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110 blood samples from clinically healthy mares of English breed, half-blood and the Wielkopolska race were tested for the presence of EHV-1 and EHV-2 using nested PCR. 15 samples were EHV-1-positive, 24 samples were EHV-2-positive, whereas only 4 samples were both EHV-1 and EHV-2 positive. The virus was isolated from PBLs in equine dermal cell cultures by co-cultivation or by culture inoculation with cell lysates derived from PBLs. A total of 14 strains were isolated from EHV-2 and EHV-1/EHV-2-positive samples. However, all of them were identified by nPCR as being type 2. Since no EHV-1 was isolated, even from dually infected leukocytes, it was concluded that the presence of EHV-2 does not stimulate in-vitro isolation of EHV-1 from infected leukocytes. It is tempting to speculate that such stimulatory effects in-vivo may involve EHV-2-induced immunosupression. Despite any possible mechanism of EHV-1 stimulation, it seems that EHV-2 does not play a significant role in the epidemiology of EHV-1-caused miscarriages in horses since mixed infections are rather rare.
Coronavirus 800 (18) propagated in the lung cells of the calf foetus (16) was purified and concentrated by: a) differential centrifugation, b) precipitation with PEG-6000, c) repeated haemadsorption-elution assay (9). The samples were examined under a transmissive microscope after prior negative staining. There were found many virions (Fig 1 and 2) with a typical morphology of coronavirus (2, 12, 13, 14, 15, 17). Besides, there were observed some particles with internal nucleoprotein in the form of a spisal or S-shape, and empty particles (Fig 5) with bilateral concave surfaces (fig 6). Unusual peplomers forming short (Fig 7, 8) and long filaments (Fig 9, 10) were also seen. However, to identify these unusual structures as corona- viruses or coronavirus substructures (Fig 11, 12) it is necessary to employ a specific method using labelled antibodies.
The nested PCR technique (Borchers and Slater, 1993) was applied for the diagnosis of EHV-1 infections. DNA samples were isolated from livers of miscarried fetuses or dead foals. After amplification we detected EHV-1 specific sequences in 9 out of 15 fetuses and in 2 out of 6 foals. PCR results were compared to results of routinely performed immunofluorescent detection of viral antigen in cryosections of tissues. Six tested fetuses were FAT-positive, 7 FAT-negative and 2 were questionable. One FAT-negative fetus and two questionable were PCR-positive. All foals were FAT-negative. We have demonstrated that PCR is more sensitive than FAT and allows verification of inconclusive results of antigen detection. PCR is also suitable for detection of EHV-1 specific sequences in archival samples frozen up to 3 years.
Equine herpesvirus type 1 (EHV-1) is one of the major horse diseases, causing considerable worldwide losses. A variety of techniques, including nested PCR, have been used to diagnose EHV-1 infections. In this paper, a real-time PCR assay that uses non-specific SYBR Green I® fluorochrome for the detection of EHV-1 DNA is described. This method does not require post-amplification manipulations, thereby reducing the risk of cross-contamination. The assay was sensitive enough to detect EHV-1 sequences in neuronal cell cultures and also different clinical samples. The technique is specific: it was not reactive with other herpesviruses or opportunistic bacterial pathogens such as Escherichia coli, Staphylococcus epidermidis and Enterococcus faecium. In comparison to virus isolation or the nested PCR used previously, the test was more sensitive and should be useful for the common diagnosis based on its specificity and rapidity.
Producing high quality offspring of good physiological performance, able to survive to independence and, then, to reproductive maturity is a major component of life history strategies. The ability of nestling altricial birds to develop a good physiological condition depends to a large extent on the amount and quality of food provided by parents, as well as other aspects of parental care. We hypothesized that experimental changes to the original brood size should affect both parental Blue Tits and their offspring, resulting in corresponding changes in the body condition of the nestlings. Over two breeding seasons, using two habitat sites, we conducted an experiment with two manipulative treatments applied to broods of three-day-old nestlings — the reduction or enlargement of broods by three nestlings, and one non-manipulative control treatment. Our aim was to test whether the experiment would affect a number of different measures of nestling condition: blood concentrations of hemoglobin and glucose, heterophil-to-lymphocyte ratio and morphometric condition index, all being analyzed when the nestlings were 13 days old. We found no effect in the case of hemoglobin, despite the fact that it had previously been shown to be sensitive to large-scale differences in trophic conditions between habitats and years and to the experimental removal of nest parasites. All the remaining variables, i.e. heterophil-to-lymphocyte ratio, glucose concentration and morphometric condition index, responded to the experimental treatments, showing different but reasonable patterns of variation. We suggest that an experimental increase in brood size definitely hinders the development of nestling physiological condition, but even an experimental reduction of broods can affect some physiological indicators (glucose), probably because of readjustments in the feeding rate.
Amount of food supplied to nestlings by their parents is considered to affect the development of nestling physiological condition. In this study we supplied parental Great Tits Parus major with extra food, larvae of Tenebrio molitor, put into feeders close to nest-boxes, assuming that this should facilitate parental care and, as a consequence, nestling nutrition. The following nestling characteristics measured 13 days after hatching were analysed: body mass, haematocrit, blood concentrations of haemoglobin, glucose and triglycerides, heterophil-to-lymphocyte ratio (H/L), and patagium swelling after PHA injection. Nestlings from extra food broods were significantly heavier than control ones. They also had lower H/L, which indicated lower stress. No other variable was significantly affected by the experiment. Possibly, the rainy weather and non-restrictive natural trophic conditions during the experiment caused weakening of the net benefits from extra food.
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