpads occurs in severe cases. Single or multiple feet can be affected. Affected dogs walked on their severely mutilated feet without evidence of pain, lameness, or ataxia. The majority of the dogs were euthanized within days to months of diagnosis.     

Germination of <Emphasis Type="Italic">Gibberella zeae</Emphasis> ascospores as affected by age of spores after discharge and environmental factors

The effects of age of ascospores (0–18 days after discharge), photon flux density (0–494 mol m^–2 s^–1 PAR), temperature (4–30 °C), frost (–15 °C for 30 min), relative humidity (RH; 0–100%), pH (2.5–6.5) and dryness (0 and 53% RH for up to 40 min) on the germination of the ascospores of the mycotoxin-producing fungus Gibberella zeae (anamorph Fusarium graminearum) were studied. Freshly discharged ascospores germinated within 4 h at 20 °C and 100% RH. The rate of germination and the percentage of viable ascospores decreased over time after the spores were discharged from perithecia. The time course of ascospore germination was not significantly affected by photon flux density. The period of time required to obtain 50% germinated ascospores at 100% RH was 26.90 h at 4 °C, 10.40 h at 14 °C, 3.44 h at 20 °C and 3.31 h at 30 °C. There was no significant effect of frost on the percentage of viable ascospores. A small percentage (6.6 ± 3.8%) of the ascospores germinated at 53% RH. At RH 84% and 20 °C almost 100% of the freshly discharged ascospores germinated. The time course of ascospore germination was affected by pH. The maximum rate of ascospore germination was estimated to be at pH 3.76. Ascospores lost their ability to germinate following exposure to 0% RH almost instantaneously. No germinating spores were detected after an incubation period of 1 min at 0% RH. Incubating the ascospores at 53% RH decreased the percentage of viable spores from 93 to 6% within 10 min. The data demonstrate that age of spores, relative humidity, temperature and pH, but not photon flux density, are key factors in germination of G. zeae ascospores.     

Unsaturated fatty acids from zoospores of <Emphasis Type="Italic">Sclerospora graminicola</Emphasis> induce resistance in pearl millet

Downy mildew of pearl millet, caused by Sclerospora graminicola, is a devastating disease, resulting in high economic losses in the semi-arid regions of the world. Recently, induction of host plant resistance using biotic and abiotic inducers are included among disease management practices as an eco-friendly approach. Unsaturated fatty acids are considered as a new generation of plant disease resistance inducers. In the present study, six unsaturated fatty acids, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), linolenic acid, linoleic acid and oleic acid, all originally detected in the zoospores of S. graminicola,were applied to seeds of susceptible cultivars of pearl millet to examine their ability to protect against downy mildew under greenhouse and field conditions. In greenhouse experiments, EPA and AA induced a maximum of 78.6% and 76.5% protection, whereas linoleic acid, DHA and linolenic acid provided up to 66.3%, 61.2% and 24.5% protection, respectively. Oleic acid was not effective in protecting pearl millet (only 5.1% protection). A time interval of four days between treatment of seeds and challenge inoculation was required to obtain optimum protection. Plants raised from treated seeds and challenge inoculated at the tillering and inflorescence stages showed enhanced resistance, resulting in higher grain yield compared to untreated plants of the same cultivar. Chitinase activity was found to be higher in susceptible seedlings of pearl millet after treatment with the fatty acids and pathogen inoculation than in seedlings only inoculated with the pathogen. This indicates that host defence responses are activated and thus that induced resistance is involved in the protection observed. The role of unsaturated fatty acids as activators of resistance against downy mildew in pearl millet is discussed.     

Investigation of changes in the middle latency auditory evoked potential during anesthesia with sevoflurane in dogs

OBJECTIVE: To investigate the middle latency auditory evoked potential (MLAEP) in awake dogs and dogs anesthetized with 2 concentrations of sevoflurane. ANIMALS: 10 adult Beagles. PROCEDURE: The MLAEP was recorded while dogs were awake and anesthetized with sevoflurane (end-tidal concentration, 2.7% or 3.5%).Three needle electrodes were inserted SC, and click stimuli were delivered biaurally. Signal acquisition, averaging, and analysis were performed by use of computer software developed in-house. Signals were recorded for 128 milliseconds, and the responses to 1,024 stimuli were averaged. Waveforms from 10 recordings in each dog were averaged, and latencies of peaks were measured. Data acquired for awake dogs and dogs anesthetized with high and low sevoflurane concentrations were compared statistically. RESULTS: Sevoflurane anesthesia attenuated the MLAEP so that only peaks P0, Na, and Pa could be identified. The MLAEP changes were maximal at the lower concentration of sevoflurane evaluated. The latencies of these peaks were significantly shorter in awake dogs, compared with values in anesthetized dogs. No difference in the peak latency was detected between the sevoflurane concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: In terms of CNS responsiveness, the effects of anesthesia with sevoflurane are similar to those of anesthesia with isoflurane. Data suggest that sevoflurane is not the inhalant agent of choice in a research setting where electroencephalographic measurements are to be recorded during anesthesia. The depression of the MLAEP waveform by sevoflurane also suggests that the MLAEP is not a suitable tool with which to monitor anesthetic depth during sevoflurane anesthesia in dogs.     

Biochemical analysis of the articular cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis

OBJECTIVE: To assess whether site-related changes in biochemical composition are present in the cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis. SAMPLE POPULATION: Right metacarpophalangeal joints from 59 mature warmblood horses. PROCEDURE: Biochemical data (cross-link, amino acid, DNA, and ash contents; denatured collagen and glycosaminoglycan [GAG] concentrations; bone mineral density; and mineral composition) were obtained from 2 differently loaded sites of phalanx I cartilage and subchondral and trabecular bone samples; data were compared with previously published values from nonosteoarthritic equine joints. RESULTS: Compared with findings in nonosteoarthritic joints, GAG concentration was lower in cartilage from osteoarthritic joints and there was a loss of site differences in cellularity and lysylpyridinoline (LP) cross-link content. In subchondral bone, LP cross-link content was decreased overall and there was a loss of site differences in osteoarthritic joints; ash content was higher in the osteoarthritic joints. Hydroxyproline content in trabecular bone from osteoarthritic joints was greater than that in nonosteoarthritic trabecular bone. In all 3 layers and at both sites, the linear increase of the pentosidine cross-link content with age had diminished or was not apparent in the horses with osteoarthritic joints. CONCLUSIONS AND CLINICAL RELEVANCE: In equine metacarpophalangeal joints with early osteoarthritis, distinct biochemical changes were detected in the cartilage and subchondral and trabecular bone. The dissimilarity in response of the different tissues and differences between the sites that are affected may be related to differences in biomechanical loading and transmission and dissipation of force.     

Reproductive Disorders of Crossbred Dairy Cows in the Central Highlands of Ethiopia and Their Effect on Reproductive Performance

The study was conducted to estimate the proportion of reproductive disorders and to determine factors affecting reproductive performance of crossbred dairy cows under four different production systems in the central highlands of Ethiopia. The principal postpartum reproductive disorders were retained fetal membranes (14.7%) and uterine infection (15.5%). Anoestrus was the major postpartum reproductive problem in the mixed crop–livestock production system (38.6%) and was significantly associated with this production system. Apart from anoestrus, the occurrence of reproductive disorders was not significantly associated with a production system. Most of the reproductive disorders occurred as a complex rather than as a single abnormality. Two or more abnormal conditions were seen in 11.4% of the cases. Each reproductive trait measured was affected adversely by reproductive disorders. Cows with reproductive disorders in each production system, lactation group and suckling and non-suckling group had longer intervals from calving to first service and to conception (p < 0.001) and required more services per conception (p < 0.001). Pregnancy rate and conception to first service were 84.7% and 51.7%, respectively, for cows without reproductive health problems; and 64.2% and 15.1%, respectively, for cows with reproductive disorders (p < 0.001). Overall, intervals from calving to first service were shorter (p < 0.05) than in younger cows. Intervals from calving to first service and to conception were longer in suckling than in non-suckling cows (p > 0.05). Cows with a good body condition score (>3.5) at calving had shorter calving to first service and conception intervals than cows in poor condition (p < 0.001). The results showed that reproductive abnormalities, coupled with poor body condition, are important factors that contributed to reproductive inefficiency. An appropriate reproductive health management, a reliable artificial insemination service and supplementary feeding could be the management options to reduce or alleviate some of the problems.     

Diagnostic tests for small ruminant lentiviruses

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.     

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: a first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n = 207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09–22.99, P < 0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002–2003, O.R. 3.69; 95% CI 1.27–11.9, P < 0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.     

Reduced Milk Production in Udder Quarters with Subclinical Mastitis and Associated Economic Losses in Crossbred Dairy Cows in Ethiopia

The objective of the study was to estimate the losses associated with subclinical mastitis (SCM) in crossbred dairy cows in the Central Highlands of Ethiopia. A split udder investigation was performed with 30 cows to determine production losses associated with SCM. Each quarter of the study cows was examined using the California Mastitis Test (CMT) and quarter milk production was measured over a period of 8 days. Production losses were determined for different CMT scores by comparing production of quarters with CMT score 0 to quarters with CMT scores trace, 1, 2 and 3, respectively. Using data from a recently published study, economic losses were determined for different farm sizes and production subsystems by multiplying the prevalence of the respective CMT scores with the production losses associated with these CMT scores. Mean quarter milk production was 0.82 + 0.40 kg per milking in the split udder trial. Milk production was reduced by 1.2%, 6.3%, and 33% in quarters with CMT scores 1+, 2+, and 3+, respectively. Using data from the published study, a quarter with SCM lost an average of 17.2% of its milk production. Production losses associated with SCM were estimated at 5.6% for the Addis Ababa Milk Shed. Stratified losses were highest (9.3%) in urban dairy farms (UDF) and small-scale farms (6.3%). The estimates of the financial losses ranged from US$29.1 in dairy herds in secondary towns (DHIST) to US$66.6 in UDF. A total loss of US$38 was estimated for each cow per lactation. Reducing mastitis in UDF (highest prevalence) to the level of DHIST (lowest prevalence) could reduce the loss by US$35. As this does not include costs associated with treatment or culling of diseased cows, this figure probably underestimates the possible benefits of control measures.